Filamentous temperature-sensitive Z protein J175 regulates maize chloroplasts and amyloplasts division and development

Filamentous temperature-sensitive Z protein J175 regulates maize chloroplasts and amyloplasts division and development | 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 Filamentous temperature-sensitive Z protein J175 regulates maize chloroplasts and amyloplasts division and development Huayang Lv, Xuewu He, Hongyu Zhang, Zeting Mou, Xuerui He, Yongping Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5848477/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plastid division regulatory genes play a crucial role in the morphogenesis of chloroplasts and amyloplasts. Chloroplasts are the main sites for photosynthesis and metabolic reactions, while amyloplasts are the organelles responsible for forming and storing starch granules. The proper division of chloroplasts and amyloplasts is essential for plant growth and maintenance yield. Therefore, this study aims to examine the J175 (FtsZ2-2) gene, cloned from an ethyl methanesulphonate ( EMS)mutant involved in chloroplast and amyloplast division in maize, through map-based cloning. We found that J175 encodes a cell division protein, FtsZ (filamentous temperature-sensitive z). The FtsZ family of proteins is widely distributed in plants and may be related to the division of chloroplasts and amyloplasts. J175 is localized in plastids and expressed across various tissues. From the seedling stage, the leaves of the j175 mutant exhibited white stripes, while the division of chloroplasts was inhibited, leading to a significant increase in volume and a reduction in their number. Measurement of the photosynthetic rate showed a significant decrease in the photosynthetic efficiency of j175 . Additionally, the division of amyloplasts in j175 grains at different stages was impeded, resulting in irregular polygonal starch granules. RNA-seq analyses of leaves and kernels also showed that multiple genes affecting plastid division, such as FtsZ1, ARC3, ARC6, PDV1-1, PDV2 , and MinE1 , were significantly downregulated. This study demonstrates that the maize gene j175 is essential for maintaining the division of chloroplasts and amyloplasts, ensuring normal plant growth, and providing an important gene resource for maize molecular breeding. Maize Chloroplast Amyloplast FtsZ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Maize, a vital C4 plant, serves as a food crop and an important industrial raw material. Chloroplasts are the primary sites for photosynthesis and numerous metabolic reactions. Chloroplast division is crucial for the survival and reproduction of various plant species in nature (Chen et al. 2018a , Glynn et al. 2007 ). The division of amyloplasts significantly influences the size, morphology, and yield of starch granules (Kawagoe 2009 , Zhao et al. 2018 ). In cells with varying physiological and developmental states, chloroplasts and amyloplasts undergo different degrees of division. This process is governed by multiple pathways that require further refinement (Osteryoung and Mcandrew 2001 , Yang et al. 2008 ). Chloroplasts are photosynthetic organelles in plants that convert energy. They have a double-layered membrane structure (Osteryoung et al. 2003; Martin and Kowallik 1999). They are believed to have evolved from cyanobacteria and replicate through a process called binary fission (Chen et al. 2018b , Miyagishima, Osteryoung et al. 2014). The mechanism responsible for chloroplast division mainly involves four ring-like structures: the FtsZ ring (Z ring), inner and outer plastid-dividing rings, and the DRP5B ring. These components contract and compress the division site to facilitate chloroplast division (Miyagishima, 2005 , Osteryoung et al. 2014). The Z ring, formed by chloroplast FtsZ proteins of cyanobacterial origin, is the first ring structure formed during chloroplast division (Miyagishima et al. 2001 ); it belongs to the GTPase family, which is associated with tubulin and acts as a scaffold protein for the division complex (Chen et al. 2018a , Osteryoung and Mcandrew 2001 ). In plants, FtsZ proteins are composed of two subfamilies: FtsZ1 and FtsZ2. These proteins assemble into a polymer and form a ring at the cleavage site (Sun et al. 2023 , Vitha and S. 2001). The FtsZ proteins in chloroplasts are anchored to the inner membrane through an interaction between the C-terminal domain of FtsZ2 and N-terminal domain of the chloroplast transmembrane protein accumulation and replication (ARC6) (Miyagishima 2011 ). In arc6 mutants, filamentous FtsZ proteins are fragmented, indicating that ARC6 is crucial for the assembly and stability of FtsZ rings (Chen et al. 2018b ). The chloroplast outer envelope membrane protein PDV2 and its paralog PDV1 recruit the cytosolic dynamin-related GTPase ARC5 (accumulation and replication of chloroplasts 5) to the chloroplast division site (Miyagishima et al. 2006 ), which determines the rate of chloroplast division (Liu et al. 2024 , Okazaki et al. 2009). Studies have demonstrated that the size of chloroplasts can be altered through either the overexpression or antisense repression of FtsZ (Mcandrew et al. 2001 , Osteryoung et al. 1998 , Stokes et al. 2000). Overexpression or antisense repression of AtFtsZ1 or AtFtsZ2 in Arabidopsis resulted in the formation of one or few large chloroplasts per cell. A threefold increase in AtFtsZ1-1 protein levels inhibited chloroplast division (Mcandrew et al. 2001 , Stokes et al. 2000). Higher AtFtsZ1-1 protein levels resulted in more severe phenotypes. The defects in chloroplast division caused by AtFtsZ1-1 overproduction may reflect a stoichiometric imbalance among the components necessary for chloroplast division (Mcandrew et al. 2001 ). Furthermore, a decrease in StFtsZ1 protein levels in potato leaves resulted in the reduction of chloroplasts in guard cells (de Pater et al. 2006 ). The role of the FtsZ protein in Z ring assembly during chloroplast division in maize requires further investigation. The amyloplast reaction in cereals can coordinate with photosynthesis occurring in the leaves, allowing the products of both reactions to be mutually transformed under light induction (Balmer et al. 2006 , Zhu et al. 1984 ). Ultrastructural, molecular, and genetic data indicate that the components required for the division process are similar across all plastid types (Mcandrew et al. 2001 , Osteryoung and Mcandrew 2001 ). The mutant parc6, which affects the splitting of wheat chloroplasts and amyloplasts, exhibited enlarged plastids in the leaves and endosperm. Additionally, the endosperm amyloplasts of this mutant contained a higher proportion of A-type and B-type starch particles compared to the wild type (Esch et al. 2023 ). An increase in StFtsZ1 protein levels results in a significant reduction in the number of starch granules and an increase in their size in the tubers (de Pater et al. 2006 ). Further analysis is needed to determine whether FtsZ in maize affects grain starch splitting. Therefore, this study aims to examine the J175 genes, which regulate chloroplast and amyloplast division in maize. Using map-based cloning, we identified that J175 encodes the cell division protein FtsZ (filamentous temperature-sensitive Z). During the seedling stage, white stripes began to emerge on the leaves of the j175 mutant, with a significant decrease in photosynthesis. Electron microscopy observations revealed abnormal division of chloroplasts and amyloplasts in the mutant, resulting in a significantly lower grain weight compared to that of the wild type. These research findings will not only enhance our understanding of the molecular mechanism underlying chloroplast and amyloplast division in maize but also provide valuable genetic resources for improving maize germplasm through molecular regulation. 2. Materials and methods 2.1 Cultivation of materials and investigation of agronomic traits The leaf color-related mutant, isolated from the EMS mutant library with an RP125 background, was temporarily named j175 after undergoing continuous self-selection to stabilize its traits. The material was cultivated and managed in Wenjiang, Chengdu, Sanya, and Hainan provinces. We observed the phenotypes of j175 plants at different stages. During the maturity period, we measured agronomic traits for mutant and wild-type plants, including plant height, ear height, weight, leaf length, leaf width, leaf thickness, ear length, ear thickness, ear weight, ear shaft thickness, ear shaft weight, grain length, grain width, grain thickness, and hundred-grain weight. The data on these agronomic traits were analyzed using Prism 10.2 software. 2.2 Chlorophyll Content Measurement To determine the chlorophyll content, we collected 0.1 g of green, white, and wild-type leaves of j175 at the heading stage. The leaves were washed dried, and their midribs were removed. Subsequently, the leaves were sliced into thin filaments with a blade and placed in a 10 mL centrifuge tube. Furthermore, 10 mL of a mixture of acetone and anhydrous ethanol in a ratio of 1:1 was then added to the tube. The tube was kept in the dark until the green color of the leaves completely faded before measuring chlorophyll using a UV-visible spectrophotometer. We used the following equations to calculate the chlorophyll content: Chl a (mg g − 1 ) =(12.7D 663 -2.69D 645 )×(100/0.1×1,000) Chl b (mg g − 1 )=(22.9D 645 -4.68D 663 )×(100/0.1×1,000) CTChlorophyll(mg g − 1 )=Chl a (mg g − 1 ) + Chl b (mg g − 1 ) 2.3 Determining photosynthetic rate The portable photosynthesis analyzer (Li-COR, Li-4800, NE, USA) was used to measure the photosynthetic rates of j175 white, green, and wild-type leaves during the jointing stage. Measurements included the transpiration rate, net photosynthetic rate, stomatal conductance, and intercellular CO 2 concentration. Three biological replicates were established for each measurement. 2.4 Enzyme activity assessment The white, green, and wild-type ear leaves of j175 were collected during the jointing stage. The following kits were utilized to measure the enzyme activity in the leaves: Plant soluble sugar content detection kit (BC0030, Solaibao, China), Malondialdehyde (MDA) content detection kit (BC0020, Solaibao, China), Catalase (CAT) activity detection kit (BC0200, Solaibao, China), Peroxidase (POD) activity detection kit (BC0090, Solaibao, China), Superoxide dismutase (SOD) activity detection kit (BC0170, Solaibao, China), and Proline (Pro) content detection kit (BC0290, Solaibao, China). 