Characterization of Waxy Rice with Floury-Core Endosperm and Its Impact on Rice Balls

Characterization of Waxy Rice with Floury-Core Endosperm and Its Impact on Rice Balls | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization of Waxy Rice with Floury-Core Endosperm and Its Impact on Rice Balls Jun Fang, Haihong Wang, Hui Zhang, Jinlong Peng, Liuyin Ma, Yuting Dai, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9480499/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Waxy rice is an important raw material in food processing, particularly for traditional Asian products such as rice balls. High-quality waxy rice powder, characterized by low levels of damaged starch and small particle size, is typically produced via wet grinding. However, this method generates substantial wastewater and consumes lots of energy, raising environmental concerns. In non-waxy rice, floury endosperm mutants have enabled high-quality rice powder through dry grinding, but this approach has not been extended to waxy rice. Here, a waxy rice mutant with floury-core endosperm was developed by an insertion mutation of the soluble starch synthase IIIa ( ssIIIa ) gene. Compared with the ssIIIa mutant of non-waxy rice, the waxy rice mutant exhibited distinct agronomic characteristics, including reduced yield-related traits. Nevertheless, the floury-core endosperm reduced starch damage and particle size in dry-ground flour, enhancing its suitability for waxy rice ball production. The mutant also contained more short chains and smaller molecules in amylopectin, associated with the upregulation of multiple ssIIIa -related genes. Those changes further resulted in decreased crystallinity and altered pasting and thermal properties. Waxy rice powder of the mutant was obtained via dry grinding and exhibited favorable processing and eating qualities. Dough was harder and easier to handle, while boiled balls were softer, less sticky, and easier to chew and swallow. Collectively, the floury-core endosperm modified agronomic traits, physicochemical properties, and gene expression in waxy rice, ultimately improving product quality. The dry grinding method using the mutant offers an environmentally friendly alternative to conventional wet grinding for high-quality waxy rice powder. ssIIIa mutation agronomic and physicochemical traits starch structures starch synthesis gene expression waxy rice processing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Waxy rice has considerable value in the food industry and is widely utilized in products such as rice ball, low-fat ice-cream and salad dressings 1 . In Asia, waxy rice balls are traditional delicacies commonly sold frozen and consumed boiled, especially during the Lantern Festival 2 . High-quality rice balls require powder with minimal damaged starch and small particle size, typically achieved through wet milling 3 . However, this process results in large amounts of wastewater and consumes considerable energy, raising environmental concerns 4 . Additionally, soluble nutrients such as proteins, vitamins, and minerals are lost during processing. In contrast, dry grinding offers a more sustainable alternative, but produces more damaged starch 3 . In non-waxy rice, floury-core endosperm mutants have successfully reduced starch damage during dry milling 5 . This phenotype appears as white core and can be triggered by either environmental conditions or genetic mutations 6 . For instance, high temperatures during early grain-filling stage often induce white-core endosperm 7 . Mutations in genes related to starch synthesis, regulatory pathways and transport processes can also generate white-core phenotypes. Mutants affecting storage proteins and grain lipids have been reported as well. Starch synthesis gene mutants include soluble starch synthase IIIa (ssIIIa) 8 , UDP-glucose pyrophosphorylase (flo8) 9 and ADP-glucose pyrophosphorylase large subunit 2 (agp-l2) 10 . Regulator and transporter gene mutants include AP2/ethylene responsive element binding protein family transcription factor (rsr1) 11 , basic leucine zipper 10 (bzip10) 12 , glyoxalase (flo15) 13 , and ADP-glucose transporter (bt1) 14 . Storage protein gene mutants include heat shock protein 70 (flo11) 15 , plant-unique phox-homology domain-containing protein (gpa5) 16 , small GTPase (sar1c, gpa13) 17 , pyruvate orthophosphate dikinase (flo4) 18 , alanine aminotransferase 1 (flo12) 19 , pyruvate dehydrogenase complex E1 component subunit Alpha 1 (flo19) 20 and class I glutamine amidotransferase (flo19-1, flo19-2) 21 . In addition, the grain lipid mutation, pyruvate kinase (pkpα1) , has been associated with white-core endosperm 22 . These mutations alter starch granule (SG) and amyloplast formation, thereby producing floury-core phenotype. Rice SG and amyloplast are mainly composed of amylose and amylopectin 10 . Previous studies on floury endosperm have primarily focused on translucent non-waxy rice containing more than 10% amylose 6 . Whereas, little research has been conducted on non-translucent waxy rice with less than 2% amylose. The extremely low apparent amylose content (AAC) in waxy rice results from deficiency of granules-bound starch synthase I (gbssI) gene, which is responsible for amylose and extra-long chains of amylopectin 23 . This makes waxy rice an ideal model to investigate floury-core endosperm formation with minimal amylose interference. However, identifying floury phenotype in non-translucent waxy rice remains challenging. In this study, a floury-core endosperm mutant was identified in waxy rice, caused by ssIIIa mutation. This mutant displayed altered agronomic traits, gene expression, and amylopectin molecular structures. More importantly, dry-grinding powder from this mutant showed reduced starch damage, smaller particle size, and influenced physicochemical properties. Furthermore, the floury-core endosperm improved the processing and eating qualities of waxy rice balls. A promising strategy for producing high-quality waxy rice powder was provided through an environmentally friendly dry grinding method. 2. Materials and methods 2.1 Plant materials An elite waxy rice, Yannuo12 ( Oryza sativa japonica), was irradiated with a 250 Gy electron beam using a linear accelerator facility (Nuctech™ IS1020, China). Floury-core phenotype was screened in the M2 generation by grain sectioning. Half seeds containing embryos with floury-core phenotype were cultured on MS medium. Survival seedlings were transplanted into paddy fields and self-pollinated for three generations to obtain a stable floury-core line designated WFC (waxy floury core). For genetic analysis, WFC plants were crossed with indica rice cultivar 9311. Then F2 mapping population were developed from self-pollinated F1 plants. 2.2 Map-based cloning and genetic complementation Gene mapping was performed using polymorphic simple sequence repeat (SSR) markers between Nipponbare and 9311 (Fig. 1 a, Table S1 ). Both parents and recessive seeds were used for preliminary mapping. And additional genetic markers were employed for fine mapping (Table S2 ). The WFC genome was sequenced, and the candidate region was compared to the Nipponbare genome (NCBI GCF_034140825.1). For complementation analysis, the full-length cDNA of wild type was cloned into the pCAMBIA1300 vector under its native promoter (2 kb upstream from the start codon 24 ) (Figure S1 ). The construct was introduced into the Agrobacterium tumefaciens strain LBA4404 and transform into WFC callus. Transgenic plants were identified by PCR amplification of hygromycin-resistance gene. Primers for construct generation, sequencing, and transgenic plant identification were listed in Table S3. 2.3 Agronomic traits To investigate the effects of the mutated locus in waxy rice, agronomic traits were compared between WFC and a complementation line, thereby eliminated the influence of other background mutations. Three plots (1.0 m 2 each) were used to evaluate plant height and panicle numbers per plant during the maturity stage. Panicles from each plant were collected to determine panicle length, grains per panicle and seed-setting rate. Grains and brown rice thickness were measured with using digital caliper (Nscing ES-1, China). While their width, length, and 1000-grain weight were analyzed using a QM3 rice analyzer system (Vibe Bnei Brak, Israel). Ten replicates were applied for traits. 2.4 Expression of ssIIIa -related genes Several genes involved starch synthesis were co-expressed with ssIIIa in rice 11 . These expression levels were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR) using QuantStudio 6 Flex system (Applied Biosystem, USA). Total RNA was isolated from developing seeds at 7 days after fertilization (DAF), and cDNA was synthesized by reverse transcription. The rice actin gene served as internal reference. Primers were listed in Table S4. Each reaction was performed in triplicate. 2.5 Chemical composition and physical properties of milled rice Floury-core endosperm in waxy rice was observed in transverse sections of brown rice using a stereo microscope (Leica EZ4W, Germany). Total starch content and damaged starch were measured with assay kits (Megazyme Wicklow, Ireland). AAC was determined using a Segmented Flow analyzer (SEAL AA3, USA). Protein content was quantified with a nitrogen and protein analyzer (Elementar Rapid N exceed, Germany). Crude fat was analyzed by hydrolysis, extraction, and evaporation using a Soxhlet extractor (Drawell Scientific DW-SXT-06, China). Ash content was determined after heating rice at 900 ℃ in a muffle furnace (Thermo Scientific Thermolyne, USA). Hardness of milled rice was measured using a texture analyzer with a P5 probe (Texture technologies TA-XT plus, USA). Rice powder was prepared by dry grinding milled rice (IKA A11 basic, Germany) and passing powder through a 100-mesh sieve. Particle size distribution in rice powder was analyzed with a laser particle size analyzer (Malvern Panalytical Mastersizer 2000, UK). Each measurement was performed with three replicates, except for chemical composition (five replicates) and hardness (ten replicates). 2.6 Starch structures SGs and amyloplasts were observed in rice transverse sections using a scanning electron microscopy (Hitachi TM4000Plus, Japan). Starch crystallinity was estimated with an X-ray diffractometry (Malvern Panalytical X’Pert Pro MPD, UK). For degree of polymerization (DP), starch was extracted from rice powder and debranched with iso-amylase. The dried starch was dissolved in a solution of 8-Aminopyrene-1,3,6-trisulfonic acid trisodium salt and urea. The filtered sample was analyzed using a high-performance anion exchange chromatography (Merck Sephacryl S-1000SF, Germany) with a pulsed amperometric detection. Branching degree was examined using a nuclear magnetic resonance (Bruker, Fourier80, Germany). Molecular weight of amylopectin was measured using a liquid chromatography with a differential refraction detector (Wyatt Optilab T-rEX, USA) and a multi-angle static light scattering detector (Wyatt Dawn Heleos II, USA). Three replicates were perform each assay. 2.7 Gelatinization and pasting properties of rice powders Gelatinization properties were measured with a differential scanning calorimeter (DSC, Mettler Toledo DSC1, USA). Samples were heat from 30–95 ℃ at a rate of 10 ℃/min. Pasting properties were examined using a rapid viscosity analyzer (shorted for RVA, Perten Tecmaster, Sweden) following the AACC standard method 25 . Each assay was conducted in triplicate. 2.8 Textural properties of waxy rice balls Dough was prepared by mixing 100 g of rice flour and 70 g of water and was allowed to rest for 20 min at room temperature (RT). Small rice balls (~ 1.5 g each) were formed and frozen at -18 ℃ for 2 days. These balls were thawed and drained at RT for 2 h. A portion of the dough was used for textural analysis, while the others were boiled in water for 5 min, then taken out and cooled to RT. Textural properties were measured using a P/36R probe and the texture profile analysis (TPA) method 2 . The pre-test, test, and post-test speeds were set to 2, 1, and 2 mm/s, respectively, with a trigger force of 5 g. Compression strain was set as 70% of sample thickness with a 5-s interval between two compressions. Five replicates were applied for both dough or boiled balls. 2.9 Statistical analysis Data on agronomic traits and physicochemical properties was analyzed with Student’s t -test in Statistical Product and Service Solutions (SPSS) software. Values were presented as means ± standard deviation (SD). Statistical significance was indicated by one asterisk (*) for P < 0.05 and double asterisks (**) for P < 0.01. 3. Results 3.1 The mutation locus encoded the SSIIIa enzyme A floury-core endosperm mutant was identified in the M2 population. In the F2 mapping population, 1,082 seeds showed normal endosperm, whereas 295 seeds displayed the floury-core phenotype, fitting to a segregation ratio of approximately 3:1. This indicated that the phenotype was controlled by a single recessive nuclear gene. The wfc locus was first mapped to chromosome 8 between markers RM5428 and R8M10. Then fine mapping further narrowed the candidate region to a 97 kb interval between RM22544 and RM22547 (Fig. 1 b), which harbored 10 open reading frames (Table 1 ). Genome sequencing revealed a 1 bp insertion in the third exon of ssIIIa , while no change of protein sequences was observed in the other genes. This insertion led to a frameshift and introduced a premature stop codon, resulting in a truncated polypeptide with 332 N-terminal amino acids (aa) instead of the full-length 1,788 aa. Furthermore, complementation test generated 25 positive lines containing the hygromycin-resistance gene. Among them, 23 lines rescued the floury-core endosperm, confirming that ssIIIa was responsible for the WFC mutant phenotype. Those results confirmed that ssIIIa was responsible for the floury-core endosperm of WFC, providing a genetic basis for following analysis of agronomic traits. Table 1 Candidate genes in the fine mapping region No length description LOC4344848 4108 disease resistance RPP13-like protein 3 LOC136351517 1100 predicted gene LOC136351420 20887 predicted gene LOC4344849 3694 myosin-2 LOC4344850 2244 cysteine-rich PDZ-binding protein LOC4344852 2377 protein PIN-LIKES 2 LOC9270306 7408 aconitate hydratase LOC4344854 3974 phosphoribosylamine–glycine ligase LOC9268758 10822 starch synthase 3a LOC4344857 2107 autophagy-related protein 8C 3.2 The ssIIIa mutation altered agronomic traits To assess the effects of the on agronomic traits, WFC plants were compared with a transgenic complementation line. Among the rescued lines, 21 lines displayed similar agronomic traits. One line (the seventh, short for L7) was randomly chosen for detailed comparison. In the field, WFC plants showed phenotypic differences compared with L7 (Fig. 2 a). Statistical analysis revealed significant reductions in plant height (-5%), grains per panicle (-11%), and seed-setting rate (-7%) in WFC (Table 2 ). Other traits, including panicle number per plant and panicle length, showed no significant differences. Width and thickness of brown rice were increased by more than 12% in WFC, whereas its length and weight remained unchanged. These findings indicated that ssIIIa mutation altered specific agronomic traits in rice. To seek mechanisms in trait variation, expression of ssIIIa -related genes was analyzed. Table 2 Agronomic traits of plants and grains Sample Plant height (cm) Panicle numbers per plant Panicle Length (cm) Grains per panicle Seed-setting rate (%) WFC 96.7 ± 1.5* 5.7 ± 1.2 15.5 ± 1.5 98.3 ± 5.1* 84.7 ± 2.5* L7 101.3 ± 2.1 6.3 ± 0.6 17.4 ± 0.9 111.3 ± 4 91.3 ± 2.1 Sample 1000-grain weight (g) Grain length (mm) Grain width (mm) Grain thickness (mm) WFC 24.9 ± 1.6 5.01 ± 0.30 3.58 ± 0.13 2.53 ± 0.50 L7 26.1 ± 1.3 4.89 ± 0.28 3.66 ± 0.17 2.21 ± 0.23 Sample 1000-brown rice weight (g) Brown rice length (mm) Brown rice width (mm) Brown rice thickness (mm) WFC 20.6 ± 1.6 4.61 ± 0.31 3.22 ± 0.30* 2.04 ± 0.17* L7 19.7 ± 1.8 4.73 ± 0.32 2.86 ± 0.28 1.81 ± 0.20 3.3 Expression of several ssIIIa -related genes up-regulated in WFC endosperm Expression of ssIIIa gene in WFC developing endosperm was reduced by 85% (Fig. 3 a). As expected, mature gbssI mRNA levels were low in both waxy rice lines. Interestingly, gbssI pre-mRNA levels were significantly higher in WFC ( p < 0.01) (Fig. 3 b). Except for branching enzyme II ( beIIb ), the other starch synthesis genes were significantly up-regulated in WFC, including ADP-glucose pyrophosphorylase small subunit 1 ( agp-s1) and its large subunit 2 ( agp-l2 ), soluble starch synthase I ( ssI ), soluble starch synthase IIa ( ssIIa ), and branching enzyme I ( beI ). Notably, ssI expression increased 2.5-fold, while two agp genes increased about 1.5-fold in WFC. Moreover, debranching enzyme genes in WFC, pullulanase ( pul ) and isoamylase2 ( isa2 ) increased 1.9- and 3.0-fold, respectively. Whereas, isoamylase1 ( isa1 ) expression was comparable between WFC and L7. Additionally, a pre-amylopectin synthesis gene, starch phosphorylase ( phol ), increased by 30% in WFC. These transcriptional changes may reflect compensatory regulation of starch metabolism in response to ssIIIa deficiency. To figure out variation influence on WFC rice, its properties were analyzed. 