2.5 Chloroplast morphology observation The white, green, and wild-type ear leaves of j175 were collected during the jointing stage and subjected to a series of processes, including Fixation, dehydration, infiltration, embedding, ultrathin sectioning, and staining. Finally, the fixed leaves were examined under a transmission electron microscope (JEM-1400FLASH, Japan) at various magnifications (200×, 500×, and 1000×). 2.6 Amyloplast and starch granule morphology observation The seeds of wild-type and mutant plants were pollinated for 12 and 20 days, respectively. The endosperms were then placed in a culture dish, and a separation solution was added. Furthermore, the endosperm was cut into small particles using a blade and filtered. A pipette was used to absorb the homogenate surrounding the endosperm, and this process was repeated twice before centrifuging the filtrate. The supernatant was discarded, and 5 mL of the separation solution was slowly added while gently flipping the centrifuge tube to suspend the precipitate and this process was repeated once or twice. Following centrifugation, the precipitate obtained was amyloplasts. Subsequently, 2 mL of the separation solution was added dropwise, followed by a drop of 0.5% I2-KI solution, and the sample was observed under a fluorescence microscope (Denyer and Pike 2008 , Echeverria et al. 1985 , Matsushima and Hisano 2019 ). Mature seeds of J175 and wild-type plants were selected from the same location. The seeds were cut in half using a blade and then fixed onto an operating plate. Gold was sputtered onto the seeds, and they were subjected to a vacuum. Additionally, the sections were observed at different magnifications (500×, 1000×, and 2000×) using a scanning electron microscope (Sigma 500, Zeiss, Germany). 2.7 Map-based cloning and allelic test We hybridize the j175 mutant with B73 to generate an F2 segregating population. Genetic analysis was performed by calculating the segregation rate between plants with the mutant phenotype and wild-type plants in the F2 generation, using a chi-squared test to confirm the fit. DNA from isolated single plants in the F2 generation was used for positional cloning. For the initial mapping of the J175 gene, an isolated F2 population was utilized to select 50 leaves with significant phenotypes and 50 leaves without phenotypes, creating mixed dominant and recessive pools for BSA-seq analysis. The population was subsequently expanded, and new polymorphic molecular markers were screened to enable more precise mapping of linkage genes related to the mutant phenotype. Molecular markers were specifically designed to localize accurately based on polymorphisms between the reference genomes of B73 and RP125. To confirm the candidate gene obtained by map-based cloning, we performed an allelic test. An EMS-mutagenized allelic mutant (EMS5-099e01, j175-1 ; EMS4-0e87c8, j175-2) of j175 was obtained from maizeEMSDB ( http://maizeems.qlnu.edu.cn/ ), and the allelic test was conducted using j175 , 175-1 , and j175-2 . 2.8 Protein sequence analysis Protein sequences were aligned using the ClustalW model of MEGA11 (Tamura et al. 2021 ). The gene structure diagram was generated using the online website GSDS2.0 ( https://gsds.gao-lab.org/)(Hu et al. 2015) . The input protein sequence of the structural domain was conserved for Batch CD Search in NCBI, and the output file was processed for visualization analysis using the TBools-II tool (Chen et al. 2023a ). The protein motif structures were predicted using MEME ( https://meme-suite.org/meme/)(Bailey et al. 2015) . 2.9 Subcellular localization To determine the subcellular localization of the J175 protein, its full-length coding sequence (excluding stop codons) was amplified and cloned into the pCAMBIA2300 subcellular localization expression vector, which includes eGFP tags to construct the C-terminal fusion protein J175-eGFP. The 35S: J175 eGFP and 35S: eGFP plasmids were then introduced into maize protoplasts via a polyethylene glycol (PEG)-mediated transformation method. eGFP fluorescence was subsequently detected using a ZEISS LSM 700 laser confocal scanning microscope (Germany). 2.10 RNA-seq analysis For RNA sequencing, samples were collected from the middle of the white, green, and wild-type spike leaves of the jointing mutant. The mutant and wild-type kernels at 10 days after pollination (DAP) were selected, and the kernel coat was removed. Three independent replicates were obtained for each sample. The samples were sequenced and analyzed by Grand Omics (Wuhan, China). 3. Results 3.1 Phenotypic identification of j175 The j175 mutant was derived from EMS mutagenesis of the inbred line RP125 and was continuously backcrossed for purification.. The leaves of the j175 mutant seedlings were yellowed and grew weaker than those of the wild type (Fig. S1 ). During the jointing stage, the leaves changed from yellow to green. At this stage, clear white stripes emerged on the leaves of j175 , and the j17 5 plants exhibited a shorter stature compared to that of the wild type (Fig. 1 A, B). The mature ears of j175 are much shorter than those of the wild type, and the kernels are significantly smaller than those of the wild type (Fig. 1 C, D). The leaf length, width, and thickness of j175 are significantly lower than those of the wild type (Fig. S2). Additionally, the height of the plant, ear height, hundred-kernel weight, kernel length, kernel width, and kernel thickness of j175 were all reduced to varying degrees. However, no significant change was observed in the number of kernels per ear in j175 (Fig. 2 ). 3.2 Chloroplast division defect in j175 We observed the chloroplasts in the leaf mesophyll cells of j175 mutant and wild-type plants. Normal mature mesophyll cells contained multiple chloroplasts of varying sizes (Fig. 1 E). The number of chloroplasts decreased, while the volume increased in the green leaves of the j175 mutant (Fig. 1 F). Furthermore, the white leaves of the j175 mutant had almost no chloroplasts (Fig. 1 G). This suggests that the chloroplast division ability of the j175 mutant is significantly reduced, particularly in the white leaves. The chloroplasts of j175 leaf mesophyll cells during the seedling and mature stages are much larger than those of WT, indicating that this division defect accompanies the entire growth period of the plant(Fig. 1 H,I; Fig. S3 ). 3.3 Chlorophyll content of j175 decreases The photosynthetic capacity of leaves is closely related to their chlorophyll content. Almost no chlorophyll was detected in the white leaves of j175 . In contrast, chlorophyll a, chlorophyll b, and total chlorophyll content in the green leaves of j175 were significantly reduced, with chlorophyll a and b decreasing by 15.43% and 27.95%, respectively (Fig. 3 A). 3.4 j175 photosynthetic rate decreases The net photosynthetic rate refers to the organic matter accumulated during photosynthesis in plants. The net photosynthetic rate of the green leaves of j175 exhibited a decrease of 22.26%, while the rate for the white leaves decreased substantially by 97.93% (Fig. 3 B). Transpiration rate refers to the capacity of plants to regulate water loss through leaf stomata as they adapt to their natural environment. The transpiration rates of j175 green and white leaves exhibited a decrease of 26.50% and 94.60%, respectively (Fig. 3 C). The size of stomatal conductance indicates the extent of stomatal opening in plant leaves, which influences the ability of plants to absorb CO 2 for photosynthesis. The stomatal conductance of j175 green and white leaves decreased by 30.10% and 95.11%, respectively (Fig. 3 D). The intercellular CO 2 concentration is inversely proportional to photosynthesis. Compared to the wild-type leaves, no significant change was observed in intercellular CO 2 concentration in the green leaves of j175. However, the intercellular CO 2 concentration in the white leaves increased by 504.38% (Fig. 3 E). In summary, the mutant showed a significant decrease in the photosynthetic capacity of its green leaves, while photosynthetic capacity was nearly absent in the white leaves. 3.5 Determinations in enzyme activity of j175 The detection of soluble sugar content, MOD content, and CAT activity showed no significant difference between the green leaves of j175 and the wild type. In the white leaves, the soluble sugar content, MOD content, and CAT activity decreased by 75.60%, 69.70%, and 61.80%, respectively. In contrast, the POD activity in the green and white leaves of j175 decreased significantly by 49.77% and 53.85%, respectively. Additionally, no significant change was observed in the activity of SOD dismutase and Pro content (Fig. 3 F-K). These results showed a decrease in the ability of j175 to resist abiotic stress. 3.6 Positional cloning and allelic testing confirmed that J175 encodes FtsZ2-2 protein The homozygous j175 mutant was crossed with the B73 inbred line, and F1 j175 / + plants were self-crossed to create the F2 mapping population. The chi-square test of the F2 population confirmed that the mutant phenotype was a recessive mutation controlled by a single gene (Fig. S4). BSA-seq analysis, conducted on F2 wild-type and mutant segregant populations, identified the location of the gene near the Bin value of 10.07 on chromosome 10 (RefGen_v4) (Fig. S5). Subsequently, using the surrounding insertion-deletion (In Del) markers from the 962 individuals in the F2 mapping population, the candidate interval was narrowed down to 144kb (Fig. 4 A; S6). This region contains four coding genes. PCR amplification and sequencing identified a single-base mutation (G to A) in the third exon of Zm00001d026669, resulting in an amino acid change from glycine (GGG) to arginine (AGG), which altered the function of the gene. This functional change likely contributes to the emergence of the j175 phenotype (Fig. 4 A, B). To determine the candidate gene Zm00001d026669, we conducted allele test. EMS mutants j175-1 and j175-2 of this gene were obtained from maizeEMSDB. The mutations in j175-1 and j175-2 are both C-to-T substitutions, occurring on the exons 1 and exons 6, respectively, which results in the transformation of a arginine and asparagine residue to a premature stop codon leading to the loss of some functional domains in Zm00001d026669 (Fig. 4 A, B). The leaves of self pollinated homozygous j175-1 and j175-2 exhibit the same white striped