3.4 Grain composition and physical properties altered in WFC In WFC, floury endosperm was not visible under top lighting, but appeared opaque back lighting (Fig. 2 b). Transverse sections revealed that the core region appeared floury white in WFC, while the outer region resembled L7. Compared with L7, WFC had significantly lower total starch content ( P < 0.05 ) but higher AAC ( P < 0.01 ) (Table 3 ). Protein, lipid, and ash contents were comparable between WFC and L7. Damaged starch content in WFC was approximately 35% lower than that in L7. Moreover, WFC rice exhibited a hardness reduction of more than 20%. Particle size distribution showed that D90 and D50 values of WFC powder were significantly smaller than those of L7 ( P < 0.05). Size of the smallest 10% of WFC particles was less than 15 µm. These evidences suggested that WFC SG and amyloplastic formation was disrupted. Table 3 Rice composition, hardness, and particle size distribution Sample Total starch (%) Amylose (%) Protein (%) Lipid (%) Ash (%) WFC 77.5 ± 3.8* 2.9 ± 1.1** 7.8 ± 1.2 0.7 ± 0.1 1.5 ± 0.1 L7 82.4 ± 2.9 0.8 ± 0.5 9.0 ± 1.2 0.8 ± 0.2 1.6 ± 0.1 Sample Damage starch (%) Hardness (kg) Dx (10) Dx (50) Dx (90) WFC 7.7 ± 1.1** 5.7 ± 0.4** 14.8 ± 2.9** 45.1 ± 8.1* 104.8 ± 9.3* L7 11.9 ± 2.1 7.3 ± 0.6 30.4 ± 3.4 74.6 ± 9.2 127.9 ± 8.5 3.5 Floury core contained loosely packed SGs and amyloplasts Observation of SEM revealed structural differences between WFC and L7 grains. In floury core region (position 1), WFC performed loosely packed amyloplasts and SGs with large air spaces. In contrast, WFC amyloplasts were compact and dense in the peripheral region (position 3), but still distinguishable by clear boundaries (Fig. 2 c). In WFC floury core, small spherical and polyhedral SGs surrounded large amyloplasts. These amyloplasts had diameters of ~ 15 µm and were coated by membranes. The intermediate region (position 2) contained both compact and partially separated amyloplasts. In L7, amyloplasts were flattened and tightly packed, making individual structures difficult to distinguish. These differences likely reflected alterations in starch chain-length distribution. 3.6 Short-chain enrichment and disrupted starch structure in WFC Both WFC and L7 exhibited similar overall chain-length distribution patterns (Fig. 4 a), but differences were observed in specific fractions (Fig. 4 b). In WFC, chains with DP 10–15 and DP 22–34 increased significantly by 10% and 7%, respectively (Table 4 ). Whereas chains with DP 6–9 displayed no significantly difference. Other chains were reduced significantly by 6.2% and 15% at DP 16–21, and DP 35–75, respectively. In terms of chain categories, WFC contained more A chains (DP 6–12) and B2 chains (DP 25–36), unchanged B1 chains (DP 13–24) and fewer B3 (DP ≥ 37). Accordingly, amylopectin linkage analysis revealed reductions of 4% in α-1,4 linkages and 20% in α-1,6 linkages, resulting in a significantly lower branching degree( p < 0.05) (Table 5 ). Those alterations in WFC also led to a ~ 3% reduction in molecular size ( R z ) and 5%-8% reduction in molecular weights ( M n , M w and M z ). Molecular weight of WFC also displayed a similar peak ( M p ) and a wider polydispersity ( P < 0.05). Disrupted starch molecules led to impaired crystal structures. X-ray diffraction (XRD) patterns revealed an A-type crystal form with peaks at 15°, 17°, 18°, and 23° ( 2θ ) (Fig. 5 a). In WFC, the peaks at 17° and 18° were weaken, indicating impaired crystal structures. Relative crystallinity of WFC was reduced by approximately 15% compared with L7 (Fig. 5 b). Together, these results demonstrated that ssIIIa mutation enriched short chains which changed starch molecule and crystallization. Table 4 Chain length distribution (%) of amylopectin Chain length Distribution (%) WFC L7 DP6-9 10.82 ± 0.27 11.23 ± 0.16 DP10-15 38.95 ± 0.96* 35.30 ± 0.37 DP16-21 22.99 ± 0.56* 24.52 ± 0.34 DP22-34 18.80 ± 0.46* 17.52 ± 0.24 DP35-75 10.87 ± 0.43** 12.80 ± 0.18 A chain (DP6-12) 31.25 ± 0.76* 29.63 ± 0.30 B1 chain (DP13-24) 49.23 ± 1.21 48.80 ± 0.68 B2 chain (DP25-36) 12.29 ± 0.30* 11.38 ± 0.16 B3 chain (DP37-75) 9.66 ± 0.39** 11.55 ± 0.16 Table 5 Molecule parameters of rice starch Sample 1,4-linkage 1,6-linkage Branching degree (%) M w (kDa) M p (kDa) WFC 6551 ± 121* 187 ± 9* 2.77 ± 0.08* 80926 ± 1462* 130148 ± 963 L7 6846 ± 47 235 ± 16 3.31 ± 0.24 84767 ± 1363 132307 ± 1374 Sample M n (kDa) M z (kDa) Polydispersity ( M w / M n ) R z (nm) WFC 68745 ± 708** 87041 ± 1776* 1.18 ± 0.01* 102 ± 1.1* L7 74704 ± 920 92035 ± 1196 1.13 ± 0.01 105 ± 1.3 M w : Weight average molecular weight, M p : Peak molecular weight, M n : Number average molecular weight, M z : average molecular weight of size, R z : the average rotation radius of size. 3.7 WFC powder exhibited reduced thermal stability and pasting viscosity DSC showed significantly lower values of onset ( T 0 ) and peak ( T p ) gelatinization temperatures and enthalpy ( ∆H ) in WFC ( P < 0.05) (Table 6 ). Value of WFC enthalpy was reduced by at least 22%. Although gelatinization temperatures were lower in WFC, pasting temperatures did not differ between WFC and L7. RVA analysis revealed that pasting curves of both WFC and L7 displayed similar shapes (Fig. 6 a). From onset to the end, viscosity reached a peak and then decreased, followed by a similar stabilization. However, WFC curve reached its peak earlier than L7 ( P < 0.05). WFC viscosities were reduced by more than 41%, 55% and 56% at the peak, trough, and final positions, respectively (Fig. 6 b). Those reductions resulted in significant decreases in breakdown and setback values ( P < 0.05). These changes indicate that floury-core endosperm had directly implications for pasting and thermal properties. Those influences implied that rice foods made of WFC powder could displayed various textures. Table 6 Thermal properties with DSC dectection Sample Oneset ( T o , ℃) Peak ( T p , ℃) Conclusion ( T c , ℃) Enthalpy ( ∆H ) WFC 63.8 ± 0.5* 68.3 ± 0.3* 76.3 ± 0.5 9.1 ± 0.8* L7 65.2 ± 0.3 70.5 ± 0.6 76.5 ± 0.4 11.7 ± 0.7 3.8 Textural properties altered in WFC doughs and rice balls Textural analysis revealed a few differences between WFC and L7 products (Table 7 ). Both WFC and L7 doughs exhibited low stickiness and elasticity. Dough adhesiveness was lower than − 4 g.s, while dough cohesiveness and springiness were less than 0.1. Hardness of WFC doughs increased 4.2-fold compared with L7. Whereas hardness reduced by 51% for WFC boiled balls, and this reduction decreased gumminess and chewiness by 44% and 37%, respectively. Moreover, boiled balls showed less stickiness and higher elasticity. Compared with L7 balls, WFC balls displayed 43% lower adhesiveness of reduced, 50% higher springiness and significantly increased cohesiveness ( P < 0.01). These results demonstrated that floury-core endosperm impacted both raw and cooked rice product quality, providing practical implications for rice-based food processing. Table 7 Texture properties of rice balls Texture property dough boiled ball WFC L7 WFC L7 Hardness (g) 3226.24 ± 12.34** 767.74 ± 60.49 964.57 ± 42.81** 1968.68 ± 145.90 Adhesiveness (g.s) -1.50 ± 0.12** -3.55 ± 1.11 -171.72 ± 39.24** -304.30 ± 30.30 Springiness 0.066 ± 0.008 0.058 ± 0.010 0.804 ± 0.018** 0.719 ± 0.019 Cohesiveness 0.090 ± 0.007** 0.060 ± 0.003 0.550 ± 0.011** 0.475 ± 0.009 Gumminess 289.93 ± 17.46** 46.36 ± 6.15 529.93 ± 21.72** 936.56 ± 77.53 Chewiness 19.21 ± 2.76** 2.70 ± 0.74 425.76 ± 15.74** 674.48 ± 73.60 Resilience 0.047 ± 0.004** 0.022 ± 0.002 0.250 ± 0.007** 0.166 ± 0.006 4 Discussion The identification of the WFC mutant provides new insights into the role of ssIIIa in waxy rice endosperm development. The SSIIIa enzyme is the second major starch synthase in rice endosperm 10 , and its catalytic domain (residues 1,353–1,705) 24 loss led to non-functional activity. This result not only altered morphological traits and starch molecular structure, but also affected gene expression, physicochemical features, and final food properties. 4.1 Morphological and compositional differences in ssIIIa mutant of waxy rice The WFC mutant displayed altered agronomic traits at both plant and grain levels compared with L7 (Table 2 ). This contrasted previous studies reporting that ssIIIa deficiency alone in non-waxy rice had little influence on these traits 8 , 26 , 27 . In WFC, plant height, grains per panicle, and seed-setting rate were significantly reduced ( P < 0.05). Although WFC plants were slightly shorter, their height still remained within the range of ideal rice architecture 28 . However, the seed-setting rate and grains per panicle decreased by 7–12% in WFC, which might lead to lower yield. Floury endosperm typically reduced grain weight 29 . Whereas Brown rice weight and grain traits were unchanged in WFC, likely due to the increased width and thickness. Total starch content was decreased in WFC, consistent with ssIIIa RNAi line in non-waxy rice 30 . Unexpectedly, AAC increased slightly in WFC despite gbssI deficiency. This enhancement was not observed in ssIIIa mutants of potato and waxy maize 31 , 32 . In non-waxy rice, AAC enhancement was attributed to ssIIIa mutations upregulating gbssI expression 8 , 26 , 27 , but this explanation seemed unconvincing in waxy crops. Those results suggested that AAC variations in ssIIIa mutation might be species-specific, with mechanisms yet to be fully understood. Interestingly, the beIIb mutation also increased AAC in waxy rice 33 , implying that disruption of starch synthesis networks might underlie AAC enhancement in WFC. Floury-core endosperm in WFC contributed to lower grain hardness, reduced damaged starch, and finer particle size (Table 3 ). They were consistent with ssIIIa mutants of non-waxy rice 5 , 34 . Lower grain hardness facilitated dry-grinding powder 35 . Moreover, the size of the smallest 10% of WFC powder particles corresponded well with amyloplast size in the floury core (Fig. 2 c), further linking the phenotype to the property. 4.2 Distinct SGs and amyloplasts from other mutations Floury endosperm is commonly associated with altered amyloplasts and SGs morphology. In L7, amyloplasts appeared flattened, a feature similar with wild-type endosperm covered by thick enveloped SGs 36 . By contrast, the ssIIIa mutation in WFC increased air space between amyloplasts in floury core and generated both polyhedral and spherical SGs (Fig. 2 ). Polyhedral SGs were previously observed in ssIIIa mutants of non-waxy rice 26 , whereas ssI suppression or ssIVb deficiency promoted spherical SGs 36 , 37 . These findings suggest that gbssI deficiency was essential for spherical SGs formation in WFC. Amyloplast compactness varied across endosperm regions in WFC, from loose packing in the core (position 1) to dense structures at the periphery (position 3). This suggested that starch synthesis was initially disrupted during early endosperm development and partially recovered later. However, incomplete recovery was evident from joints structures between compact amyloplasts at periphery region in WFC. This pattern differed from the ss3a-1 mutant of non-waxy rice, where peripheral amyloplasts resembled wild type. Such distinctions were potentially due to the loss of epistatic effect of gbssI gene on ssIIIa 38 . These findings demonstrated that ssIIIa deficiency in waxy rice induced heterogeneous SG morphology and amyloplast arrangements. 4.3 Short chains enhancement led to smaller molecule and impaired crystal structure WFC amylopectin displayed increased short chains of DP 10–15 and DP 22–34, and decreased long chains of DP ≥ 35 (Table S5). This distribution closely resembles that of ssIIIa mutants in non-waxy rice, which exhibited elevated DP 9–15 and DP 20–32 8, 26, 36, 39 . In WFC, A chains and B2 chains increased, B1 chains remained stable, and B3 chains decreased. In contrast, only B1 chains increased in maize gbssI/ssIIIa double mutant, and rice ssIIIa RNAi line under high temperature 27 , 40 . Those evidences highlighted that chain length distribution was shaped by both genetic background and environmental conditions. The enhancement of short chains resulted in decreased molecular weight (5%) and molecular size (3%) (Table 5 ). In comparison, the ss3a-1 mutant in non-waxy rice exhibited a 25% reduction in molecular weight 26 . This smaller impact on WFC amylopectin suggested that amylopectin in waxy rice was more resistant to structural disruption due to their higher weight and bigger size 41 . The peak of molecular weight ( M p ) was similar between WFC and L7, and smaller WFC molecules led to the wider range. Short chains were considered to form shorter crystallites and decrease crystalline order, and amylose disrupted crystalline packing 42 . Therefore, increased short chains and AAC leading to lower relative crystallinity in WFC. These results indicate that disrupted starch structures in WFC were attributed to more short chains and smaller molecules. 4.4 Gene expression alterations promoted short chain accumulation Amylopectin chain length was regulated by starch synthesis-related genes 27 . The ssI and gbssI were increased in ssIIIa mutants of non-waxy rice 26 . In WFC, the upregulation of ssI resembled that in non-waxy rice, contributing to the accumulation of short amylopectin chains with DP 8–12 and DP 20–30. Interestingly, gbssI pre-mRNA was elevated in WFC, while mature mRNA remained comparable to L7. This result reflected post-transcriptional regulation: the pre-mRNA levels of gbssI were similar in waxy and non-waxy rice, and post-transcriptional procedure handled these pre-mRNA to mature mRNA at different levels 43 . Besides, both ssIIIa and gbssI participate in long-chain synthesis 10 , and their deficiency favored the accumulation of shorter amylopectin chains in WFC. In addition, the beI gene cooperated with ssIIIa and ssIIa to synthesize long chains. Although ssIIa and beI expressions increased in WFC, the contribution of beI may be limited due to the ssIIIa deficiency and low SSIIa activity in japonica rice. In WFC, expressions of agp-s1 and agp-l2 genes were upregulated, consistent with observations in ssIIIa RNAi lines under high temperature 27 . Enhanced AGPase activity likely provided more ADP-glucose substrate for chain biosynthesis, but disrupted function of long-chain synthesis caused short chain accumulation. Debranching enzyme, isa1 , works association with isa2 and pul to complete amylopectin structures 10 . However, isa1 expression remained unchanged in WFC, indicating that the upregulation of isa2 and pul likely had minimal impact on amylopectin structures. The phol gene was essential for pre-amylopectin synthesis from maltose 44 , and its overexpression led to floury endosperm and more short and DP 25–35 chains in non-waxy rice 45 , 46 . Enhanced phol expression in WFC might partially contribute to the floury-core formation and increased proportions of DP 25–34 chains. 