phenotype as j175 , and their agronomic traits have also been measured to have varying degrees of reduction (Fig. 4 C).The chloroplast division of the mesophyll cells of j175-1 and j175-2 was also inhibited; and the morphology of starch granules was also non rough polygon, which was quite different from the smooth spherical wild type (Fig. S7). Using j175 to hybridize with j175-1 and j175-2 , the offspring j175/175-1 and j175/j175-2 both exhibit a white striped phenotype (Fig. 4 D). These results confirmed that Zm00001d026669 is the causative gene for j175 . Genome sequencing revealed that the protein encoded by J175 consists of 467 amino acids and has a molecular weight of 57.67 kDa. In MaizeGDB, the Zm00001d026669 gene is annotated as a cell division protein FtsZ homolog (Jiao et al. 2017 ). In this study, Zm00001d026669 is referred to as J175. Protein sequence analysis indicated that Zm00001d026669 encodes FtsZ2-2 (J175), a homologous protein of the FtsZ family, which is highly conserved in evolution. Maize contains only three FtsZ proteins: FtsZ1, FtsZ2-1, and FtsZ2-2. FtsZ2-2 shares a high degree of sequence similarity (77.8%) with FtsZ2-1, indicating similar functions. J175 exhibits high sequence similarity (79.80%) to AtFtsZ2-2 (Fig. 5 A; S8). AtFtsZ2-2 is involved in chloroplast division, indicating that J175 may play a role in the process of chloroplast division in maize (Mcandrew et al. 2001 ). Additionally, conservative domain analysis indicated that the missense mutation site(Gly to Arg)of J175 is located at a highly conserved site in the FtsZ protein (Fig. 5 B-E), with the mutated SNP occurring in the second motif. The glycine at this site is highly conserved. We speculate that the mutation at this site affects the function of the protein, leading to the inhibition of chloroplast division and potentially resulting in a complete loss of division ability. 3.7 Constitutive expression and subcellular localization of j175 The expression of the J175 gene in different tissues of wild-type RP125 at the V13 stage (the time when the plant rapidly transitions from vegetative to reproductive growth) was examined using qRT-PCR. J175 was expressed across various tissues, with the highest expression levels in the bracts and female tassels and the lowest in the male tassels (Fig. 6 A). Analysis of j175 expression in seeds at different days after pollination reveals a trend characterized by an initial increase followed by a subsequent decrease, with the peak expression level observed at 10 DAP (Fig. 6 B). To determine the subcellular localization of J175, we fused J175 with GFP and expressed the fusion protein in maize leaf protoplast. Furthermore, as a control, signals from 35: GFP were detected in the nucleus and cytoplasm. In contrast, the signal of 35S: J175 GFP overlaps with the plastid marker (Fig. 6 C), suggesting that J175 functions within the plastid. 3.8 j175 had abnormal development of amyloplasts and starch granules Amyloplasts and chloroplasts are types of plastids that share similarities in their division processes (Mcandrew et al. 2001 ). Endosperm cells were selected for observation using fluorescence microscopy. The wild-type endosperm amyloplasts at 12 DAP displayed a smooth spherical shape, whereas the mutant endosperm amyloplasts exhibited a typical bead-on-a-string shape during the powder-making division. However, some of the amyloplasts displayed relatively regular polygon shapes with smooth surfaces (Fig. 7 A-C). At 20 DAP, the j175 endosperm amyloplasts developed further into irregular polygons with multiple starch particles clustered together, significantly reducing the number of amyloplasts (Fig. 7 D-F). Scanning electron microscopy was utilized to observe the endosperm starch in mature grains of wild-type and j175 . The wild-type starch granules exhibited smooth, spherical shapes arranged in a regular and orderly manner (Fig. 7 G-I). In contrast, starch granules of j175 existed in the form of oligomers, with a smooth surface and compact arrangement, and their number was significantly reduced (Fig. 7 J-L). These results indicate that the mutation of the J175 gene affects amyloplast division in the grain, ultimately forming a complex of oligomers. 3.9 Transcriptome profiling of j175 To analyze the gene expression differences between j175 and WT, RNA-seq was performed on ear leaves and kernel. Overall, 35,375 genes were detected. In the comparison of j175-green-leaf with WT-leaf, 769 genes showed differential expression, with 389 upregulated and 380 downregulated genes. In the j 175-white-leaf vs. WT-leaf comparison, 2,850 genes were differentially expressed, with 2,178 upregulated and 672 downregulated genes. Between j175 -green-leaf and j175 -white-leaf, 3,529 genes showed differential expression, with 2,552 upregulated and 977 downregulated genes. For WT-kernel vs. j 175-kernel, 528 genes were differentially expressed, with 172 upregulated and 356 downregulated genes (Fig. 9 -A). A clustering heatmap analysis of the differential genes revealed that replicates from each sample are grouped closely, indicating similar expression patterns among replicates (Fig. S9). The gene ontology (GO) pathway enrichment analysis of differentially expressed genes in leaves revealed that the main enriched biological processes included "carbohydrate metabolism processes" and "photosynthesis." Functional categories of GO molecules, including "iron ion binding," "oxidoreductase activity," and "UDP glycosyltransferase activity," alongside the component categories of GO cells, such as "photosystem I" and "photosystem II," were significantly enriched (Fig. 8 A; Appendix G). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that differentially expressed genes were mainly concentrated in metabolic and photosynthetic pathways, including "sucrose and starch metabolism," "carbon metabolism," and "photosynthesis," with downregulation being more prevalent than upregulation. The upregulated genes were primarily concentrated in pathways such as plant-pathogen interactions, plant hormone signal transduction, and the MAPK signaling pathway plant. The different leaves of J175 will have varying effects on plant metabolism, hormone signal transduction, and MAPK plant disease resistance signaling pathways (Fig. 8 C, S6). The GO pathway enrichment analysis of differentially expressed genes in kernels indicated that the main biological processes enriched were "immune response" and "Protein morphology." The functional categories of GO molecules, such as "defense response," "protein stabilization," and "response to heat," along with the component categories of GO cells, such as "protein folding chaperone complex" and "protein aggregate center," were significantly enriched (Fig. 8 B). Similar to the leaf transcriptome, KEGG analysis of the kernel transcriptome showed that differentially expressed genes are in the pathway sets of "plant hormone signal transduction" and "plant pathway interaction." However, the difference is that the kernel differential genes are largely enriched in "protein processing in endoplasmic reticulum" (Fig. 8 D). The expression of plastid division-related genes in j175 green and white leaves was inconsistent. Most plastid division-related genes, including j175 , were downregulated in j175 green leaves and kernel, while the upregulation of FtsZ2-1 may partially complement the J175 gene mutation. Additionally, the expression levels of ARC3 and GC1 , which are negative regulators of chloroplast division, were significantly increased in the mutant. To further verify the reliability of the transcriptome data, we conducted qPCR validation of several genes, and the results were consistent with the transcriptome data (Appendix H). Many essential genes involved in kernel development were also significantly differentially expressed between the WT kernels and j175 kernels. Among these, genes such as smk9 (small kernel opaque endosperm2), o2 (opaque endosperm2), fl2 (floury endosperm2), fl3, fl4 , bt2 (brittle endosperm) were downregulated in j175 kernel. However, genes such as betl3 (basal endosperm transfer layer3), betl4, betl9, betl10 , and bap2 (basal layer antifungal protein 2) were upregulated in j175 kernel (Fig. 9 C). To explore the significant differences in chlorophyll content between the WT and j175 , we analyzed the expression levels of key enzyme genes involved in the chlorophyll synthesis pathway. In Fig. 9 D, the expression of the CHLH gene, which encodes the H subunit of magnesium chelatase, was significantly downregulated in J175 white leaves. Together, CHLH, CHLI, and CHLD form a magnesium-chelating enzyme (MgCh) that catalyzes the transformation of protoporphyrin IX into magnesium protoporphyrin IX. This enzyme is crucial in chlorophyll synthesis, and the mutation in the J 175 gene also inhibits this process. 4. Discussion The leaf is the site where photosynthetic products are synthesized, determining the production of assimilates and acting as the "source" of yield formation (Hofius and Börnke 2007 ). As an important C4 plant, 90% of corn yield is derived from leaf photosynthesis (Khanna-Chopra 2000 , Zhu et al. 2010 ). Grain serves as the "reservoir" for materials stored in plants, with starch being the most important storage material in grains, accounting for approximately 70% of the total content (Luchese et al. 2017 ). We discovered that a SNP mutation in the exon of j175 (Fig. 4 A) prevented chloroplast division and inhibited chlorophyll synthesis (Fig. 1 E-G, 3 A). Thus, this reduced the photosynthetic efficiency in j175 (Fig. 3 B-E). Simultaneously, this mutation limited the division of the amyloplasts (Fig. 7 A-F), altering the shape of starch granules (Fig. 7 G-L). Therefore, the j175 mutation affects the "source" and "sink" of yield composition, resulting in a significant reduction in most agronomic traits of this material (Fig. 2 ).As the allelic mutants of j175 , j175-1 and j175-2 , due to the higher position of the j175-1 mutation site, more structural domain functions are lost, resulting in a more severe reduction in agronomic traits for j175-2 (Fig. 4 C). This result further proves that j175 mutation will have a certain impact on the agronomic traits of maize. Chloroplast division mutants are essential genetic materials for studying the underlying mechanisms of chloroplast division in plants (Glynn et al. 2007 , Vitha and S. 2001). Partial genes regulating chloroplast division have been identified, and their regulatory mechanisms have been elucidated through the studies on Arabidopsis mutants deficient in chloroplast division (Okazaki et al. 2009, Pyke et al. 1994 , Robertson et al. 1996 , Yoder et al. 2007 ). This includes FtsZ1, ARC5, ARC6, and PDV1 (plastid division 1). In Arabidopsis, AtFtsZ directly or indirectly affects the localization and assembly of other proteins in the division complex, thereby inhibiting chloroplast division (Miyagishima et al. 2006 , Osteryoung et al. 2014). An increase in AtFtsZ1-1 protein levels inhibits chloroplast division, with higher AtFtsZ1-1 protein levels resulting in more severe phenotypes (Stokes et al. 2000). Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant (Schmitz et al. 2009), we found through evolutionary tree analysis that AtFtsZ2-1 and AtFtsZ2-2 have the highest similarity of 83.55%, while ZmFtsZ2-2 and ZmFtsZ2-1 have only 63.11% similarity. ZmFtsZ2-1 has the highest sequence similarity with SbFtsZ2-2 in sorghum. Does this imply that maize ZmFtsZ2 has different functions compared to Arabidopsis AtFtsZ2. (Fig. 5 ). The j175 mutant gene found in maize not only affects the chloroplast division as observed in other related genes, but also causes the yellowing of the j175 seedlings, weak leaf growth, and the formation of white stripes on the leaves, affecting plant growth (Fig. 1 A, F; S1, 2 , 3 ). This suggests that the j175 gene may serve different functions across various species. The high expression of j175 gene in green tissues such as the ears, ligules, and bracts further indicates its potential role in regulating plant growth and development (Fig. 6 -A). In the mesophyll cells of j175 , the chloroplasts are larger and fewer compared to those of the wild-type chloroplasts (Fig. 1 E-G), suggesting that mutations in the FtsZ 2–2 protein encoded by j175 causes damage to the Z-ring structure during maize chloroplast division, thereby hindering the division process. Furthermore, the RNA-seq and qPCR results demonstrated that most genes related to chloroplast division are inhibited to varying degrees in the j175 green leaves (Fig. 9 D; S11), supporting this hypothesis. The interference with chloroplast development may affect the chlorophyll synthesis, and genes involved in chlorophyll biosynthesis (including ZmHEMD, ZmCHLH, ZmCHLD, and ZmPORD ) are inhibited in the mutants (Fig. 9 C). As the production sites of salicylic and jasmonic acid, which are important mediators of plant immunity, chloroplasts also participate in PAMP-induced defense gene expression, playing a vital role in plant immunity (Nomura et al. 2012 , Zhang et al. 2024 ). Therefore, in the GO and KEGG pathway analyses, several genes related to hormone signal transduction and plant-pathogen interactions were downregulated (Fig. 8 A, C; S10). Additionally, the significant reduction in peroxidase activity observed in the enzyme activity assay could also indicate that the stress resistance of j175 was weakened (Fig. 3 I). Thus, mutations in the genes related to chloroplast division may reduce plant immunity.Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant, Unlike the compound starch granules found in the rice endosperm, maize endosperm contains simple starch granules, with only one starch granule per amyloplast (Kawagoe 2013 , Shannon et al. 1987 ). Changes in the expression levels of amyloplast division proteins can alter starch granule synthesis (Yun MinSoo et al. 2011 ). Moreover, the membrane structure of amyloplasts is also affected by the levels of plastid division proteins (Wang et al. 2020 ). In the j175 mutant, amyloplast division is inhibited during growth and maturity, and amyloplasts in the mature grain endosperm existed in the form of oligomers, with a significant decrease in quantity (Fig. 7 A-L). An increase in StFtsZ1 protein levels in potatoes results in a significant reduction in the number and size of starch granules in tubers (Li et al. 2016 , Sack 1991 , 1997 ). Similarly, in Arabidopsis FtsZ deficient mutants, amyloplasts did not proliferate (Fujiwara et al. 2024 , Osteryoung and Pyke 2014 ), while the j175 mutant in maize exhibited a change in amyloplast shape, implying that the regulation of the maize j175 gene on amyloplasts differs from that of Arabidopsis and potato. Normal amyloplast division, such as during gravity sensing, is essential for plant growth and development (Li et al. 2016 , Sack 1991 , 1997 ). Amyloplasts in Arabidopsis are essential for root gravity sensing and for repolarizing LAZY proteins through sedimentation (Chen et al. 2023b , Kiss and Sack 1989 , Nishimura et al. 2023 , Zhang et al. 2023 ). Whether the j175 gene in maize regulates gravity sensing in plants still requires further study. The formation of vitreous/opaque endosperm depends on the close interactions between proteosomes (storage gliadin) and amyloplasts (Wang et al. 2020 ). Most opaque and floury genes in maize regulate zein synthesis by influencing the regulatory or structural genes of zein (Gillikin et al. 1997 , Kim et al. 2004 , Schmidt et al. 1987 , Wang et al. 2014 ). The upregulation of o2, fl2, fl4, de30 , and other genes in the kernels of the j175 mutant may serve as compensation for the deletion of the j175 gene, allowing stable synthesis of gliadin (Fig. 9 C). However, this specific interaction requires further investigation. The expression of all basal endosperm transfer layer genes and basal antifungal protein genes in maize was downregulated in the j175-kernel (Fig. 9 C). This finding implies that the j175 gene plays a critical role in regulating the development and immunity of the maize endosperm transfer layer, which may contribute to the smaller kernel size observed in j175 (Fig. 2 ). 5. Conclusion The mutation of maize white stripe is caused by a single nucleotide substitution in the exon of the filamentous temperature-sensitive Z protein gene, j175 . This mutation affects the division of chloroplasts and amyloplasts, thereby affecting agronomic traits. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This study was supported by the National Natural Science Foundation of China (32272184). Authorship contribution statement Huayang Lv: Writing – original draft, Data curation.Xuewu He: Methodology. Hongyu Zhang: Investigation. Zeting Mou: Investigation. Xuerui He: Investigation. Hanmei Liu: Software, Visualization. Yangping Li: Investigation. Yinghong Liu: Investigation. Yufeng Hu: Investigation. Zhiming Zhang: Resources. Yubi Huang: Conceptualization. Junjie Zhang: Conceptualization, Supervision, Funding acquisition. References Aaron J. Schmitza b, c, Jonathan M. Glynna,d, Bradley J.S.C. Olsona,e,f, Kevin D. Stokesa,g, Osteryounga K W. 2009. Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant, but FtsZ-based plastid division is not essential for chloroplast partitioning or plant growth and development. Molecular plant, 2 , 1211-1222. Bailey T L, Johnson J, Grant C E, Noble W S. 2015. The MEME suite. Nucleic acids research, 43 , W39-W49. Balmer Y, Vensel W, Cai N, Manieri W, Schurmann P, Hurkman W, Buchanan B. 2006. A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts. Proceedings of the National Academy of Sciences, 103 , 2988-2993. Chen C, MacCready J S, Ducat D C, Osteryoung K W. 2018a. The molecular machinery of chloroplast division. Plant physiology, 176 , 138-151. Chen C, Maccready J S, Ducat D C, Osteryoung K W. 2018b. The Molecular Machinery of Chloroplast Division. Plant physiology, 176 , 138. Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, Liu Y, Feng J, Chen H, He Y. 2023a. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Molecular plant, 16 , 1733-1742. Chen J, Yu R, Li N, ZhaoguoZhang, XinxinZhao, YaranQu, ChengfuYuan, YanfangPan, ZhexianZhou, YangyangLi, KunlunWang, JiajunChen, ZhirenWang, XiaoyiWang, XiaolianHe, Shu-NanDong, JuanDeng, Xing WangChen, Haodong. 2023b. Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants. Cell, 186 , 4788-+. de Pater S, Caspers M, Kottenhagen M, Meima H, Ter Stege R, de Vetten N. 2006. Manipulation of starch granule size distribution in potato tubers by modulation of plastid division. Plant Biotechnology Journal, 4 , 123-134. Denyer K, Pike M. 2008. Isolation of amyloplasts. Current protocols in cell biology, 38 , 3.28. 21-23.28. 15. Echeverria E, Boyer C, Liu K-C, Shannon J. 1985. Isolation of amyloplasts from developing maize endosperm. Plant physiology, 77 , 513-519. Esch L, Ngai Q Y, Barclay J E, Mcnelly R, Hayta S, Smedley M, Smith A M, Seung D. 2023. Increasing amyloplast size in wheat endosperm through mutation of PARC6 affects starch granule morphology. The New phytologist . Fujiwara M T, Yoshioka Y, Kazama Y, Hirano T, Niwa Y, Moriyama T, Sato N, Abe T, Yoshida S, Itoh R D. 2024. Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana. Plant physiology , kiae314. Gillikin J W, Zhang F, Coleman C E, Bass H W, Larkins B A, Boston R S. 1997. A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane. Plant physiology, 114 , 345-352. Glynn J M, Miyagishima S y, Yoder D W, Osteryoung K W, Vitha S. 2007. Chloroplast division. Traffic, 8 , 451-461. Hofius D, Börnke F A. 2007. Photosynthesis, carbohydrate metabolism and source–sink relations. In: Potato biology and biotechnology . Elsevier. pp. 257-285. Hu B, Jin J, Guo A-Y, Zhang H, Luo J, Gao G. 2015. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics, 31 , 1296-1297. Jiao Y, Peluso P, Shi J, Liang T, Stitzer M C, Wang B, Campbell M S, Stein J C, Wei X, Chin C-S. 2017. Improved maize reference genome with single-molecule technologies. Nature, 546 , 524-527. Kawagoe Y. 2013. The characteristic polyhedral, sharp-edged shape of compound-type starch granules in rice endosperm is achieved via the septum-like structure of the amyloplast. Journal of Applied Glycoscience, 60 , 29-36. Kawagoe Y Y. 2009. Amyloplast Division Progresses Simultaneously at Multiple Sites in the Endosperm of Rice. Plant & Cell Physiology, 50 , 1617. Khanna-Chopra R. 2000. Photosynthesis in relation to crop productivity. Probing Photosynthesis: Mechanisms, Regulation and Adaptations , 263-280. Kim C S, Hunter B G, Kraft J, Boston R S, Yans S, Jung R, Larkins B A. 2004. A defective signal peptide in a 19-kD α-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant. Plant physiology, 134 , 380-387. Kiss J Z, Sack H F D. 1989. Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana. Planta, 177 , 198-206. Li, Han-Hai, Xue, Shan, Le, Jie, Zou, Jun-Jie, Wang, Yu-Ren. 2016. The role of Arabidopsis Actin-Related Protein 3 in amyloplast sedimentation and polar auxin transport in root gravitropism. Journal of Experimental Botany . Liu M, Yu J, Yang M, Cao L, Chen C. 2024. Adaptive evolution of chloroplast division mechanisms during plant terrestrialization. Cell Reports, 43 . Luchese C L, Spada J C, Tessaro I C. 2017. Starch content affects physicochemical properties of corn and cassava starch-based films. Industrial Crops and Products, 109 , 619-626. Matsushima R, Hisano H. 2019. Imaging amyloplasts in the developing endosperm of barley and rice. Scientific reports, 9 , 3745. Mcandrew R S, Froehlich J E, Vitha S, Osteryoung S K W. 2001. Colocalization of Plastid Division Proteins in the Chloroplast Stromal Compartment Establishes a New Functional Relationship between FtsZ1 and FtsZ2 in Higher Plants. Plant physiology, 127 , 1656-1666. Miyagishima S Y. Mechanism of Plastid Division: From a Bacterium to an Organelle1. Miyagishima S Y. 2005. Origin and evolution of the chloroplast division machinery. Journal of Plant Research, 118 , 295-306. Miyagishima S Y. 2011. Mechanism of Plastid Division: From a Bacterium to an Organelle. Plant physiology, 155 , 1533-1544. Miyagishima S Y, Froehlich J E, Osteryoung K W. 2006. PDV1 and PDV2 Mediate Recruitment of the Dynamin-Related Protein ARC5 to the Plastid Division Site. THE PLANT CELL ONLINE, 18 , 2517-2530. Miyagishima S Y, Takahara M, Mori T, Kuroiwa H, Kuroiwa T. 2001. Plastid Division Is Driven by a Complex Mechanism That Involves Differential Transition of the Bacterial and Eukaryotic Division Rings. The Plant Cell, 13 , 2257-2268. Nishimura T, Mori S, Shikata H, Nakamura M, Hashiguchi Y, Abe Y, Hagihara T, Yoshikawa H Y, Toyota M, Higaki T. 2023. Cell polarity linked to gravity sensing is generated by LZY translocation from statoliths to the plasma membrane. Science, 381 , 1006-1010. Nomura H, Komori T, Uemura S, Kanda Y, Shimotani K, Nakai K, Furuichi T, Takebayashi K, Sugimoto T, Sano S. 2012. Chloroplast-mediated activation of plant immune signalling in Arabidopsis. Nature communications, 3 , 926. Okazaki, Kumiko, Kabeya, Yukihiro, Suzuki, Kenji, Mori, Toshiyuki, Ichikawa, Takanari. 2009. The PLASTID DIVISION1 and 2 Components of the Chloroplast Division Machinery Determine the Rate of Chloroplast Division in Land Plant Cell Differentiation. Plant Cell . Osteryoung, Katherine, W., Pyke, Kevin, A. 2014. Division and Dynamic Morphology of Plastids. Annual review of plant biology, 65 , 443-472. Osteryoung K W, Mcandrew R S. 2001. THE PLASTID DIVISION MACHINE. Annual Review of Plant Physiology & Plant Molecular Biology, 52 , 315. Osteryoung K W, Pyke K A. 2014. Division and dynamic morphology of plastids. Annual review of plant biology, 65 , 443-472. Osteryoung K W, Stokes K D, Rutherford S M, Lee P W Y. 1998. Chloroplast Division in Higher Plants Requires Members of Two Functionally Divergent Gene Families with Homology to Bacterial ftsZ. THE PLANT CELL ONLINE, 10 , 1991-2004. Pyke K A, Rutherford S M, Robertson E J, Leech R M. 1994. arc6, A Fertile Arabidopsis Mutant with Only Two Mesophyll Cell Chloroplasts. Plant physiology, 106 , 1169-1177. Robertson E J, Rutherford S M, Leech R M. 1996. Characterization of Chloroplast Division Using the Arabidopsis Mutant arc5. Plant physiology . Sack F D. 1991. Plant Gravity Sensing. International Review of Cytology, 127 , 193-252. Sack F D. 1997. Plastids and gravitropic sensing. Planta, 203 , S63-S68. Schmidt R J, Burr F A, Burr B. 1987. Transposon tagging and molecular analysis of the maize regulatory locus opaque-2. Science, 238 , 960-963. Shannon J C, Echeverria E, Boyer C. 1987. [22] Isolation of amyloplasts from developing endosperm of maize (Zea mays L.). In: Methods in enzymology . Elsevier. pp. 226-234. Stokes, McAndrew, Figueroa, Vitha. 2000. Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. Plant physiology . Sun Q, Cao X, Liu Z, An C, Hu J, Wang Y, Qiao M, Gao T, Cheng W, Zhang Y. 2023. Structural and functional insights into the chloroplast division site regulators PARC6 and PDV1 in the intermembrane space. Proceedings of the National Academy of Sciences, 120 , e2215575120. Tamura K, Stecher G, Kumar S. 2021. MEGA11: molecular evolutionary genetics analysis version 11. Molecular biology and evolution, 38 , 3022-3027. Vitha, S. 2001. Ftsz Ring Formation at the Chloroplast Division Site in Plants. Journal of Cell Biology, 153 , 111-120. Wang G, Qi W, Wu Q, Yao D, Zhang J, Zhu J, Wang G, Wang G, Tang Y, Song R. 2014. Identification and characterization of maize floury4 as a novel semidominant opaque mutant that disrupts protein body assembly. Plant physiology, 165 , 582-594. Wang H, Huang Y, Xiao Q, Huang X, Li C, Gao X, Wang Q, Xiang X, Zhu Y, Wang J. 2020. Carotenoids modulate kernel texture in maize by influencing amyloplast envelope integrity. Nature communications, 11 , 5346. Yang Y, Glynn J M, Olson B J, Schmitz A J, Osteryoung K W. 2008. Plastid division: across time and space. Current opinion in plant biology, 11 , 577-584. Yoder D W, Kadirjan-Kalbach D, Olson B J S C, Miyagishima S Y, Deblasio S L, Hangarter R P, Osteryoung, W. K. 2007. Effects of Mutations in Arabidopsis FtsZ1 on Plastid Division, FtsZ Ring Formation and Positioning, and FtsZ Filament Morphology in Vivo. Plant & Cell Physiology, 48 , 775. Yun MinSoo Y M, Umemoto T, Kawagoe Y. 2011. Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis. Zhang N, Guil S, Wang Y. 2023. LAZing around: The intricate dance of amyloplast sedimentation and gravity sensing in plants. Molecular Plant , 016. Zhang R, Wu Y, Qu X, Yang W, Wu Q, Huang L, Jiang Q, Ma J, Zhang Y, Qi P. 2024. The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function. Nature communications, 15 , 6905. Zhao F, Jing L, Wang D, Bao F, Wang G. 2018. Grain and starch granule morphology in superior and inferior kernels of maize in response to nitrogen. Scientific Reports, 8 , 6343. Zhu X-G, Long S P, Ort D R. 2010. Improving photosynthetic efficiency for greater yield. Annual review of plant biology, 61 , 235-261. Zhu Y S, Merkle-Lehman D L, Kung S D. 1984. Light-Induced Transformation of Amyloplasts into Chloroplasts in Potato Tubers. Plant physiology, 75 , 142-145. Supplementary Files RevisedSupplementarywithTrackChanges.docx Cite Share Download PDF Status: Posted Version 1 posted 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-5848477","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446615615,"identity":"d5f7c46b-4de7-4f2e-95b9-8b037d2a0965","order_by":0,"name":"Huayang Lv","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huayang","middleName":"","lastName":"Lv","suffix":""},{"id":446615616,"identity":"92297f4c-cfd5-4de0-9501-a507e347394d","order_by":1,"name":"Xuewu He","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xuewu","middleName":"","lastName":"He","suffix":""},{"id":446615617,"identity":"660509e1-66ff-4e78-8da0-24bcb05d57c3","order_by":2,"name":"Hongyu Zhang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Zhang","suffix":""},{"id":446615618,"identity":"e799ebe7-7ed7-4eaa-bfe8-4f90b3b0ba03","order_by":3,"name":"Zeting Mou","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zeting","middleName":"","lastName":"Mou","suffix":""},{"id":446615619,"identity":"727ed8a6-d39b-4733-b8e5-e1a27c10f636","order_by":4,"name":"Xuerui He","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xuerui","middleName":"","lastName":"He","suffix":""},{"id":446615620,"identity":"24386f4e-f23f-4046-97e7-8ea7c19102b3","order_by":5,"name":"Yongping Li","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yongping","middleName":"","lastName":"Li","suffix":""},{"id":446615621,"identity":"688287b3-c976-4c30-a9b9-853046def9e3","order_by":6,"name":"Yinghong Liu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yinghong","middleName":"","lastName":"Liu","suffix":""},{"id":446615622,"identity":"197eb1fe-c8a0-4900-bed3-8e2ec8392c50","order_by":7,"name":"Hanmei Liu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hanmei","middleName":"","lastName":"Liu","suffix":""},{"id":446615623,"identity":"5ea01e32-5a8f-413e-b4a1-6cf289186222","order_by":8,"name":"Yufeng Hu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yufeng","middleName":"","lastName":"Hu","suffix":""},{"id":446615624,"identity":"7eea4782-71e8-4194-be66-ccbd36568bb9","order_by":9,"name":"Zhiming Zhang","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhiming","middleName":"","lastName":"Zhang","suffix":""},{"id":446615625,"identity":"22c0f272-c255-48cb-a5f9-ad9435214897","order_by":10,"name":"Yubi Huang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yubi","middleName":"","lastName":"Huang","suffix":""},{"id":446615626,"identity":"236ff616-3d08-4c68-8b8e-58e890a3ce3e","order_by":11,"name":"junjie zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYBACxgYGBoMEBoYEEPsBgwFEVIJYLcwGRGmBAZAWNrhKvFqYZ+QeKHhQUZfHP7v9WuWPAjt5gwPMB2/zMNjl4XTYjLwEg4QzbMUSd86U3eYxSDbccIAt2ZqHIbkYt5YcA4PENp7Ehhs5abcZDA4wbjjAYybNw3AgsQGvln8SifOBWgp/GByw33CA/xsRWhoMEjfcSD/GwGNwIBFoCxt+LT1vDAwSjiUkbryRwywN9EvyzMNsxpZzDJJxajFszzEz/FFTlzjvRvrDjz/+2Nn2HW9+eONNhR1uLQ0MbND444HSzCDCAId6IJAHKnkAYbI/wK1sFIyCUTAKRjQAAEhTWfFiS4qNAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4781-6862","institution":"Sichuan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"junjie","middleName":"","lastName":"zhang","suffix":""}],"badges":[],"createdAt":"2025-01-17 10:50:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5848477/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5848477/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81309839,"identity":"90e168da-edf4-4f1c-a605-e98d8d3868a5","added_by":"auto","created_at":"2025-04-24 15:15:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":822983,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic observation of \u003cem\u003ej175. \u003c/em\u003eA, Ear leaf of \u003cem\u003ej175\u003c/em\u003eand wild type at jointing stage (bar: 4 cm). B, \u003cem\u003ej175\u003c/em\u003e and wild type plants at the jointing stage (bar: 20 cm). C, \u003cem\u003ej175\u003c/em\u003e and wild type kernels (bar: 1 cm). D, Ear size of \u003cem\u003ej175 \u003c/em\u003eand wild type (bar: 2 cm). E, Wild type mesophyll cells. F\u003cem\u003e, j175\u003c/em\u003e green leaf mesophyll cells. G, \u003cem\u003ej175\u003c/em\u003ewhite leaf mesophyll cells; cp, chloroplast (bar: 2 cm).H, Statistical analysis of chloroplast surface area in WT and j175 leaf mesophyll cells during seedling stage under projection electron microscopy. I, Statistical analysis of chloroplast surface area in mature WT and j175 mesophyll cells under projection electron microscopy. Data are presented as mean ± SD and were statistically analyzed using Student's t-test with N=20. * (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) denotes a significant difference between WT and \u003cem\u003ej175\u003c/em\u003e, while ** (p\u0026lt;0.01) indicates a highly significant difference between them.