4.5 Thermal and pasting properties reflected changes of floury-core endosperm and molecules Thermal properties were strongly influenced by amylopectin chain length 10 . Gelatinization properties were negative with A chains and positive with B1 chains in waxy rice. In WFC, enhanced A chains and unchanged B1 chains decreased onset ( T 0 ,), peak ( T p ), and enthalpy ( ∆H ) values (Table 6 ). This result was consistence with ssIIIa mutants in non-waxy rice 8 , 26 , but opposite to gbssI/ssIIIa double mutation in maize 40 . It was likely due to differences of ssIIa activity across genetic backgrounds. Although ssIIa expression was upregulated in WFC, its low activity limited influence on gelatinization temperature in WFC. Typically, AAC positively influenced pasting and thermal properties in non-floury endosperm 10 , but opposite result was performed in ssIIIa-1 26 and WFC mutants. It implied that floury endosperm had a greater impact on those properties. The floury region played a key role in pasting behavior: less energy was required to disrupt separated amyloplasts and SGs. RVA profiles revealed characteristic patterns of waxy rice 47 in both WFC and L7 (Fig. 6 a). In WFC, viscosity peak was earlier, consistent with less required energy. Chain length distribution showed no significant correlation with pasting viscosity in non-floury rice 10 . However, reduction of molecular size and weight contributed to lower viscosity 48 , whereas these reduction due to floury core decreased viscosity in WFC. In contrast, beIIb/gbssI double mutation increased viscosity with depleted chains in DP 6–14 and enriched chains in DP ≥ 15 33 , implying that short chains accumulation was positive related with pasting viscosity. Notably, the setback value of waxy rice was lower than that of non-waxy rice 49 , whereas WFC displayed an even lower setback, indicating reduced retrogradation tendency and improved food stability. 4.4 Improved processing and tasting quality in WFC-based products The textural properties of waxy rice dough and boiled rice balls are critical for both processing performance and consumer acceptance. Dough containing less damaged starch resulted in lower solubility and higher water-holding capacity 3 . When mixed with the same amount of water, WFC dough exhibited greater hardness and easier shaping. In contrast, L7 dough containing excess soluble fraction led to softer texture and reduced shaping ability. Short chains enrichment, finer particle size and less damaged starch improved quality properties in rice-based foods 23 , 50 , 51 . After boiling, WFC rice balls displayed desirable qualities, including softer, less stickiness, and more elasticity. Furthermore, WFC rice balls showed reduced gumminess and chewiness, allowing faster chewing and swallowing. This property was particularly suitable for children and elderly consumers. Those findings demonstrated practical advantages of the floury-core endosperm in waxy rice. Conclusion The effects of the floury-core endosperm on agronomic traits, physicochemical properties, gene expression, and textural characteristics were systematically investigated in waxy rice. The WFC mutant exhibited reduced grains per panicle and seed-setting rate, which could negatively impact yield. Therefore, the ssIIIa mutation should be introgressed into waxy rice cultivars with higher seed-setting rates and more grain numbers to maintain optimal yield in future breeding programs. At the molecular level, increased short chains resulted in smaller molecular size in WFC. It likely underlain the formation of the floury-core endosperm. Upregulation of multiple ssIIIa -related genes in WFC indicated that the endosperm starch synthesis network partially compensated for ssIIIa deficiency. This compensation likely contributed to the formation of compact starch granules in the peripheral endosperm and enhanced rate of milled rice. From a processing perspective, WFC powder produced by dry grinding had smaller particle size and lower starch damage, resulting in favorable thermal and pasting properties. These characteristics enhanced processing performance and consumer-relevant qualities of WFC-based products. Overall, the floury-core endosperm of WFC demonstrates a promising trait for improving waxy rice processing. Dry-grinding powder derived from WFC mutant might serve as a suitable alternative to conventionally wet-grinding powder in food applications. Declarations Ethics approval Not applicable. Competing interests All the authors agreed on the contents of the paper and post no conflicting interest. Funding This work was supported by the Research Funds from Germplasm Innovation Group (No. JCYJ261201), SAAS Excellent Research Group (No. NKC2017A05) and Special Rice Variety Group of Wine Clean Production (No. JS01202XM25001). Author Contribution J. F. performed designed the experiments, gene expression and wrote the manuscript, H. W. performed phenotype analysis and gene cloning, Z. H. and J. P. performed rice powder preparation, L. M. performed gene mapping, Y. Z. and J. J. performed agronomic traits, Y. D. and T. S. performed data analysis, W. Y. performed the mutation development, C. C. and F. N. performed pasting and thermal properties, J. Z. and B. S. performed ball preparation and texture properties, H. C. and L. C. designed the experiments and revised the manuscript. All the authors reviewed the manuscript, agreeded on the contents of the paper, and posted no conflicting interest. Acknowledgements Not applicable. Availability of data and material The datasets supporting the conclusions of this article are provided within the article and its additional files. References Bryant RJ, Kadan RS, Champagne ET, Vinyard BT, Boykin D (2001) Functional and digestive characteristics of extruded rice flour. Cereal Chem 78:131–137 Wang H, Xiao N, Wang X, Zhao X, Zhang H (2019) Effect of pregelatinized starch on the characteristics, microstructures, and quality attributes of glutinous rice flour and dumplings. Food Chem 283:248–256 Fang S, Chen M, Xu F, Liu F, Zhong F (2023) The possibility of replacing wet-milling with dry-milling in the production of waxy rice flour for the application in waxy rice ball. Foods 12:1–17 Tian Y, Sun J, Li J, Wang A, Nie M, Gong X, Wang L, Liu L, Wang F, Tong L (2024) Effects of milling methods on rice flour properties and rice product quality: A review. Rice Sci 31:33–46 Kang JW, Lee SK, Shim SH, Shin D, Cho JH, Lee JY, Ko JM, Ji HYS, Park HM, Ahn EK, Lee JH, Jeon JS (2023) Dry-milled flour rice 'Seolgaeng' harbors a mutated fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase2. Front Plant Sci 14:1–11 Mo Y, Jeung J (2020) The use of floury endosperm mutants to develop rice cultivars suitable for dry milling. Plant Biotechnol Rep 14:185–191 Wu HM, Ren YL, Dong H, Xie C, Zhao L, Wang X, Zhang FL, Zhang BL, Jiang XK, Huang YS, Jing RN, Wang J, Miao R, Bao XH, Yu MZ, Nguyen T, Mou CL, Wang YL, Wang YH, Lei CL, Cheng ZJ, Jiang L, Wan JM (2024) Floury endosperm24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice. New Phytol 242:2635–2651 Ryoo N, Yu C, Park CS, Baik MY, Park IM, Cho MH, Bhoo SH, An G, Hahn TR, Jeon JS (2007) Knockout of a starch synthase gene OsSSIIIa/Flo5 causes white-core floury endosperm in rice (Oryza sativa L). Plant Cell Rep 26:1083–1095 Long W, Dong B, Wang Y, Pan P, Wang Y, Liu L, Chen X, Liu X, Liu S, Tian Y, Chen L, Wan J (2017) Floury endosperm8, encoding the UDP-glucose pyrophosphorylase 1, affects the synthesis and structure of starch in rice endosperm. J Plant Biology 60:513–522 Bao J (2019) Rice starch. Rice Chemistry and Technology (Fourth Edition). Elsevier Inc.: AACC International,, pp 55–108 Fu F, Xue H (2010) Coexpression analysis identifies rice starch regulator1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator. Plant Physiol 154:927–938 Jiang M, Zhang HL, Song Y, Chen JL, Bai JJ, Tang JH, Wang Q, Fotopoulos V, Zhu QH, Yang RF, Li RQ (2024) Transcription factor OsbZIP10 modulates rice grain quality by regulating OsGIF1. Plant J 119:2181–2198 You XM, Zhang WW, Hu JL, Jing RN, Cai Y, Feng ZM, Kong F, Zhang J, Yan HG, Chen WW, Chen XG, Ma J, Tang XJ, Wang P, Zhu SS, Liu LL, Jiang L, Wan JM (2019) Floury endosperm15 encodes a glyoxalase I involved in compound granule formation and starch synthesis in rice endosperm. Plant Cell Rep 38:345–359 Li SF, Wei XJ, Ren YL, Qiu JH, Jiao GA, Guo XP, Tang SQ, Wan JM, Hu PS (2017) OsBT1 encodes an ADP-glucose transporter involved in starch synthesis and compound granule formation in rice endosperm. Sci Rep 7:1–13 Zhu X, Teng X, Wang Y, Hao Y, Jing R, Wang Y, Liu Y, Zhu J, Wu M, Zhong M, Chen X, Zhang Y, Zhang W, Wang C, Wang Y, Wan J (2018) Floury endosperm11 encoding a plastid heat shock protein 70 is essential for amyloplast development in rice. Plant Sci 277:89–99 Ren YL, Wang YH, Pan T, Wang YL, Wang YF, Gan L, Wei ZY, Wang F, Wu MM, Jing RN, Wang JC, Wan GX, Bao XH, Zhang BL, Zhang PC, Zhang Y, Ji Y, Lei CL, Zhang X, Cheng ZJ, Lin QB, Zhu SS, Zhao ZC, Wang J, Wu CY, Qiu LJ, Wang HY, Wan JM (2020) GPA5 encodes a Rab5a effector required for post-golgi trafficking of rice storage proteins. Plant Cell 32:758–777 Bao XH, Wang YF, Qi YZ, Lei CL, Wang YL, Pan T, Yu MZ, Zhang Y, Wu HM, Zhang PC, Ji Y, Yang H, Jiang XK, Jing RA, Yan MY, Zhang BL, Gu CW, Zhu JP, Hao YY, Lei J, Zhang S, Chen XL, Chen RB, Sun YL, Zhu Y, Zhang X, Jiang L, Visser RGF, Ren YL, Wang YH, Wan JM (2023) A deleterious Sar1c variant in rice inhibits export of seed storage proteins from the endoplasmic reticulum. Plant Mol Biol 111:291–307 Ha SK, Lee HS, Lee SY, Lee CM, Mo YJ, Jeung JU (2023) Characterization of flo4-6, a novel cyOsPPDKB allele conferring floury endosperm characteristics suitable for dry-milled rice flour production. Agronomy-Basel 13:1–10 Zhong M, Liu X, Liu F, Ren Y, Wang Y, Zhu J, Teng X, Duan E, Wang F, Zhang H, Wu M, Hao Y, Zhu X, Jing R, Guo X, Jiang L, Wang Y, Wan J (2019) Floury endosperm12 encoding alanine aminotransferase 1 regulates carbon and nitrogen metabolism in rice. J Plant Biology 62:61–73 Lei J, Teng X, Wang YF, Jiang XK, Zhao HH, Zheng XM, Ren YL, Dong H, Wang YL, Duan EC, Zhang YY, Zhang WW, Yang H, Chen XL, Chen RB, Zhang Y, Yu MZ, Xu SB, Bao XH, Zhang PC, Liu SJ, Liu X, Tian YL, Jiang L, Wang YH, Wan JM (2022) Plastidic pyruvate dehydrogenase complex E1 component subunit Alpha1 is involved in galactolipid biosynthesis required for amyloplast development in rice. Plant Biotechnol J 20:437–453 Lou G, Chen P, Zhou H, Li P, Xiong J, Wan S, Zheng Y, Alam M, Liu R, Zhou Y, Yang H, Tian Y, Bai J, Rao W, Tan X, Gao H, Li Y, Gao G, Zhang Q, Li X, Liu C, He Y (2021) Floury endosperm19 encoding a class I glutamine amidotransferase affects grain quality in rice. Mol Breeding 41:1–15 Cai Y, Zhang WW, Jin J, Yang XM, You XM, Yan HG, Wang L, Chen J, Xu JH, Chen WW, Chen XG, Ma J, Tang XJ, Kong F, Zhu XP, Wang GX, Jiang L, Terzaghi W, Wang CM, Wan JM (2018) OsPKpα1 encodes a plastidic pyruvate kinase that affects starch biosynthesis in the rice endosperm. J Integr Plant Biol 60:1097–1118 Honda Y, Saito Y, Katsumi N, Nishikawa M, Takagi H (2023) Physicochemical properties of starch in rice flour with different hardening rates of glutinous rice cake (mochi). J Cereal Sci 112:1–6 Mishra BP, Kumar R, Mohan A, Gill KS (2017) Conservation and divergence of starch synthase III genes of monocots and dicots. PLoS ONE 12:e0189303 AACC Approved Methods of Analysis (1999) 11th Ed. Cereals & Grains Association, St. Paul, MN, U.S.A. Fujita N, Yoshida M, Kondo T, Saito K, Utsumi Y, Tokunaga T, Nishi A, Satoh H, Park JH, Jane JL, Miyao A, Hirochika H, Nakamura Y (2007) Characterization of SSIIIa-deficient mutants of rice: the function of SSIIIa and pleiotropic effects by SSIIIa deficiency in the rice endosperm. Plant Physiol 144:2009–2023 Zhao Q, Ye Y, Han Z, Zhou L, Guan X, Pan G, Asad MA, Cheng F (2020) SSIIIa-RNAi suppression associated changes in rice grain quality and starch biosynthesis metabolism in response to high temperature. Plant Sci 294:1–12 Chen W-f, Xu Z-j, Tang L (2012) Advances and Prospects in Research on Super Rice Breeding. J Shenyang Agricultural Univ 43:643–649 Song XH, Chen ZH, Du X, Li B, Fei YY, Tao YJ, Wang FQ, Xu Y, Li WQ, Wang J, Liang GH, Zhou Y, Tan XL, Li YL, Yang J (2023) Generation of new rice germplasms with low amylose content by CRISPR/Cas9-targeted mutagenesis of the floury endosperm 2 gene. Front Plant Sci 14:1–10 Zhou H, Wang L, Liu G, Meng X, Jing Y, Shu X, Kong X, Sun J, Yu H, Smith SM, Wu D, Li J (2016) Critical roles of soluble starch synthase SSIIIa and granule-bound starch synthase Waxy in synthesizing resistant starch in rice. Natl Acad Sci U S A 113:12844–12849 Wang YJ, White P, Pollak L, Jane J (1993) Characterization of starch structures of 17 maize endosperm mutant genotypes with Oh43 inbred line background. Cereal Chem 70:171–179 Lloyd JR, Landschutze V, Kossmann J (1999) Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin. Biochem J 338:515–521 Kubo A, Akdogan G, Nakaya M, Shojo A, Suzuki S, Satoh H, Kitamura S (2010) Structure, physical, and digestive properties of starch from wx ae double-mutant rice. J Agric Food Chem 58:4463–4469 Mo YJ, Jeung JU, Shin YS, Park CS, Kang KH, Kim BK (2013) Agronomic and genetic analysis of Suweon 542, a rice floury mutant line suitable for dry milling. Rice (NY) 6:1–12 Kwak J, Yoon M-R, Lee J-S, Lee J-H, Ko S, Tai TH, Won Y-J (2017) Morphological and starch characteristics of the Japonica rice mutant variety Seolgaeng for dry-milled flour. Food Sci Biotechnol 26:43–48 Toyosawa Y, Kawagoe Y, Matsushima R, Crofts N, Ogawa M, Fukuda M, Kumamaru T, Okazaki Y, Kusano M, Saito K, Toyooka K, Sato M, Ai Y, Jane JL, Nakamura Y, Fujita N (2016) Deficiency of starch synthase IIIa and IVb alters starch granule morphology from polyhedral to spherical in rice endosperm. Plant Physiol 170:1255–1270 Fujita N, Satoh R, Hayashi A, Kodama M, Itoh R, Aihara S, Nakamura Y (2011) Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa. J Exp Bot 62:4819–4831 Yang B, Xu S, Xu L, You H, Xiang X (2018) Effects of Wx and its interaction with SSIII-2 on rice eating and cooking qualities. Front Plant Sci 9:1–10 Hayashi M, Kodama M, Nakamura Y, Fujita N (2015) Thermal and pasting properties, morphology of starch granules, and crystallinity of endosperm starch in the rice ssI and ssIIIa double-mutant. J Appl Glycosci 62:81–86 Jane J, Chen YY, Lee LF, McPherson AE, Wong KS, Radosavljevic M, Kasemsuwan T (1999) Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chem 76:629–637 Park IM, Ibáñez AM, Shoemaker CF (2007) Rice starch molecular size and its relationship with amylose content. Starch 59:69–77 You S-Y, Lim S-T, Lee JH, Chung H-J (2014) Impact of molecular and crystalline structures on in vitro digestibility of waxy rice starches. Carbohydr Polym 112:729–735 Wang Y, Dong H, Yao H, Yao J, Cheng F, Fang Y, Dai C, Li D (2008) The abundance of GBSS, SBE and its effect on amylose content. J Shandong Agricultural Univ 39:501–505 Nakamura Y, Steup M (2025) Contents and structures of branched and linear maltodextrins, malto-oligosaccharides, and sugars in the early developmental stage of phosphorylase1 mutant endosperm of rice. Carbohydr Polym 348:1–7 Nakamura Y, Ono M, Sawada T, Crofts N, Fujita N, Steup M (2017) Characterization of the functional interactions of plastidial starch phosphorylase and starch branching enzymes from rice endosperm during reserve starch biosynthesis. Plant Sci 264:83–95 Liu S, Shao G, Jiao G, Zhu M, Wu J, Cao R, Chen Y, Xie L, Sheng Z, Tang S (2021) Editing of rice endosperm plastidial phosphorylase gene OsPho1 advances its function in starch synthesis. Rice Sci 28:209–211 Kong X, Sun X, Xu F, Umemoto T, Chen H, Bao J (2014) Morphological and physicochemical properties of two starch mutants induced from a high amylose indica rice by gamma irradiation. Starch 66:157–165 Xu Y, Cheng K, Zhao S, Xiong S (2007) Molecular weight distribution of rice starches and its correlation with viscosity. Scientia Agricultura Sinica 40:566–577 Wu D, Shu Q, Xia Y (2001) Assisted-selection for early indica rice with good eating quality by RVA Profile. Acta Agron sinica 27:165–172 Qin W, Lin Z, Wang A, Xiao T, He Y, Chen Z, Wang L, Liu L, Wang F, Tong L-T (2021) Influence of damaged starch on the properties of rice flour and quality attributes of gluten-free rice bread. J Cereal Sci 101:1–8 Qin W, Lin Z, Wang A, Chen Z, He Y, Wang L, Liu L, Wang F, Tong L-T (2021) Influence of particle size on the properties of rice flour and quality of gluten-free rice bread. LWT-Food Sci Technol 151:1–9 Additional Declarations No competing interests reported. Supplementary Files WFCFigureSupplementary.doc WFCTableSupplementary.xls Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 May, 2026 Reviewers agreed at journal 17 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers invited by journal 24 Apr, 2026 Editor assigned by journal 22 Apr, 2026 Submission checks completed at journal 22 Apr, 2026 First submitted to journal 21 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9480499","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632260600,"identity":"3304c803-b8fb-4a25-96fc-9682f630df17","order_by":0,"name":"Jun Fang","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Fang","suffix":""},{"id":632260601,"identity":"53d41a4b-d2ad-4e94-8fe1-fe8095511c3e","order_by":1,"name":"Haihong Wang","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Haihong","middleName":"","lastName":"Wang","suffix":""},{"id":632260602,"identity":"bb40cb4e-0c9d-4fa7-920a-42ac991c070f","order_by":2,"name":"Hui Zhang","email":"","orcid":"","institution":"Shanghai Jinfeng Wine Co. Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zhang","suffix":""},{"id":632260603,"identity":"6bc98dba-e19e-4e98-8207-e5cf3c0d446b","order_by":3,"name":"Jinlong Peng","email":"","orcid":"","institution":"Shanghai Jinfeng Wine Co. Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Jinlong","middleName":"","lastName":"Peng","suffix":""},{"id":632260604,"identity":"49a8a99d-ab4e-4936-bc5a-fb1ec663dddf","order_by":4,"name":"Liuyin Ma","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Liuyin","middleName":"","lastName":"Ma","suffix":""},{"id":632260605,"identity":"88127199-b3ea-418a-aafc-de09493bdee2","order_by":5,"name":"Yuting Dai","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Dai","suffix":""},{"id":632260606,"identity":"ae57b222-83fb-4157-bc2d-64e6ec0f800e","order_by":6,"name":"Weiqiang Yan","email":"","orcid":"","institution":"Shanghai SN Irradiation Technology Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Weiqiang","middleName":"","lastName":"Yan","suffix":""},{"id":632260607,"identity":"20e6384b-67e7-4990-a901-e5a6c570627d","order_by":7,"name":"Yuanhong Zhu","email":"","orcid":"","institution":"Qingpu Modern Agricultural Park","correspondingAuthor":false,"prefix":"","firstName":"Yuanhong","middleName":"","lastName":"Zhu","suffix":""},{"id":632260608,"identity":"23c52a5f-8ebe-4b2c-98aa-c8efe357b262","order_by":8,"name":"Jiashun Jin","email":"","orcid":"","institution":"Qingpu Modern Agricultural Park","correspondingAuthor":false,"prefix":"","firstName":"Jiashun","middleName":"","lastName":"Jin","suffix":""},{"id":632260610,"identity":"f0e20670-866f-4465-bf81-1d58f0e3696d","order_by":9,"name":"Can Cheng","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Can","middleName":"","lastName":"Cheng","suffix":""},{"id":632260611,"identity":"558909ef-ec3a-4c02-b069-bf969c8689ab","order_by":10,"name":"Fuan Niu","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fuan","middleName":"","lastName":"Niu","suffix":""},{"id":632260612,"identity":"3109c3e5-2ea8-403f-850d-28729c5deea3","order_by":11,"name":"Jihua Zhou","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jihua","middleName":"","lastName":"Zhou","suffix":""},{"id":632260614,"identity":"7f0f71eb-4061-41d7-88e3-a269081f99f0","order_by":12,"name":"Anpeng Zhang","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Anpeng","middleName":"","lastName":"Zhang","suffix":""},{"id":632260615,"identity":"90459e91-09fa-412c-8558-870013286487","order_by":13,"name":"Bing Sun","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Sun","suffix":""},{"id":632260616,"identity":"f7c79cd0-3144-4960-9e1e-5c5815c46768","order_by":14,"name":"Huangwei Chu","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Huangwei","middleName":"","lastName":"Chu","suffix":""},{"id":632260617,"identity":"923117bd-baa9-496e-87eb-0bb882f258c3","order_by":15,"name":"Liming Cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYDACdgaGw38qbHj4+RuI1cLMwPiA50yajOSMA8RrYTbgbTtsY9CQQKQO/mbuNAnJtvM8BgwHGD98zCFCi8Rh3m0SBudu85gzNzBLztxGhBYDZqCWhLLbPJYNB9iAbGK1HGA7x2NwIIF4LZsNG9oOkKAF6JeNjxnOJPNIzjjYTJxf+Nt7NxxmqLCz5+dvPvjhIzFakABjA2nqR8EoGAWjYBTgBgBvhDNE1hdKWgAAAABJRU5ErkJggg==","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Liming","middleName":"","lastName":"Cao","suffix":""}],"badges":[],"createdAt":"2026-04-21 07:38:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9480499/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9480499/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108494194,"identity":"6e33a072-63ef-48ad-8cea-59656385909d","added_by":"auto","created_at":"2026-05-05 10:02:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91718,"visible":true,"origin":"","legend":"\u003cp\u003eMapping of the WFC locus. (a) Mapping markers distribution in rice chromosome. (b) The candidate gene was localized to a 97-kb interval on chromosome 8 between markers RM22544 and RM22547. Sequencing revealed a 1-bp insertion in the third exon of the \u003cem\u003essIIIa\u003c/em\u003egene. The box indicated the inserted nucleotide base pair.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/b9ceba3b572285c1374ab7bc.jpg"},{"id":108453975,"identity":"f50d4d8a-8fbc-42e5-87c7-3784fa64125e","added_by":"auto","created_at":"2026-05-04 20:26:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78640,"visible":true,"origin":"","legend":"\u003cp\u003eMorphologic characterization of WFC and L7. a. Plants phenotypes. b. Grain morphology under top light (left) and backlight (right), showing the floury-core phenotype in WFC. c. SEM images of transverse sections at three positions, revealing loosely packed amyloplasts in the WFC core region. Position 1, 2 and 3 were indicated with red numbers.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/cdd505e7fedac8a5de500dea.jpg"},{"id":108494051,"identity":"c52e6f43-df60-4dfc-9d62-d2f071b004c7","added_by":"auto","created_at":"2026-05-05 10:02:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41352,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of\u003cem\u003e ssIIIa\u003c/em\u003e-related genes at 7 DAF. a. Expression levels of soluble starch synthesis, \u003cem\u003eagp\u003c/em\u003e and debranching enzyme genes. b. \u003cem\u003egbssI\u003c/em\u003e pre-mRNA and mature mRNA levels, along with branching and debranching enzyme genes.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/acaf1fba9a790da2ba41e639.jpg"},{"id":108453977,"identity":"a8e2c9f8-c900-464a-aee0-78104fe1adc2","added_by":"auto","created_at":"2026-05-04 20:26:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51757,"visible":true,"origin":"","legend":"\u003cp\u003eChain length distribution of amylopectin. a. Comparison between WFC (grey square) and L7 (black square). b. Relative difference showing increased short chains (DP 10-15, 22-34) and decreased long chains (DP ≥35) in WFC.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/957303a15aed10dcaa59d04c.jpg"},{"id":108453978,"identity":"42521457-ee3e-418c-b319-536d474673eb","added_by":"auto","created_at":"2026-05-04 20:26:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30691,"visible":true,"origin":"","legend":"\u003cp\u003eStarch crystal structure. (a) X-ray diffraction patterns displaying A-type crystal from. (b) Relative crystallinity reduced in the WFC starch.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/577e28ffe9b714b9c86ddff9.jpg"},{"id":108493529,"identity":"9eb466e6-c5f3-417c-a0a9-60b3788927ca","added_by":"auto","created_at":"2026-05-05 10:00:53","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50645,"visible":true,"origin":"","legend":"\u003cp\u003eRVA analysis of the rice flour. (a) RVA curves showing similar sharps. (b) Pasting properties performing difference between WFC and L7.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/81dbe239b565c7ecc03c9f69.jpg"},{"id":108495494,"identity":"69f0c217-4fa4-4f4e-bcca-538563852adb","added_by":"auto","created_at":"2026-05-05 10:10:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":917261,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/b3cc4675-30ce-45ba-b5cc-ddfe5681365a.pdf"},{"id":108453972,"identity":"b5198f3e-fe80-471c-92f2-3e1c815781c5","added_by":"auto","created_at":"2026-05-04 20:26:05","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":102912,"visible":true,"origin":"","legend":"","description":"","filename":"WFCFigureSupplementary.doc","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/3dcb8c73db09144fd2e21ea8.doc"},{"id":108493422,"identity":"f9e8c752-c2f0-4fba-a724-537721578f1d","added_by":"auto","created_at":"2026-05-05 10:00:20","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54272,"visible":true,"origin":"","legend":"","description":"","filename":"WFCTableSupplementary.xls","url":"https://assets-eu.researchsquare.com/files/rs-9480499/v1/d3e7449ff89ae1bfd6a86141.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of Waxy Rice with Floury-Core Endosperm and Its Impact on Rice Balls","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWaxy rice has considerable value in the food industry and is widely utilized in products such as rice ball, low-fat ice-cream and salad dressings\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. In Asia, waxy rice balls are traditional delicacies commonly sold frozen and consumed boiled, especially during the Lantern Festival\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. High-quality rice balls require powder with minimal damaged starch and small particle size, typically achieved through wet milling\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. However, this process results in large amounts of wastewater and consumes considerable energy, raising environmental concerns\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Additionally, soluble nutrients such as proteins, vitamins, and minerals are lost during processing. In contrast, dry grinding offers a more sustainable alternative, but produces more damaged starch\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. In non-waxy rice, floury-core endosperm mutants have successfully reduced starch damage during dry milling\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. This phenotype appears as white core and can be triggered by either environmental conditions or genetic mutations\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. For instance, high temperatures during early grain-filling stage often induce white-core endosperm\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Mutations in genes related to starch synthesis, regulatory pathways and transport processes can also generate white-core phenotypes. Mutants affecting storage proteins and grain lipids have been reported as well. Starch synthesis gene mutants include \u003cem\u003esoluble starch synthase IIIa (ssIIIa)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eUDP-glucose pyrophosphorylase (flo8)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eADP-glucose pyrophosphorylase large subunit 2 (agp-l2)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Regulator and transporter gene mutants include \u003cem\u003eAP2/ethylene responsive element binding protein family transcription factor (rsr1)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ebasic leucine zipper 10 (bzip10)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eglyoxalase (flo15)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eADP-glucose transporter (bt1)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Storage protein gene mutants include \u003cem\u003eheat shock protein 70 (flo11)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eplant-unique phox-homology domain-containing protein (gpa5)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003esmall GTPase (sar1c, gpa13)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003epyruvate orthophosphate dikinase (flo4)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ealanine aminotransferase 1 (flo12)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003epyruvate dehydrogenase complex E1 component subunit Alpha 1 (flo19)\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eclass I glutamine amidotransferase (flo19-1, flo19-2)\u003c/em\u003e\u003csup\u003e\u003cem\u003e21\u003c/em\u003e\u003c/sup\u003e. In addition, the grain lipid mutation, \u003cem\u003epyruvate kinase (pkpα1)\u003c/em\u003e, has been associated with white-core endosperm\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. These mutations alter starch granule (SG) and amyloplast formation, thereby producing floury-core phenotype.\u003c/p\u003e \u003cp\u003eRice SG and amyloplast are mainly composed of amylose and amylopectin\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Previous studies on floury endosperm have primarily focused on translucent non-waxy rice containing more than 10% amylose\u003csup\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sup\u003e. Whereas, little research has been conducted on non-translucent waxy rice with less than 2% amylose. The extremely low apparent amylose content (AAC) in waxy rice results from deficiency of \u003cem\u003egranules-bound starch synthase I (gbssI)\u003c/em\u003e gene, which is responsible for amylose and extra-long chains of amylopectin\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. This makes waxy rice an ideal model to investigate floury-core endosperm formation with minimal amylose interference. However, identifying floury phenotype in non-translucent waxy rice remains challenging.\u003c/p\u003e \u003cp\u003eIn this study, a floury-core endosperm mutant was identified in waxy rice, caused by \u003cem\u003essIIIa\u003c/em\u003e mutation. This mutant displayed altered agronomic traits, gene expression, and amylopectin molecular structures. More importantly, dry-grinding powder from this mutant showed reduced starch damage, smaller particle size, and influenced physicochemical properties. Furthermore, the floury-core endosperm improved the processing and eating qualities of waxy rice balls. A promising strategy for producing high-quality waxy rice powder was provided through an environmentally friendly dry grinding method.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant materials\u003c/h2\u003e \u003cp\u003eAn elite waxy rice, Yannuo12 (\u003cem\u003eOryza sativa\u003c/em\u003e japonica), was irradiated with a 250 Gy electron beam using a linear accelerator facility (Nuctech\u0026trade; IS1020, China). Floury-core phenotype was screened in the M2 generation by grain sectioning. Half seeds containing embryos with floury-core phenotype were cultured on MS medium. Survival seedlings were transplanted into paddy fields and self-pollinated for three generations to obtain a stable floury-core line designated WFC (waxy floury core). For genetic analysis, WFC plants were crossed with indica rice cultivar 9311. Then F2 mapping population were developed from self-pollinated F1 plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Map-based cloning and genetic complementation\u003c/h2\u003e \u003cp\u003eGene mapping was performed using polymorphic simple sequence repeat (SSR) markers between Nipponbare and 9311 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Both parents and recessive seeds were used for preliminary mapping. And additional genetic markers were employed for fine mapping (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The WFC genome was sequenced, and the candidate region was compared to the Nipponbare genome (NCBI GCF_034140825.1). For complementation analysis, the full-length cDNA of wild type was cloned into the pCAMBIA1300 vector under its native promoter (2 kb upstream from the start codon\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The construct was introduced into the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain LBA4404 and transform into WFC callus. Transgenic plants were identified by PCR amplification of hygromycin-resistance gene. Primers for construct generation, sequencing, and transgenic plant identification were listed in Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Agronomic traits\u003c/h2\u003e \u003cp\u003eTo investigate the effects of the mutated locus in waxy rice, agronomic traits were compared between WFC and a complementation line, thereby eliminated the influence of other background mutations. Three plots (1.0 m\u003csup\u003e2\u003c/sup\u003e each) were used to evaluate plant height and panicle numbers per plant during the maturity stage. Panicles from each plant were collected to determine panicle length, grains per panicle and seed-setting rate. Grains and brown rice thickness were measured with using digital caliper (Nscing ES-1, China). While their width, length, and 1000-grain weight were analyzed using a QM3 rice analyzer system (Vibe Bnei Brak, Israel). Ten replicates were applied for traits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Expression of \u003cem\u003essIIIa\u003c/em\u003e-related genes\u003c/h2\u003e \u003cp\u003eSeveral genes involved starch synthesis were co-expressed with \u003cem\u003essIIIa\u003c/em\u003e in rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. These expression levels were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR) using QuantStudio 6 Flex system (Applied Biosystem, USA). Total RNA was isolated from developing seeds at 7 days after fertilization (DAF), and cDNA was synthesized by reverse transcription. The rice \u003cem\u003eactin\u003c/em\u003e gene served as internal reference. Primers were listed in Table S4. Each reaction was performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Chemical composition and physical properties of milled rice\u003c/h2\u003e \u003cp\u003eFloury-core endosperm in waxy rice was observed in transverse sections of brown rice using a stereo microscope (Leica EZ4W, Germany). Total starch content and damaged starch were measured with assay kits (Megazyme Wicklow, Ireland). AAC was determined using a Segmented Flow analyzer (SEAL AA3, USA). Protein content was quantified with a nitrogen and protein analyzer (Elementar Rapid N exceed, Germany). Crude fat was analyzed by hydrolysis, extraction, and evaporation using a Soxhlet extractor (Drawell Scientific DW-SXT-06, China). Ash content was determined after heating rice at 900 ℃ in a muffle furnace (Thermo Scientific Thermolyne, USA). Hardness of milled rice was measured using a texture analyzer with a P5 probe (Texture technologies TA-XT plus, USA). Rice powder was prepared by dry grinding milled rice (IKA A11 basic, Germany) and passing powder through a 100-mesh sieve. Particle size distribution in rice powder was analyzed with a laser particle size analyzer (Malvern Panalytical Mastersizer 2000, UK). Each measurement was performed with three replicates, except for chemical composition (five replicates) and hardness (ten replicates).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Starch structures\u003c/h2\u003e \u003cp\u003eSGs and amyloplasts were observed in rice transverse sections using a scanning electron microscopy (Hitachi TM4000Plus, Japan). Starch crystallinity was estimated with an X-ray diffractometry (Malvern Panalytical X\u0026rsquo;Pert Pro MPD, UK). For degree of polymerization (DP), starch was extracted from rice powder and debranched with iso-amylase. The dried starch was dissolved in a solution of 8-Aminopyrene-1,3,6-trisulfonic acid trisodium salt and urea. The filtered sample was analyzed using a high-performance anion exchange chromatography (Merck Sephacryl S-1000SF, Germany) with a pulsed amperometric detection. Branching degree was examined using a nuclear magnetic resonance (Bruker, Fourier80, Germany). Molecular weight of amylopectin was measured using a liquid chromatography with a differential refraction detector (Wyatt Optilab T-rEX, USA) and a multi-angle static light scattering detector (Wyatt Dawn Heleos II, USA). Three replicates were perform each assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Gelatinization and pasting properties of rice powders\u003c/h2\u003e \u003cp\u003eGelatinization properties were measured with a differential scanning calorimeter (DSC, Mettler Toledo DSC1, USA). Samples were heat from 30\u0026ndash;95 ℃ at a rate of 10 ℃/min. Pasting properties were examined using a rapid viscosity analyzer (shorted for RVA, Perten Tecmaster, Sweden) following the AACC standard method\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Each assay was conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Textural properties of waxy rice balls\u003c/h2\u003e \u003cp\u003eDough was prepared by mixing 100 g of rice flour and 70 g of water and was allowed to rest for 20 min at room temperature (RT). Small rice balls (~\u0026thinsp;1.5 g each) were formed and frozen at -18 ℃ for 2 days. These balls were thawed and drained at RT for 2 h. A portion of the dough was used for textural analysis, while the others were boiled in water for 5 min, then taken out and cooled to RT. Textural properties were measured using a P/36R probe and the texture profile analysis (TPA) method\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. The pre-test, test, and post-test speeds were set to 2, 1, and 2 mm/s, respectively, with a trigger force of 5 g. Compression strain was set as 70% of sample thickness with a 5-s interval between two compressions. Five replicates were applied for both dough or boiled balls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eData on agronomic traits and physicochemical properties was analyzed with Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test in Statistical Product and Service Solutions (SPSS) software. Values were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was indicated by one asterisk (*) for \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and double asterisks (**) for \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The mutation locus encoded the SSIIIa enzyme\u003c/h2\u003e \u003cp\u003eA floury-core endosperm mutant was identified in the M2 population. In the F2 mapping population, 1,082 seeds showed normal endosperm, whereas 295 seeds displayed the floury-core phenotype, fitting to a segregation ratio of approximately 3:1. This indicated that the phenotype was controlled by a single recessive nuclear gene. The \u003cem\u003ewfc\u003c/em\u003e locus was first mapped to chromosome 8 between markers RM5428 and R8M10. Then fine mapping further narrowed the candidate region to a 97 kb interval between RM22544 and RM22547 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), which harbored 10 open reading frames (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Genome sequencing revealed a 1 bp insertion in the third exon of \u003cem\u003essIIIa\u003c/em\u003e, while no change of protein sequences was observed in the other genes. This insertion led to a frameshift and introduced a premature stop codon, resulting in a truncated polypeptide with 332 N-terminal amino acids (aa) instead of the full-length 1,788 aa. Furthermore, complementation test generated 25 positive lines containing the hygromycin-resistance gene. Among them, 23 lines rescued the floury-core endosperm, confirming that \u003cem\u003essIIIa\u003c/em\u003e was responsible for the WFC mutant phenotype. Those results confirmed that \u003cem\u003essIIIa\u003c/em\u003e was responsible for the floury-core endosperm of WFC, providing a genetic basis for following analysis of agronomic traits.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCandidate genes in the fine mapping region\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003elength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edescription\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC4344848\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edisease resistance RPP13-like protein 3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC136351517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epredicted gene\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC136351420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20887\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epredicted gene\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC4344849\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3694\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emyosin-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC4344850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecysteine-rich PDZ-binding protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC4344852\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eprotein PIN-LIKES 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC9270306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7408\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eaconitate hydratase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC4344854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3974\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ephosphoribosylamine\u0026ndash;glycine ligase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC9268758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10822\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003estarch synthase 3a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC4344857\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eautophagy-related protein 8C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The \u003cem\u003essIIIa\u003c/em\u003e mutation altered agronomic traits\u003c/h2\u003e \u003cp\u003eTo assess the effects of the on agronomic traits, WFC plants were compared with a transgenic complementation line. Among the rescued lines, 21 lines displayed similar agronomic traits. One line (the seventh, short for L7) was randomly chosen for detailed comparison. In the field, WFC plants showed phenotypic differences compared with L7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Statistical analysis revealed significant reductions in plant height (-5%), grains per panicle (-11%), and seed-setting rate (-7%) in WFC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Other traits, including panicle number per plant and panicle length, showed no significant differences. Width and thickness of brown rice were increased by more than 12% in WFC, whereas its length and weight remained unchanged. These findings indicated that \u003cem\u003essIIIa\u003c/em\u003e mutation altered specific agronomic traits in rice. To seek mechanisms in trait variation, expression of \u003cem\u003essIIIa\u003c/em\u003e-related genes was analyzed.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAgronomic traits of plants and grains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlant height (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePanicle numbers per plant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePanicle Length (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eGrains per panicle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eSeed-setting rate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e98.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e84.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e111.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e91.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1000-grain weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrain length (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eGrain width (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eGrain thickness (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e3.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1000-brown rice weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown rice length (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBrown rice width (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eBrown rice thickness (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e3.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Expression of several \u003cem\u003essIIIa\u003c/em\u003e-related genes up-regulated in WFC endosperm\u003c/h2\u003e \u003cp\u003eExpression of \u003cem\u003essIIIa\u003c/em\u003e gene in WFC developing endosperm was reduced by 85% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). As expected, mature \u003cem\u003egbssI\u003c/em\u003e mRNA levels were low in both waxy rice lines. Interestingly, \u003cem\u003egbssI\u003c/em\u003e pre-mRNA levels were significantly higher in WFC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Except for \u003cem\u003ebranching enzyme II\u003c/em\u003e (\u003cem\u003ebeIIb\u003c/em\u003e), the other starch synthesis genes were significantly up-regulated in WFC, including \u003cem\u003eADP-glucose pyrophosphorylase small subunit 1\u003c/em\u003e (\u003cem\u003eagp-s1)\u003c/em\u003e and its \u003cem\u003elarge subunit 2\u003c/em\u003e (\u003cem\u003eagp-l2\u003c/em\u003e), \u003cem\u003esoluble starch synthase I\u003c/em\u003e (\u003cem\u003essI\u003c/em\u003e), \u003cem\u003esoluble starch synthase IIa\u003c/em\u003e (\u003cem\u003essIIa\u003c/em\u003e), and \u003cem\u003ebranching enzyme I\u003c/em\u003e (\u003cem\u003ebeI\u003c/em\u003e). Notably, \u003cem\u003essI\u003c/em\u003e expression increased 2.5-fold, while two \u003cem\u003eagp\u003c/em\u003e genes increased about 1.5-fold in WFC. Moreover, debranching enzyme genes in WFC, \u003cem\u003epullulanase\u003c/em\u003e (\u003cem\u003epul\u003c/em\u003e) and \u003cem\u003eisoamylase2\u003c/em\u003e (\u003cem\u003eisa2\u003c/em\u003e) increased 1.9- and 3.0-fold, respectively. Whereas, \u003cem\u003eisoamylase1\u003c/em\u003e (\u003cem\u003eisa1\u003c/em\u003e) expression was comparable between WFC and L7. Additionally, a pre-amylopectin synthesis gene, \u003cem\u003estarch phosphorylase\u003c/em\u003e (\u003cem\u003ephol\u003c/em\u003e), increased by 30% in WFC. These transcriptional changes may reflect compensatory regulation of starch metabolism in response to \u003cem\u003essIIIa\u003c/em\u003e deficiency. To figure out variation influence on WFC rice, its properties were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Grain composition and physical properties altered in WFC\u003c/h2\u003e \u003cp\u003eIn WFC, floury endosperm was not visible under top lighting, but appeared opaque back lighting (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Transverse sections revealed that the core region appeared floury white in WFC, while the outer region resembled L7. Compared with L7, WFC had significantly lower total starch content (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) but higher AAC (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Protein, lipid, and ash contents were comparable between WFC and L7. Damaged starch content in WFC was approximately 35% lower than that in L7. Moreover, WFC rice exhibited a hardness reduction of more than 20%. Particle size distribution showed that D90 and D50 values of WFC powder were significantly smaller than those of L7 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Size of the smallest 10% of WFC particles was less than 15 \u0026micro;m. These evidences suggested that WFC SG and amyloplastic formation was disrupted.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRice composition, hardness, and particle size distribution\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal starch (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmylose (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLipid (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAsh (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDamage starch (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHardness (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDx (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDx (50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDx (90)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e104.