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/b684e807bc09746d2fb3ceb7.png"},{"id":81309837,"identity":"6ba5dce2-aedb-4eac-a9af-50a76a6f0f94","added_by":"auto","created_at":"2025-04-24 15:15:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140420,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical analysis of\u003cem\u003e j175\u003c/em\u003e agronomic traits. Data are presented as mean ± SD and statistically calculated using Student's t-test, with N=20. * (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) denotes a significant difference between WT and \u003cem\u003ej175\u003c/em\u003e, while ** (p\u0026lt;0.01) indicates a highly significant difference between them.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/e9c4f2905141075ce4da2af1.png"},{"id":81309841,"identity":"5466e492-3107-4be5-b5cd-69fadf8336a5","added_by":"auto","created_at":"2025-04-24 15:15:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":214591,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of chlorophyll content, enzyme activity, and photosynthetic rate in \u003cem\u003ej175. \u003c/em\u003eA, Chlorophyll content. B–E, Photosynthetic rate related indicators: Pn (net photosynthetic rate), Tr (transpiration rate), Cond (stomatal conductance), Ci (intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration). E, Chlorophyll content. F–K, Enzyme activity assay: MOD (malondialdehyde), CAT (catalase), POD (peroxidase), SOD (superoxide dismutase), and Pro (proline). Data are presented as mean ± SD and statistically analyzed using Student's t-test, with N=4. * (p\u0026lt;0.05), ** (p\u0026lt;0.01)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/70cce0cb708cd4479f364fca.png"},{"id":81311395,"identity":"7e2e565b-c4fe-46e2-9481-4050abfe4b9c","added_by":"auto","created_at":"2025-04-24 15:31:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":348903,"visible":true,"origin":"","legend":"\u003cp\u003ePositional cloning and allelic testing A, Positional cloning and Gene structure and mutation sit; B, sequencing analysis of \u003cem\u003ej175\u003c/em\u003e, \u003cem\u003ej175-1\u003c/em\u003eand \u003cem\u003ej715-2\u003c/em\u003e; C, Partial agronomic traits of \u003cem\u003ej175-1\u003c/em\u003e and \u003cem\u003ej175-2\u003c/em\u003e; D, Allelic test of \u003cem\u003ej175.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/8d7da792f559ff6c6a055b23.png"},{"id":81309842,"identity":"417a9ddb-c8b9-4c7b-8013-dab020f99fd5","added_by":"auto","created_at":"2025-04-24 15:15:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":264553,"visible":true,"origin":"","legend":"\u003cp\u003eGene Evolution Analysis. A, Evolutionary tree analysis. B, Gene structure analysis. C, Conservative structural domain analysis. D–E, Gene motif analysis\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/1c344f2b8889f8a5ef400656.png"},{"id":81311397,"identity":"cdd84481-b3ba-466d-b609-3af6c4d31775","added_by":"auto","created_at":"2025-04-24 15:31:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":211476,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic expression and subcellular localization. A, \u003cem\u003eJ75\u003c/em\u003e group forming expression. B, Grain expression of j175 at different DAP. C, Subcellular localization (bar: 10 μm)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/655966b3bc425dfd49e2573a.png"},{"id":81310910,"identity":"4a6e6ea0-c862-4a52-954c-74736005051f","added_by":"auto","created_at":"2025-04-24 15:23:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":712950,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of endosperm amyloplasts and starch grains. A, 12DAP wild-type endosperm amyloplasts. B–C, 12DAP \u003cem\u003ej175\u003c/em\u003e endosperm amyloplasts. D, 20DAP wild-type endosperm amyloplasts. E–F, 20DAP \u003cem\u003ej175\u003c/em\u003e endosperm amyloplasts (bar: 5 μm);G–I, Wild type endosperm starch granules at different magnifications. J-L, \u003cem\u003ej175\u003c/em\u003eendosperm starch granules at different magnifications\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/ff8ea3a2783ef3bbf0deaf41.png"},{"id":81309849,"identity":"572e2332-44ce-47cc-a5e5-8da822019bd9","added_by":"auto","created_at":"2025-04-24 15:15:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":140711,"visible":true,"origin":"","legend":"\u003cp\u003eGene ontology categories and pathway enrichment of differentially expressed genes. A and B, GO and KEGG enrichment analysis of WT leaf and \u003cem\u003ej175\u003c/em\u003e green leaf. C and D, GO and KEGG enrichment analysis of WT kernel and \u003cem\u003ej175\u003c/em\u003e kernel.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/843932721ad7088fd65ce778.png"},{"id":81309846,"identity":"cb78c4f4-726b-480b-9550-257a5dc57c16","added_by":"auto","created_at":"2025-04-24 15:15:42","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":265364,"visible":true,"origin":"","legend":"\u003cp\u003ePartial differentially expressed genes. A, Scatter diagram of different genes in multiple groups. B, Expression of genes related to chloroplast division. C, expression levels of the genes involved in kernel development. D, Expression of genes related to chlorophyll synthesis\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/d339336fe363ed1e7064f36d.png"},{"id":82308737,"identity":"76254920-d423-4e99-b4f2-4d82806bf67b","added_by":"auto","created_at":"2025-05-09 01:36:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4156567,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/f30e7f43-6528-46db-bde7-6940029f79bd.pdf"},{"id":81309869,"identity":"22ac076b-c02a-494f-b3bf-a90fe9b70107","added_by":"auto","created_at":"2025-04-24 15:15:42","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4155182,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedSupplementarywithTrackChanges.docx","url":"https://assets-eu.researchsquare.com/files/rs-5848477/v1/f8ecb8e5fdb5ef4f80868417.docx"}],"financialInterests":"","formattedTitle":"Filamentous temperature-sensitive Z protein J175 regulates maize chloroplasts and amyloplasts division and development","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMaize, a vital C4 plant, serves as a food crop and an important industrial raw material. Chloroplasts are the primary sites for photosynthesis and numerous metabolic reactions. Chloroplast division is crucial for the survival and reproduction of various plant species in nature (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e, Glynn et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The division of amyloplasts significantly influences the size, morphology, and yield of starch granules (Kawagoe \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Zhao et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In cells with varying physiological and developmental states, chloroplasts and amyloplasts undergo different degrees of division. This process is governed by multiple pathways that require further refinement (Osteryoung and Mcandrew \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChloroplasts are photosynthetic organelles in plants that convert energy. They have a double-layered membrane structure (Osteryoung et al. 2003; Martin and Kowallik 1999). They are believed to have evolved from cyanobacteria and replicate through a process called binary fission (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e, Miyagishima, Osteryoung \u003cem\u003eet al.\u003c/em\u003e 2014). The mechanism responsible for chloroplast division mainly involves four ring-like structures: the FtsZ ring (Z ring), inner and outer plastid-dividing rings, and the DRP5B ring. These components contract and compress the division site to facilitate chloroplast division (Miyagishima, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Osteryoung \u003cem\u003eet al.\u003c/em\u003e 2014). The Z ring, formed by chloroplast FtsZ proteins of cyanobacterial origin, is the first ring structure formed during chloroplast division (Miyagishima et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e); it belongs to the GTPase family, which is associated with tubulin and acts as a scaffold protein for the division complex (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e, Osteryoung and Mcandrew \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In plants, FtsZ proteins are composed of two subfamilies: FtsZ1 and FtsZ2. These proteins assemble into a polymer and form a ring at the cleavage site (Sun et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Vitha and S. 2001). The FtsZ proteins in chloroplasts are anchored to the inner membrane through an interaction between the C-terminal domain of FtsZ2 and N-terminal domain of the chloroplast transmembrane protein accumulation and replication (ARC6) (Miyagishima \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In \u003cem\u003earc6\u003c/em\u003e mutants, filamentous FtsZ proteins are fragmented, indicating that ARC6 is crucial for the assembly and stability of FtsZ rings (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). The chloroplast outer envelope membrane protein PDV2 and its paralog PDV1 recruit the cytosolic dynamin-related GTPase ARC5 (accumulation and replication of chloroplasts 5) to the chloroplast division site (Miyagishima et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), which determines the rate of chloroplast division (Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Okazaki \u003cem\u003eet al.\u003c/em\u003e 2009). Studies have demonstrated that the size of chloroplasts can be altered through either the overexpression or antisense repression of \u003cem\u003eFtsZ\u003c/em\u003e (Mcandrew et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Osteryoung et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Stokes \u003cem\u003eet al.\u003c/em\u003e 2000). Overexpression or antisense repression of \u003cem\u003eAtFtsZ1\u003c/em\u003e or \u003cem\u003eAtFtsZ2\u003c/em\u003e in Arabidopsis resulted in the formation of one or few large chloroplasts per cell. A threefold increase in AtFtsZ1-1 protein levels inhibited chloroplast division (Mcandrew et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Stokes \u003cem\u003eet al.