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e127.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Floury core contained loosely packed SGs and amyloplasts\u003c/h2\u003e \u003cp\u003eObservation of SEM revealed structural differences between WFC and L7 grains. In floury core region (position 1), WFC performed loosely packed amyloplasts and SGs with large air spaces. In contrast, WFC amyloplasts were compact and dense in the peripheral region (position 3), but still distinguishable by clear boundaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In WFC floury core, small spherical and polyhedral SGs surrounded large amyloplasts. These amyloplasts had diameters of ~\u0026thinsp;15 \u0026micro;m and were coated by membranes. The intermediate region (position 2) contained both compact and partially separated amyloplasts. In L7, amyloplasts were flattened and tightly packed, making individual structures difficult to distinguish. These differences likely reflected alterations in starch chain-length distribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Short-chain enrichment and disrupted starch structure in WFC\u003c/h2\u003e \u003cp\u003eBoth WFC and L7 exhibited similar overall chain-length distribution patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), but differences were observed in specific fractions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In WFC, chains with DP 10\u0026ndash;15 and DP 22\u0026ndash;34 increased significantly by 10% and 7%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Whereas chains with DP 6\u0026ndash;9 displayed no significantly difference. Other chains were reduced significantly by 6.2% and 15% at DP 16\u0026ndash;21, and DP 35\u0026ndash;75, respectively. In terms of chain categories, WFC contained more A chains (DP 6\u0026ndash;12) and B2 chains (DP 25\u0026ndash;36), unchanged B1 chains (DP 13\u0026ndash;24) and fewer B3 (DP\u0026thinsp;\u0026ge;\u0026thinsp;37). Accordingly, amylopectin linkage analysis revealed reductions of 4% in α-1,4 linkages and 20% in α-1,6 linkages, resulting in a significantly lower branching degree(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Those alterations in WFC also led to a\u0026thinsp;~\u0026thinsp;3% reduction in molecular size (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ez\u003c/em\u003e\u003c/sub\u003e) and 5%-8% reduction in molecular weights (\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ez\u003c/em\u003e\u003c/sub\u003e). Molecular weight of WFC also displayed a similar peak (\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e) and a wider polydispersity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Disrupted starch molecules led to impaired crystal structures. X-ray diffraction (XRD) patterns revealed an A-type crystal form with peaks at 15\u0026deg;, 17\u0026deg;, 18\u0026deg;, and 23\u0026deg; (\u003cem\u003e2θ\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In WFC, the peaks at 17\u0026deg; and 18\u0026deg; were weaken, indicating impaired crystal structures. Relative crystallinity of WFC was reduced by approximately 15% compared with L7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Together, these results demonstrated that \u003cem\u003essIIIa\u003c/em\u003e mutation enriched short chains which changed starch molecule and crystallization.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChain length distribution (%) of amylopectin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChain length\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDistribution (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDP6-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDP10-15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDP16-21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDP22-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDP35-75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA chain (DP6-12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB1 chain (DP13-24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB2 chain (DP25-36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB3 chain (DP37-75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecule parameters of rice starch\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,4-linkage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,6-linkage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBranching degree (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e (kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e (kDa)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6551\u0026thinsp;\u0026plusmn;\u0026thinsp;121*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e187\u0026thinsp;\u0026plusmn;\u0026thinsp;9*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80926\u0026thinsp;\u0026plusmn;\u0026thinsp;1462*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e130148\u0026thinsp;\u0026plusmn;\u0026thinsp;963\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6846\u0026thinsp;\u0026plusmn;\u0026thinsp;47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e235\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e84767\u0026thinsp;\u0026plusmn;\u0026thinsp;1363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e132307\u0026thinsp;\u0026plusmn;\u0026thinsp;1374\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e (kDa)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ez\u003c/em\u003e\u003c/sub\u003e (kDa)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ePolydispersity (\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e/\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ez\u003c/em\u003e\u003c/sub\u003e (nm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68745\u0026thinsp;\u0026plusmn;\u0026thinsp;708**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87041\u0026thinsp;\u0026plusmn;\u0026thinsp;1776*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e102\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74704\u0026thinsp;\u0026plusmn;\u0026thinsp;920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92035\u0026thinsp;\u0026plusmn;\u0026thinsp;1196\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e105\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e: Weight average molecular weight, \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e: Peak molecular weight, \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e: Number average molecular weight, \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ez\u003c/em\u003e\u003c/sub\u003e: average molecular weight of size, \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ez\u003c/em\u003e\u003c/sub\u003e: the average rotation radius of size.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7 WFC powder exhibited reduced thermal stability and pasting viscosity\u003c/h2\u003e \u003cp\u003eDSC showed significantly lower values of onset (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e) and peak (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e) gelatinization temperatures and enthalpy (\u003cem\u003e∆H\u003c/em\u003e) in WFC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Value of WFC enthalpy was reduced by at least 22%. Although gelatinization temperatures were lower in WFC, pasting temperatures did not differ between WFC and L7. RVA analysis revealed that pasting curves of both WFC and L7 displayed similar shapes (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). From onset to the end, viscosity reached a peak and then decreased, followed by a similar stabilization. However, WFC curve reached its peak earlier than L7 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). WFC viscosities were reduced by more than 41%, 55% and 56% at the peak, trough, and final positions, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Those reductions resulted in significant decreases in breakdown and setback values (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These changes indicate that floury-core endosperm had directly implications for pasting and thermal properties. Those influences implied that rice foods made of WFC powder could displayed various textures.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThermal properties with DSC dectection\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOneset (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e, ℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePeak (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e, ℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConclusion (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sub\u003e, ℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEnthalpy (\u003cem\u003e∆H\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Textural properties altered in WFC doughs and rice balls\u003c/h2\u003e \u003cp\u003eTextural analysis revealed a few differences between WFC and L7 products (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Both WFC and L7 doughs exhibited low stickiness and elasticity. Dough adhesiveness was lower than \u0026minus;\u0026thinsp;4 g.s, while dough cohesiveness and springiness were less than 0.1. Hardness of WFC doughs increased 4.2-fold compared with L7. Whereas hardness reduced by 51% for WFC boiled balls, and this reduction decreased gumminess and chewiness by 44% and 37%, respectively. Moreover, boiled balls showed less stickiness and higher elasticity. Compared with L7 balls, WFC balls displayed 43% lower adhesiveness of reduced, 50% higher springiness and significantly increased cohesiveness (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results demonstrated that floury-core endosperm impacted both raw and cooked rice product quality, providing practical implications for rice-based food processing.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTexture properties of rice balls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTexture property\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003edough\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eboiled ball\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eL7\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHardness (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3226.24\u0026thinsp;\u0026plusmn;\u0026thinsp;12.34**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e767.74\u0026thinsp;\u0026plusmn;\u0026thinsp;60.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e964.57\u0026thinsp;\u0026plusmn;\u0026thinsp;42.81**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1968.68\u0026thinsp;\u0026plusmn;\u0026thinsp;145.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdhesiveness (g.s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-171.72\u0026thinsp;\u0026plusmn;\u0026thinsp;39.24**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-304.30\u0026thinsp;\u0026plusmn;\u0026thinsp;30.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpringiness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.066\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.058\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.804\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.719\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCohesiveness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.090\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.060\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.550\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.475\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGumminess\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e289.93\u0026thinsp;\u0026plusmn;\u0026thinsp;17.46**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.36\u0026thinsp;\u0026plusmn;\u0026thinsp;6.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e529.93\u0026thinsp;\u0026plusmn;\u0026thinsp;21.72**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e936.56\u0026thinsp;\u0026plusmn;\u0026thinsp;77.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChewiness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e425.76\u0026thinsp;\u0026plusmn;\u0026thinsp;15.74**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e674.48\u0026thinsp;\u0026plusmn;\u0026thinsp;73.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResilience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.047\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.022\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.250\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.166\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe identification of the WFC mutant provides new insights into the role of \u003cem\u003essIIIa\u003c/em\u003e in waxy rice endosperm development. The SSIIIa enzyme is the second major starch synthase in rice endosperm\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and its catalytic domain (residues 1,353\u0026ndash;1,705) \u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e loss led to non-functional activity. This result not only altered morphological traits and starch molecular structure, but also affected gene expression, physicochemical features, and final food properties.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Morphological and compositional differences in \u003cem\u003essIIIa\u003c/em\u003e mutant of waxy rice\u003c/h2\u003e \u003cp\u003eThe WFC mutant displayed altered agronomic traits at both plant and grain levels compared with L7 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This contrasted previous studies reporting that \u003cem\u003essIIIa\u003c/em\u003e deficiency alone in non-waxy rice had little influence on these traits\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. In WFC, plant height, grains per panicle, and seed-setting rate were significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although WFC plants were slightly shorter, their height still remained within the range of ideal rice architecture\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. However, the seed-setting rate and grains per panicle decreased by 7\u0026ndash;12% in WFC, which might lead to lower yield. Floury endosperm typically reduced grain weight\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Whereas Brown rice weight and grain traits were unchanged in WFC, likely due to the increased width and thickness. Total starch content was decreased in WFC, consistent with \u003cem\u003essIIIa\u003c/em\u003e RNAi line in non-waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Unexpectedly, AAC increased slightly in WFC despite \u003cem\u003egbssI\u003c/em\u003e deficiency. This enhancement was not observed in \u003cem\u003essIIIa\u003c/em\u003e mutants of potato and waxy maize\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. In non-waxy rice, AAC enhancement was attributed to \u003cem\u003essIIIa\u003c/em\u003e mutations upregulating \u003cem\u003egbssI\u003c/em\u003e expression\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, but this explanation seemed unconvincing in waxy crops. Those results suggested that AAC variations in \u003cem\u003essIIIa\u003c/em\u003e mutation might be species-specific, with mechanisms yet to be fully understood. Interestingly, the \u003cem\u003ebeIIb\u003c/em\u003e mutation also increased AAC in waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, implying that disruption of starch synthesis networks might underlie AAC enhancement in WFC. Floury-core endosperm in WFC contributed to lower grain hardness, reduced damaged starch, and finer particle size (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). They were consistent with \u003cem\u003essIIIa\u003c/em\u003e mutants of non-waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Lower grain hardness facilitated dry-grinding powder\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Moreover, the size of the smallest 10% of WFC powder particles corresponded well with amyloplast size in the floury core (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), further linking the phenotype to the property.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Distinct SGs and amyloplasts from other mutations\u003c/h2\u003e \u003cp\u003eFloury endosperm is commonly associated with altered amyloplasts and SGs morphology. In L7, amyloplasts appeared flattened, a feature similar with wild-type endosperm covered by thick enveloped SGs\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. By contrast, the \u003cem\u003essIIIa\u003c/em\u003e mutation in WFC increased air space between amyloplasts in floury core and generated both polyhedral and spherical SGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Polyhedral SGs were previously observed in \u003cem\u003essIIIa\u003c/em\u003e mutants of non-waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, whereas \u003cem\u003essI\u003c/em\u003e suppression or \u003cem\u003essIVb\u003c/em\u003e deficiency promoted spherical SGs\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. These findings suggest that \u003cem\u003egbssI\u003c/em\u003e deficiency was essential for spherical SGs formation in WFC. Amyloplast compactness varied across endosperm regions in WFC, from loose packing in the core (position 1) to dense structures at the periphery (position 3). This suggested that starch synthesis was initially disrupted during early endosperm development and partially recovered later. However, incomplete recovery was evident from joints structures between compact amyloplasts at periphery region in WFC. This pattern differed from the \u003cem\u003ess3a-1\u003c/em\u003e mutant of non-waxy rice, where peripheral amyloplasts resembled wild type. Such distinctions were potentially due to the loss of epistatic effect of \u003cem\u003egbssI\u003c/em\u003e gene on \u003cem\u003essIIIa\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. These findings demonstrated that \u003cem\u003essIIIa\u003c/em\u003e deficiency in waxy rice induced heterogeneous SG morphology and amyloplast arrangements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Short chains enhancement led to smaller molecule and impaired crystal structure\u003c/h2\u003e \u003cp\u003eWFC amylopectin displayed increased short chains of DP 10\u0026ndash;15 and DP 22\u0026ndash;34, and decreased long chains of DP\u0026thinsp;\u0026ge;\u0026thinsp;35 (Table S5). This distribution closely resembles that of \u003cem\u003essIIIa\u003c/em\u003e mutants in non-waxy rice, which exhibited elevated DP 9\u0026ndash;15 and DP 20\u0026ndash;32\u003csup\u003e\u003cem\u003e8, 26, 36, 39\u003c/em\u003e\u003c/sup\u003e. In WFC, A chains and B2 chains increased, B1 chains remained stable, and B3 chains decreased. In contrast, only B1 chains increased in maize \u003cem\u003egbssI/ssIIIa\u003c/em\u003e double mutant, and rice \u003cem\u003essIIIa\u003c/em\u003e RNAi line under high temperature\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Those evidences highlighted that chain length distribution was shaped by both genetic background and environmental conditions. The enhancement of short chains resulted in decreased molecular weight (5%) and molecular size (3%) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In comparison, the \u003cem\u003ess3a-1\u003c/em\u003e mutant in non-waxy rice exhibited a 25% reduction in molecular weight\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. This smaller impact on WFC amylopectin suggested that amylopectin in waxy rice was more resistant to structural disruption due to their higher weight and bigger size\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. The peak of molecular weight (\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e) was similar between WFC and L7, and smaller WFC molecules led to the wider range. Short chains were considered to form shorter crystallites and decrease crystalline order, and amylose disrupted crystalline packing\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Therefore, increased short chains and AAC leading to lower relative crystallinity in WFC. These results indicate that disrupted starch structures in WFC were attributed to more short chains and smaller molecules.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Gene expression alterations promoted short chain accumulation\u003c/h2\u003e \u003cp\u003eAmylopectin chain length was regulated by starch synthesis-related genes\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. The \u003cem\u003essI\u003c/em\u003e and \u003cem\u003egbssI\u003c/em\u003e were increased in \u003cem\u003essIIIa\u003c/em\u003e mutants of non-waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. In WFC, the upregulation of \u003cem\u003essI\u003c/em\u003e resembled that in non-waxy rice, contributing to the accumulation of short amylopectin chains with DP 8\u0026ndash;12 and DP 20\u0026ndash;30. Interestingly, \u003cem\u003egbssI\u003c/em\u003e pre-mRNA was elevated in WFC, while mature mRNA remained comparable to L7. This result reflected post-transcriptional regulation: the pre-mRNA levels of \u003cem\u003egbssI\u003c/em\u003e were similar in waxy and non-waxy rice, and post-transcriptional procedure handled these pre-mRNA to mature mRNA at different levels\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Besides, both \u003cem\u003essIIIa\u003c/em\u003e and \u003cem\u003egbssI\u003c/em\u003e participate in long-chain synthesis\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and their deficiency favored the accumulation of shorter amylopectin chains in WFC. In addition, the \u003cem\u003ebeI\u003c/em\u003e gene cooperated with \u003cem\u003essIIIa\u003c/em\u003e and \u003cem\u003essIIa\u003c/em\u003e to synthesize long chains. Although \u003cem\u003essIIa\u003c/em\u003e and \u003cem\u003ebeI\u003c/em\u003e expressions increased in WFC, the contribution of \u003cem\u003ebeI\u003c/em\u003e may be limited due to the \u003cem\u003essIIIa\u003c/em\u003e deficiency and low SSIIa activity in japonica rice. In WFC, expressions of \u003cem\u003eagp-s1\u003c/em\u003e and \u003cem\u003eagp-l2\u003c/em\u003e genes were upregulated, consistent with observations in \u003cem\u003essIIIa\u003c/em\u003e RNAi lines under high temperature\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Enhanced AGPase activity likely provided more ADP-glucose substrate for chain biosynthesis, but disrupted function of long-chain synthesis caused short chain accumulation. Debranching enzyme, \u003cem\u003eisa1\u003c/em\u003e, works association with \u003cem\u003eisa2\u003c/em\u003e and \u003cem\u003epul\u003c/em\u003e to complete amylopectin structures\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. However, \u003cem\u003eisa1\u003c/em\u003e expression remained unchanged in WFC, indicating that the upregulation of \u003cem\u003eisa2\u003c/em\u003e and \u003cem\u003epul\u003c/em\u003e likely had minimal impact on amylopectin structures. The \u003cem\u003ephol\u003c/em\u003e gene was essential for pre-amylopectin synthesis from maltose\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and its overexpression led to floury endosperm and more short and DP 25\u0026ndash;35 chains in non-waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Enhanced \u003cem\u003ephol\u003c/em\u003e expression in WFC might partially contribute to the floury-core formation and increased proportions of DP 25\u0026ndash;34 chains.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Thermal and pasting properties reflected changes of floury-core endosperm and molecules\u003c/h2\u003e \u003cp\u003eThermal properties were strongly influenced by amylopectin chain length\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Gelatinization properties were negative with A chains and positive with B1 chains in waxy rice. In WFC, enhanced A chains and unchanged B1 chains decreased onset (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e,), peak (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e), and enthalpy (\u003cem\u003e∆H\u003c/em\u003e) values (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This result was consistence with \u003cem\u003essIIIa\u003c/em\u003e mutants in non-waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, but opposite to \u003cem\u003egbssI/ssIIIa\u003c/em\u003e double mutation in maize\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. It was likely due to differences of \u003cem\u003essIIa\u003c/em\u003e activity across genetic backgrounds. Although \u003cem\u003essIIa\u003c/em\u003e expression was upregulated in WFC, its low activity limited influence on gelatinization temperature in WFC. Typically, AAC positively influenced pasting and thermal properties in non-floury endosperm\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, but opposite result was performed in \u003cem\u003essIIIa-1\u003c/em\u003e\u003csup\u003e\u003cem\u003e26\u003c/em\u003e\u003c/sup\u003e and WFC mutants. It implied that floury endosperm had a greater impact on those properties. The floury region played a key role in pasting behavior: less energy was required to disrupt separated amyloplasts and SGs. RVA profiles revealed characteristic patterns of waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e in both WFC and L7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In WFC, viscosity peak was earlier, consistent with less required energy. Chain length distribution showed no significant correlation with pasting viscosity in non-floury rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. However, reduction of molecular size and weight contributed to lower viscosity\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, whereas these reduction due to floury core decreased viscosity in WFC. In contrast, \u003cem\u003ebeIIb/gbssI\u003c/em\u003e double mutation increased viscosity with depleted chains in DP 6\u0026ndash;14 and enriched chains in DP\u0026thinsp;\u0026ge;\u0026thinsp;15\u003csup\u003e\u003cem\u003e33\u003c/em\u003e\u003c/sup\u003e, implying that short chains accumulation was positive related with pasting viscosity. Notably, the setback value of waxy rice was lower than that of non-waxy rice\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, whereas WFC displayed an even lower setback, indicating reduced retrogradation tendency and improved food stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Improved processing and tasting quality in WFC-based products\u003c/h2\u003e \u003cp\u003eThe textural properties of waxy rice dough and boiled rice balls are critical for both processing performance and consumer acceptance. Dough containing less damaged starch resulted in lower solubility and higher water-holding capacity\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. When mixed with the same amount of water, WFC dough exhibited greater hardness and easier shaping. In contrast, L7 dough containing excess soluble fraction led to softer texture and reduced shaping ability. Short chains enrichment, finer particle size and less damaged starch improved quality properties in rice-based foods\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. After boiling, WFC rice balls displayed desirable qualities, including softer, less stickiness, and more elasticity. Furthermore, WFC rice balls showed reduced gumminess and chewiness, allowing faster chewing and swallowing. This property was particularly suitable for children and elderly consumers. Those findings demonstrated practical advantages of the floury-core endosperm in waxy rice.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe effects of the floury-core endosperm on agronomic traits, physicochemical properties, gene expression, and textural characteristics were systematically investigated in waxy rice. The WFC mutant exhibited reduced grains per panicle and seed-setting rate, which could negatively impact yield. Therefore, the \u003cem\u003essIIIa\u003c/em\u003e mutation should be introgressed into waxy rice cultivars with higher seed-setting rates and more grain numbers to maintain optimal yield in future breeding programs. At the molecular level, increased short chains resulted in smaller molecular size in WFC. It likely underlain the formation of the floury-core endosperm. Upregulation of multiple \u003cem\u003essIIIa\u003c/em\u003e-related genes in WFC indicated that the endosperm starch synthesis network partially compensated for \u003cem\u003essIIIa\u003c/em\u003e deficiency. This compensation likely contributed to the formation of compact starch granules in the peripheral endosperm and enhanced rate of milled rice. From a processing perspective, WFC powder produced by dry grinding had smaller particle size and lower starch damage, resulting in favorable thermal and pasting properties. These characteristics enhanced processing performance and consumer-relevant qualities of WFC-based products. Overall, the floury-core endosperm of WFC demonstrates a promising trait for improving waxy rice processing. Dry-grinding powder derived from WFC mutant might serve as a suitable alternative to conventionally wet-grinding powder in food applications.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAll the authors agreed on the contents of the paper and post no conflicting interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Research Funds from Germplasm Innovation Group (No. JCYJ261201), SAAS Excellent Research Group (No. NKC2017A05) and Special Rice Variety Group of Wine Clean Production (No. JS01202XM25001).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ. F. performed designed the experiments, gene expression and wrote the manuscript, H. W. performed phenotype analysis and gene cloning, Z. H. and J. P. performed rice powder preparation, L. M. performed gene mapping, Y. Z. and J. J. performed agronomic traits, Y. D. and T. S. performed data analysis, W. Y. performed the mutation development, C. C. and F. N. performed pasting and thermal properties, J. Z. and B. S. performed ball preparation and texture properties, H. C. and L. C. designed the experiments and revised the manuscript. All the authors reviewed the manuscript, agreeded on the contents of the paper, and posted no conflicting interest.