\u003c/em\u003e 2000). Higher AtFtsZ1-1 protein levels resulted in more severe phenotypes. The defects in chloroplast division caused by AtFtsZ1-1 overproduction may reflect a stoichiometric imbalance among the components necessary for chloroplast division (Mcandrew et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Furthermore, a decrease in StFtsZ1 protein levels in potato leaves resulted in the reduction of chloroplasts in guard cells (de Pater et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The role of the FtsZ protein in Z ring assembly during chloroplast division in maize requires further investigation.\u003c/p\u003e \u003cp\u003eThe amyloplast reaction in cereals can coordinate with photosynthesis occurring in the leaves, allowing the products of both reactions to be mutually transformed under light induction (Balmer et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Zhu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Ultrastructural, molecular, and genetic data indicate that the components required for the division process are similar across all plastid types (Mcandrew et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Osteryoung and Mcandrew \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The mutant parc6, which affects the splitting of wheat chloroplasts and amyloplasts, exhibited enlarged plastids in the leaves and endosperm. Additionally, the endosperm amyloplasts of this mutant contained a higher proportion of A-type and B-type starch particles compared to the wild type (Esch et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). An increase in StFtsZ1 protein levels results in a significant reduction in the number of starch granules and an increase in their size in the tubers (de Pater et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Further analysis is needed to determine whether FtsZ in maize affects grain starch splitting.\u003c/p\u003e \u003cp\u003eTherefore, this study aims to examine the \u003cem\u003eJ175\u003c/em\u003e genes, which regulate chloroplast and amyloplast division in maize. Using map-based cloning, we identified that \u003cem\u003eJ175\u003c/em\u003e encodes the cell division protein FtsZ (filamentous temperature-sensitive Z). During the seedling stage, white stripes began to emerge on the leaves of the \u003cem\u003ej175\u003c/em\u003e mutant, with a significant decrease in photosynthesis. Electron microscopy observations revealed abnormal division of chloroplasts and amyloplasts in the mutant, resulting in a significantly lower grain weight compared to that of the wild type. These research findings will not only enhance our understanding of the molecular mechanism underlying chloroplast and amyloplast division in maize but also provide valuable genetic resources for improving maize germplasm through molecular regulation.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cultivation of materials and investigation of agronomic traits\u003c/h2\u003e \u003cp\u003eThe leaf color-related mutant, isolated from the EMS mutant library with an RP125 background, was temporarily named \u003cem\u003ej175\u003c/em\u003e after undergoing continuous self-selection to stabilize its traits. The material was cultivated and managed in Wenjiang, Chengdu, Sanya, and Hainan provinces. We observed the phenotypes of \u003cem\u003ej175\u003c/em\u003e plants at different stages. During the maturity period, we measured agronomic traits for mutant and wild-type plants, including plant height, ear height, weight, leaf length, leaf width, leaf thickness, ear length, ear thickness, ear weight, ear shaft thickness, ear shaft weight, grain length, grain width, grain thickness, and hundred-grain weight. The data on these agronomic traits were analyzed using Prism 10.2 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Chlorophyll Content Measurement\u003c/h2\u003e \u003cp\u003eTo determine the chlorophyll content, we collected 0.1 g of green, white, and wild-type leaves of j175 at the heading stage. The leaves were washed dried, and their midribs were removed. Subsequently, the leaves were sliced into thin filaments with a blade and placed in a 10 mL centrifuge tube. Furthermore, 10 mL of a mixture of acetone and anhydrous ethanol in a ratio of 1:1 was then added to the tube. The tube was kept in the dark until the green color of the leaves completely faded before measuring chlorophyll using a UV-visible spectrophotometer. We used the following equations to calculate the chlorophyll content:\u003c/p\u003e \u003cp\u003eChl \u003cem\u003ea\u003c/em\u003e(mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) =(12.7D\u003csub\u003e663\u003c/sub\u003e-2.69D\u003csub\u003e645\u003c/sub\u003e)\u0026times;(100/0.1\u0026times;1,000)\u003c/p\u003e \u003cp\u003eChl \u003cem\u003eb\u003c/em\u003e(mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)=(22.9D\u003csub\u003e645\u003c/sub\u003e-4.68D\u003csub\u003e663\u003c/sub\u003e)\u0026times;(100/0.1\u0026times;1,000)\u003c/p\u003e \u003cp\u003eCTChlorophyll(mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)=Chl \u003cem\u003ea\u003c/em\u003e(mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;+\u0026thinsp;Chl \u003cem\u003eb\u003c/em\u003e(mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determining photosynthetic rate\u003c/h2\u003e \u003cp\u003eThe portable photosynthesis analyzer (Li-COR, Li-4800, NE, USA) was used to measure the photosynthetic rates of \u003cem\u003ej175\u003c/em\u003e white, green, and wild-type leaves during the jointing stage. Measurements included the transpiration rate, net photosynthetic rate, stomatal conductance, and intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration. Three biological replicates were established for each measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Enzyme activity assessment\u003c/h2\u003e \u003cp\u003eThe white, green, and wild-type ear leaves of j175 were collected during the jointing stage. The following kits were utilized to measure the enzyme activity in the leaves: Plant soluble sugar content detection kit (BC0030, Solaibao, China), Malondialdehyde (MDA) content detection kit (BC0020, Solaibao, China), Catalase (CAT) activity detection kit (BC0200, Solaibao, China), Peroxidase (POD) activity detection kit (BC0090, Solaibao, China), Superoxide dismutase (SOD) activity detection kit (BC0170, Solaibao, China), and Proline (Pro) content detection kit (BC0290, Solaibao, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Chloroplast morphology observation\u003c/h2\u003e \u003cp\u003eThe white, green, and wild-type ear leaves of j175 were collected during the jointing stage and subjected to a series of processes, including Fixation, dehydration, infiltration, embedding, ultrathin sectioning, and staining. Finally, the fixed leaves were examined under a transmission electron microscope (JEM-1400FLASH, Japan) at various magnifications (200\u0026times;, 500\u0026times;, and 1000\u0026times;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Amyloplast and starch granule morphology observation\u003c/h2\u003e \u003cp\u003eThe seeds of wild-type and mutant plants were pollinated for 12 and 20 days, respectively. The endosperms were then placed in a culture dish, and a separation solution was added. Furthermore, the endosperm was cut into small particles using a blade and filtered. A pipette was used to absorb the homogenate surrounding the endosperm, and this process was repeated twice before centrifuging the filtrate. The supernatant was discarded, and 5 mL of the separation solution was slowly added while gently flipping the centrifuge tube to suspend the precipitate and this process was repeated once or twice. Following centrifugation, the precipitate obtained was amyloplasts. Subsequently, 2 mL of the separation solution was added dropwise, followed by a drop of 0.5% I2-KI solution, and the sample was observed under a fluorescence microscope (Denyer and Pike \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Echeverria et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1985\u003c/span\u003e, Matsushima and Hisano \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMature seeds of J175 and wild-type plants were selected from the same location. The seeds were cut in half using a blade and then fixed onto an operating plate. Gold was sputtered onto the seeds, and they were subjected to a vacuum. Additionally, the sections were observed at different magnifications (500\u0026times;, 1000\u0026times;, and 2000\u0026times;) using a scanning electron microscope (Sigma 500, Zeiss, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Map-based cloning and allelic test\u003c/h2\u003e \u003cp\u003eWe hybridize the \u003cem\u003ej175\u003c/em\u003e mutant with B73 to generate an F2 segregating population. Genetic analysis was performed by calculating the segregation rate between plants with the mutant phenotype and wild-type plants in the F2 generation, using a chi-squared test to confirm the fit. DNA from isolated single plants in the F2 generation was used for positional cloning. For the initial mapping of the \u003cem\u003eJ175\u003c/em\u003e gene, an isolated F2 population was utilized to select 50 leaves with significant phenotypes and 50 leaves without phenotypes, creating mixed dominant and recessive pools for BSA-seq analysis. The population was subsequently expanded, and new polymorphic molecular markers were screened to enable more precise mapping of linkage genes related to the mutant phenotype. Molecular markers were specifically designed to localize accurately based on polymorphisms between the reference genomes of B73 and RP125. To confirm the candidate gene obtained by map-based cloning, we performed an allelic test. An EMS-mutagenized allelic mutant (EMS5-099e01, \u003cem\u003ej175-1\u003c/em\u003e; EMS4-0e87c8, j175-2) of \u003cem\u003ej175\u003c/em\u003e was obtained from maizeEMSDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://maizeems.qlnu.edu.cn/\u003c/span\u003e\u003cspan address=\"http://maizeems.qlnu.