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eThe datasets supporting the conclusions of this article are provided within the article and its additional files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBryant RJ, Kadan RS, Champagne ET, Vinyard BT, Boykin D (2001) Functional and digestive characteristics of extruded rice flour. Cereal Chem 78:131\u0026ndash;137\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Xiao N, Wang X, Zhao X, Zhang H (2019) Effect of pregelatinized starch on the characteristics, microstructures, and quality attributes of glutinous rice flour and dumplings. Food Chem 283:248\u0026ndash;256\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang S, Chen M, Xu F, Liu F, Zhong F (2023) The possibility of replacing wet-milling with dry-milling in the production of waxy rice flour for the application in waxy rice ball. Foods 12:1\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian Y, Sun J, Li J, Wang A, Nie M, Gong X, Wang L, Liu L, Wang F, Tong L (2024) Effects of milling methods on rice flour properties and rice product quality: A review. Rice Sci 31:33\u0026ndash;46\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang JW, Lee SK, Shim SH, Shin D, Cho JH, Lee JY, Ko JM, Ji HYS, Park HM, Ahn EK, Lee JH, Jeon JS (2023) Dry-milled flour rice 'Seolgaeng' harbors a mutated fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase2. Front Plant Sci 14:1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMo Y, Jeung J (2020) The use of floury endosperm mutants to develop rice cultivars suitable for dry milling. Plant Biotechnol Rep 14:185\u0026ndash;191\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu HM, Ren YL, Dong H, Xie C, Zhao L, Wang X, Zhang FL, Zhang BL, Jiang XK, Huang YS, Jing RN, Wang J, Miao R, Bao XH, Yu MZ, Nguyen T, Mou CL, Wang YL, Wang YH, Lei CL, Cheng ZJ, Jiang L, Wan JM (2024) Floury endosperm24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice. New Phytol 242:2635\u0026ndash;2651\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyoo N, Yu C, Park CS, Baik MY, Park IM, Cho MH, Bhoo SH, An G, Hahn TR, Jeon JS (2007) Knockout of a starch synthase gene OsSSIIIa/Flo5 causes white-core floury endosperm in rice (Oryza sativa L). Plant Cell Rep 26:1083\u0026ndash;1095\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLong W, Dong B, Wang Y, Pan P, Wang Y, Liu L, Chen X, Liu X, Liu S, Tian Y, Chen L, Wan J (2017) Floury endosperm8, encoding the UDP-glucose pyrophosphorylase 1, affects the synthesis and structure of starch in rice endosperm. J Plant Biology 60:513\u0026ndash;522\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao J (2019) Rice starch. Rice Chemistry and Technology (Fourth Edition). Elsevier Inc.: AACC International,, pp 55\u0026ndash;108\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu F, Xue H (2010) Coexpression analysis identifies rice starch regulator1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator. Plant Physiol 154:927\u0026ndash;938\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang M, Zhang HL, Song Y, Chen JL, Bai JJ, Tang JH, Wang Q, Fotopoulos V, Zhu QH, Yang RF, Li RQ (2024) Transcription factor OsbZIP10 modulates rice grain quality by regulating OsGIF1. Plant J 119:2181\u0026ndash;2198\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYou XM, Zhang WW, Hu JL, Jing RN, Cai Y, Feng ZM, Kong F, Zhang J, Yan HG, Chen WW, Chen XG, Ma J, Tang XJ, Wang P, Zhu SS, Liu LL, Jiang L, Wan JM (2019) Floury endosperm15 encodes a glyoxalase I involved in compound granule formation and starch synthesis in rice endosperm. Plant Cell Rep 38:345\u0026ndash;359\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi SF, Wei XJ, Ren YL, Qiu JH, Jiao GA, Guo XP, Tang SQ, Wan JM, Hu PS (2017) OsBT1 encodes an ADP-glucose transporter involved in starch synthesis and compound granule formation in rice endosperm. Sci Rep 7:1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu X, Teng X, Wang Y, Hao Y, Jing R, Wang Y, Liu Y, Zhu J, Wu M, Zhong M, Chen X, Zhang Y, Zhang W, Wang C, Wang Y, Wan J (2018) Floury endosperm11 encoding a plastid heat shock protein 70 is essential for amyloplast development in rice. Plant Sci 277:89\u0026ndash;99\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen YL, Wang YH, Pan T, Wang YL, Wang YF, Gan L, Wei ZY, Wang F, Wu MM, Jing RN, Wang JC, Wan GX, Bao XH, Zhang BL, Zhang PC, Zhang Y, Ji Y, Lei CL, Zhang X, Cheng ZJ, Lin QB, Zhu SS, Zhao ZC, Wang J, Wu CY, Qiu LJ, Wang HY, Wan JM (2020) GPA5 encodes a Rab5a effector required for post-golgi trafficking of rice storage proteins. Plant Cell 32:758\u0026ndash;777\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao XH, Wang YF, Qi YZ, Lei CL, Wang YL, Pan T, Yu MZ, Zhang Y, Wu HM, Zhang PC, Ji Y, Yang H, Jiang XK, Jing RA, Yan MY, Zhang BL, Gu CW, Zhu JP, Hao YY, Lei J, Zhang S, Chen XL, Chen RB, Sun YL, Zhu Y, Zhang X, Jiang L, Visser RGF, Ren YL, Wang YH, Wan JM (2023) A deleterious Sar1c variant in rice inhibits export of seed storage proteins from the endoplasmic reticulum. Plant Mol Biol 111:291\u0026ndash;307\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHa SK, Lee HS, Lee SY, Lee CM, Mo YJ, Jeung JU (2023) Characterization of flo4-6, a novel cyOsPPDKB allele conferring floury endosperm characteristics suitable for dry-milled rice flour production. Agronomy-Basel 13:1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong M, Liu X, Liu F, Ren Y, Wang Y, Zhu J, Teng X, Duan E, Wang F, Zhang H, Wu M, Hao Y, Zhu X, Jing R, Guo X, Jiang L, Wang Y, Wan J (2019) Floury endosperm12 encoding alanine aminotransferase 1 regulates carbon and nitrogen metabolism in rice. J Plant Biology 62:61\u0026ndash;73\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei J, Teng X, Wang YF, Jiang XK, Zhao HH, Zheng XM, Ren YL, Dong H, Wang YL, Duan EC, Zhang YY, Zhang WW, Yang H, Chen XL, Chen RB, Zhang Y, Yu MZ, Xu SB, Bao XH, Zhang PC, Liu SJ, Liu X, Tian YL, Jiang L, Wang YH, Wan JM (2022) Plastidic pyruvate dehydrogenase complex E1 component subunit Alpha1 is involved in galactolipid biosynthesis required for amyloplast development in rice. Plant Biotechnol J 20:437\u0026ndash;453\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLou G, Chen P, Zhou H, Li P, Xiong J, Wan S, Zheng Y, Alam M, Liu R, Zhou Y, Yang H, Tian Y, Bai J, Rao W, Tan X, Gao H, Li Y, Gao G, Zhang Q, Li X, Liu C, He Y (2021) Floury endosperm19 encoding a class I glutamine amidotransferase affects grain quality in rice. Mol Breeding 41:1\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai Y, Zhang WW, Jin J, Yang XM, You XM, Yan HG, Wang L, Chen J, Xu JH, Chen WW, Chen XG, Ma J, Tang XJ, Kong F, Zhu XP, Wang GX, Jiang L, Terzaghi W, Wang CM, Wan JM (2018) OsPKpα1 encodes a plastidic pyruvate kinase that affects starch biosynthesis in the rice endosperm. J Integr Plant Biol 60:1097\u0026ndash;1118\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHonda Y, Saito Y, Katsumi N, Nishikawa M, Takagi H (2023) Physicochemical properties of starch in rice flour with different hardening rates of glutinous rice cake (mochi). J Cereal Sci 112:1\u0026ndash;6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra BP, Kumar R, Mohan A, Gill KS (2017) Conservation and divergence of starch synthase III genes of monocots and dicots. PLoS ONE 12:e0189303\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAACC Approved Methods of Analysis (1999) 11th Ed. \u003cem\u003eCereals \u0026amp; Grains Association, St. Paul, MN, U.S.A.\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujita N, Yoshida M, Kondo T, Saito K, Utsumi Y, Tokunaga T, Nishi A, Satoh H, Park JH, Jane JL, Miyao A, Hirochika H, Nakamura Y (2007) Characterization of SSIIIa-deficient mutants of rice: the function of SSIIIa and pleiotropic effects by SSIIIa deficiency in the rice endosperm. Plant Physiol 144:2009\u0026ndash;2023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Q, Ye Y, Han Z, Zhou L, Guan X, Pan G, Asad MA, Cheng F (2020) SSIIIa-RNAi suppression associated changes in rice grain quality and starch biosynthesis metabolism in response to high temperature. Plant Sci 294:1\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen W-f, Xu Z-j, Tang L (2012) Advances and Prospects in Research on Super Rice Breeding. J Shenyang Agricultural Univ 43:643\u0026ndash;649\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong XH, Chen ZH, Du X, Li B, Fei YY, Tao YJ, Wang FQ, Xu Y, Li WQ, Wang J, Liang GH, Zhou Y, Tan XL, Li YL, Yang J (2023) Generation of new rice germplasms with low amylose content by CRISPR/Cas9-targeted mutagenesis of the floury endosperm 2 gene. Front Plant Sci 14:1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou H, Wang L, Liu G, Meng X, Jing Y, Shu X, Kong X, Sun J, Yu H, Smith SM, Wu D, Li J (2016) Critical roles of soluble starch synthase SSIIIa and granule-bound starch synthase Waxy in synthesizing resistant starch in rice. Natl Acad Sci U S A 113:12844\u0026ndash;12849\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang YJ, White P, Pollak L, Jane J (1993) Characterization of starch structures of 17 maize endosperm mutant genotypes with Oh43 inbred line background. Cereal Chem 70:171\u0026ndash;179\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLloyd JR, Landschutze V, Kossmann J (1999) Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin. Biochem J 338:515\u0026ndash;521\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKubo A, Akdogan G, Nakaya M, Shojo A, Suzuki S, Satoh H, Kitamura S (2010) Structure, physical, and digestive properties of starch from \u003cem\u003ewx ae\u003c/em\u003e double-mutant rice. J Agric Food Chem 58:4463\u0026ndash;4469\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMo YJ, Jeung JU, Shin YS, Park CS, Kang KH, Kim BK (2013) Agronomic and genetic analysis of Suweon 542, a rice floury mutant line suitable for dry milling. Rice (NY) 6:1\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwak J, Yoon M-R, Lee J-S, Lee J-H, Ko S, Tai TH, Won Y-J (2017) Morphological and starch characteristics of the Japonica rice mutant variety Seolgaeng for dry-milled flour. Food Sci Biotechnol 26:43\u0026ndash;48\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyosawa Y, Kawagoe Y, Matsushima R, Crofts N, Ogawa M, Fukuda M, Kumamaru T, Okazaki Y, Kusano M, Saito K, Toyooka K, Sato M, Ai Y, Jane JL, Nakamura Y, Fujita N (2016) Deficiency of starch synthase IIIa and IVb alters starch granule morphology from polyhedral to spherical in rice endosperm. Plant Physiol 170:1255\u0026ndash;1270\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujita N, Satoh R, Hayashi A, Kodama M, Itoh R, Aihara S, Nakamura Y (2011) Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa. J Exp Bot 62:4819\u0026ndash;4831\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang B, Xu S, Xu L, You H, Xiang X (2018) Effects of \u003cem\u003eWx\u003c/em\u003e and its interaction with SSIII-2 on rice eating and cooking qualities. Front Plant Sci 9:1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayashi M, Kodama M, Nakamura Y, Fujita N (2015) Thermal and pasting properties, morphology of starch granules, and crystallinity of endosperm starch in the rice \u003cem\u003essI\u003c/em\u003e and \u003cem\u003essIIIa\u003c/em\u003e double-mutant. J Appl Glycosci 62:81\u0026ndash;86\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJane J, Chen YY, Lee LF, McPherson AE, Wong KS, Radosavljevic M, Kasemsuwan T (1999) Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chem 76:629\u0026ndash;637\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark IM, Ib\u0026aacute;\u0026ntilde;ez AM, Shoemaker CF (2007) Rice starch molecular size and its relationship with amylose content. Starch 59:69\u0026ndash;77\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYou S-Y, Lim S-T, Lee JH, Chung H-J (2014) Impact of molecular and crystalline structures on in vitro digestibility of waxy rice starches. Carbohydr Polym 112:729\u0026ndash;735\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Dong H, Yao H, Yao J, Cheng F, Fang Y, Dai C, Li D (2008) The abundance of GBSS, SBE and its effect on amylose content. J Shandong Agricultural Univ 39:501\u0026ndash;505\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura Y, Steup M (2025) Contents and structures of branched and linear maltodextrins, malto-oligosaccharides, and sugars in the early developmental stage of phosphorylase1 mutant endosperm of rice. Carbohydr Polym 348:1\u0026ndash;7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura Y, Ono M, Sawada T, Crofts N, Fujita N, Steup M (2017) Characterization of the functional interactions of plastidial starch phosphorylase and starch branching enzymes from rice endosperm during reserve starch biosynthesis. Plant Sci 264:83\u0026ndash;95\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Shao G, Jiao G, Zhu M, Wu J, Cao R, Chen Y, Xie L, Sheng Z, Tang S (2021) Editing of rice endosperm plastidial phosphorylase gene OsPho1 advances its function in starch synthesis. Rice Sci 28:209\u0026ndash;211\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong X, Sun X, Xu F, Umemoto T, Chen H, Bao J (2014) Morphological and physicochemical properties of two starch mutants induced from a high amylose indica rice by gamma irradiation. Starch 66:157\u0026ndash;165\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Y, Cheng K, Zhao S, Xiong S (2007) Molecular weight distribution of rice starches and its correlation with viscosity. Scientia Agricultura Sinica 40:566\u0026ndash;577\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu D, Shu Q, Xia Y (2001) Assisted-selection for early indica rice with good eating quality by RVA Profile. Acta Agron sinica 27:165\u0026ndash;172\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin W, Lin Z, Wang A, Xiao T, He Y, Chen Z, Wang L, Liu L, Wang F, Tong L-T (2021) Influence of damaged starch on the properties of rice flour and quality attributes of gluten-free rice bread. J Cereal Sci 101:1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin W, Lin Z, Wang A, Chen Z, He Y, Wang L, Liu L, Wang F, Tong L-T (2021) Influence of particle size on the properties of rice flour and quality of gluten-free rice bread. LWT-Food Sci Technol 151:1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ssIIIa mutation, agronomic and physicochemical traits, starch structures, starch synthesis gene expression, waxy rice processing","lastPublishedDoi":"10.21203/rs.3.rs-9480499/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9480499/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWaxy rice is an important raw material in food processing, particularly for traditional Asian products such as rice balls. High-quality waxy rice powder, characterized by low levels of damaged starch and small particle size, is typically produced via wet grinding. However, this method generates substantial wastewater and consumes lots of energy, raising environmental concerns. In non-waxy rice, floury endosperm mutants have enabled high-quality rice powder through dry grinding, but this approach has not been extended to waxy rice. Here, a waxy rice mutant with floury-core endosperm was developed by an insertion mutation of the \u003cem\u003esoluble starch synthase IIIa\u003c/em\u003e (\u003cem\u003essIIIa\u003c/em\u003e) gene. Compared with the ssIIIa mutant of non-waxy rice, the waxy rice mutant exhibited distinct agronomic characteristics, including reduced yield-related traits. Nevertheless, the floury-core endosperm reduced starch damage and particle size in dry-ground flour, enhancing its suitability for waxy rice ball production. The mutant also contained more short chains and smaller molecules in amylopectin, associated with the upregulation of multiple \u003cem\u003essIIIa\u003c/em\u003e-related genes. Those changes further resulted in decreased crystallinity and altered pasting and thermal properties. Waxy rice powder of the mutant was obtained via dry grinding and exhibited favorable processing and eating qualities. Dough was harder and easier to handle, while boiled balls were softer, less sticky, and easier to chew and swallow. Collectively, the floury-core endosperm modified agronomic traits, physicochemical properties, and gene expression in waxy rice, ultimately improving product quality. The dry grinding method using the mutant offers an environmentally friendly alternative to conventional wet grinding for high-quality waxy rice powder.\u003c/p\u003e","manuscriptTitle":"Characterization of Waxy Rice with Floury-Core Endosperm and Its Impact on Rice Balls","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 20:25:57","doi":"10.21203/rs.3.rs-9480499/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T05:46:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250283855348687288786968346758951904709","date":"2026-05-18T03:35:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29065043770069940078324259356831145757","date":"2026-04-30T07:53:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-24T05:19:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-22T12:25:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-22T12:24:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Rice","date":"2026-04-21T07:32:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4913b22d-1047-47d0-8d0a-bbc971f24078","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T05:46:31+00:00","index":20,"fulltext":""},{"type":"reviewerAgreed","content":"250283855348687288786968346758951904709","date":"2026-05-18T03:35:03+00:00","index":19,"fulltext":""},{"type":"reviewerAgreed","content":"29065043770069940078324259356831145757","date":"2026-04-30T07:53:45+00:00","index":15,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T20:25:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 20:25:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9480499","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9480499","identity":"rs-9480499","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}