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the allelic test was conducted using \u003cem\u003ej175\u003c/em\u003e, \u003cem\u003e175-1\u003c/em\u003e, and \u003cem\u003ej175-2\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Protein sequence analysis\u003c/h2\u003e \u003cp\u003eProtein sequences were aligned using the ClustalW model of MEGA11 (Tamura et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The gene structure diagram was generated using the online website GSDS2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gsds.gao-lab.org/)(Hu et al. 2015)\u003c/span\u003e\u003cspan address=\"https://gsds.gao-lab.org/)(Hu et al. 2015)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The input protein sequence of the structural domain was conserved for Batch CD Search in NCBI, and the output file was processed for visualization analysis using the TBools-II tool (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). The protein motif structures were predicted using MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/)(Bailey et al. 2015)\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/)(Bailey et al. 2015)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Subcellular localization\u003c/h2\u003e \u003cp\u003eTo determine the subcellular localization of the J175 protein, its full-length coding sequence (excluding stop codons) was amplified and cloned into the pCAMBIA2300 subcellular localization expression vector, which includes eGFP tags to construct the C-terminal fusion protein J175-eGFP. The 35S: J175 eGFP and 35S: eGFP plasmids were then introduced into maize protoplasts via a polyethylene glycol (PEG)-mediated transformation method. eGFP fluorescence was subsequently detected using a ZEISS LSM 700 laser confocal scanning microscope (Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 RNA-seq analysis\u003c/h2\u003e \u003cp\u003eFor RNA sequencing, samples were collected from the middle of the white, green, and wild-type spike leaves of the jointing mutant. The mutant and wild-type kernels at 10 days after pollination (DAP) were selected, and the kernel coat was removed. Three independent replicates were obtained for each sample. The samples were sequenced and analyzed by Grand Omics (Wuhan, China).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Phenotypic identification of \u003cem\u003ej175\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ej175\u003c/em\u003e mutant was derived from EMS mutagenesis of the inbred line RP125 and was continuously backcrossed for purification.. The leaves of the \u003cem\u003ej175\u003c/em\u003e mutant seedlings were yellowed and grew weaker than those of the wild type (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). During the jointing stage, the leaves changed from yellow to green. At this stage, clear white stripes emerged on the leaves of \u003cem\u003ej175\u003c/em\u003e, and the \u003cem\u003ej17\u003c/em\u003e5 plants exhibited a shorter stature compared to that of the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). The mature ears of \u003cem\u003ej175\u003c/em\u003e are much shorter than those of the wild type, and the kernels are significantly smaller than those of the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). The leaf length, width, and thickness of \u003cem\u003ej175\u003c/em\u003e are significantly lower than those of the wild type (Fig. S2). Additionally, the height of the plant, ear height, hundred-kernel weight, kernel length, kernel width, and kernel thickness of \u003cem\u003ej175\u003c/em\u003e were all reduced to varying degrees. However, no significant change was observed in the number of kernels per ear in \u003cem\u003ej175\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Chloroplast division defect in \u003cem\u003ej175\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWe observed the chloroplasts in the leaf mesophyll cells of j175 mutant and wild-type plants. Normal mature mesophyll cells contained multiple chloroplasts of varying sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The number of chloroplasts decreased, while the volume increased in the green leaves of the j175 mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Furthermore, the white leaves of the j175 mutant had almost no chloroplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). This suggests that the chloroplast division ability of the j175 mutant is significantly reduced, particularly in the white leaves. The chloroplasts of j175 leaf mesophyll cells during the seedling and mature stages are much larger than those of WT, indicating that this division defect accompanies the entire growth period of the plant(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH,I; Fig. S3 ).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Chlorophyll content of \u003cem\u003ej175\u003c/em\u003e decreases\u003c/h2\u003e \u003cp\u003eThe photosynthetic capacity of leaves is closely related to their chlorophyll content. Almost no chlorophyll was detected in the white leaves of \u003cem\u003ej175\u003c/em\u003e. In contrast, chlorophyll a, chlorophyll b, and total chlorophyll content in the green leaves of \u003cem\u003ej175\u003c/em\u003e were significantly reduced, with chlorophyll a and b decreasing by 15.43% and 27.95%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cem\u003ej175\u003c/em\u003e photosynthetic rate decreases\u003c/h2\u003e \u003cp\u003eThe net photosynthetic rate refers to the organic matter accumulated during photosynthesis in plants. The net photosynthetic rate of the green leaves of \u003cem\u003ej175\u003c/em\u003e exhibited a decrease of 22.26%, while the rate for the white leaves decreased substantially by 97.93% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Transpiration rate refers to the capacity of plants to regulate water loss through leaf stomata as they adapt to their natural environment. The transpiration rates of \u003cem\u003ej175\u003c/em\u003e green and white leaves exhibited a decrease of 26.50% and 94.60%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The size of stomatal conductance indicates the extent of stomatal opening in plant leaves, which influences the ability of plants to absorb CO\u003csub\u003e2\u003c/sub\u003e for photosynthesis. The stomatal conductance of \u003cem\u003ej175\u003c/em\u003e green and white leaves decreased by 30.10% and 95.11%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration is inversely proportional to photosynthesis. Compared to the wild-type leaves, no significant change was observed in intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration in the green leaves of j175. However, the intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration in the white leaves increased by 504.38% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In summary, the mutant showed a significant decrease in the photosynthetic capacity of its green leaves, while photosynthetic capacity was nearly absent in the white leaves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Determinations in enzyme activity of \u003cem\u003ej175\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe detection of soluble sugar content, MOD content, and CAT activity showed no significant difference between the green leaves of \u003cem\u003ej175\u003c/em\u003e and the wild type. In the white leaves, the soluble sugar content, MOD content, and CAT activity decreased by 75.60%, 69.70%, and 61.80%, respectively. In contrast, the POD activity in the green and white leaves of \u003cem\u003ej175\u003c/em\u003e decreased significantly by 49.77% and 53.85%, respectively. Additionally, no significant change was observed in the activity of SOD dismutase and Pro content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-K). These results showed a decrease in the ability of \u003cem\u003ej175\u003c/em\u003e to resist abiotic stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Positional cloning and allelic testing confirmed that J175 encodes FtsZ2-2 protein\u003c/h2\u003e \u003cp\u003eThe homozygous \u003cem\u003ej175\u003c/em\u003e mutant was crossed with the B73 inbred line, and F1 \u003cem\u003ej175\u003c/em\u003e/ + plants were self-crossed to create the F2 mapping population. The chi-square test of the F2 population confirmed that the mutant phenotype was a recessive mutation controlled by a single gene (Fig. S4). BSA-seq analysis, conducted on F2 wild-type and mutant segregant populations, identified the location of the gene near the Bin value of 10.07 on chromosome 10 (RefGen_v4) (Fig. S5). Subsequently, using the surrounding insertion-deletion (In Del) markers from the 962 individuals in the F2 mapping population, the candidate interval was narrowed down to 144kb (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; S6). This region contains four coding genes. PCR amplification and sequencing identified a single-base mutation (G to A) in the third exon of Zm00001d026669, resulting in an amino acid change from glycine (GGG) to arginine (AGG), which altered the function of the gene. This functional change likely contributes to the emergence of the \u003cem\u003ej175\u003c/em\u003e phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the candidate gene Zm00001d026669, we conducted allele test. EMS mutants \u003cem\u003ej175-1\u003c/em\u003e and \u003cem\u003ej175-2\u003c/em\u003e of this gene were obtained from maizeEMSDB. The mutations in \u003cem\u003ej175-1\u003c/em\u003e and \u003cem\u003ej175-2\u003c/em\u003e are both C-to-T substitutions, occurring on the exons 1 and exons 6, respectively, which results in the transformation of a arginine and asparagine residue to a premature stop codon leading to the loss of some functional domains in Zm00001d026669 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). The leaves of self pollinated homozygous \u003cem\u003ej175-1\u003c/em\u003e and \u003cem\u003ej175-2\u003c/em\u003e exhibit the same white striped phenotype as \u003cem\u003ej175\u003c/em\u003e, and their agronomic traits have also been measured to have varying degrees of reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).The chloroplast division of the mesophyll cells of j175-1 and j175-2 was also inhibited; and the morphology of starch granules was also non rough polygon, which was quite different from the smooth spherical wild type (Fig. S7). Using \u003cem\u003ej175\u003c/em\u003e to hybridize with \u003cem\u003ej175-1\u003c/em\u003e and \u003cem\u003ej175-2\u003c/em\u003e, the offspring \u003cem\u003ej175/175-1\u003c/em\u003e and \u003cem\u003ej175/j175-2\u003c/em\u003e both exhibit a white striped phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results confirmed that Zm00001d026669 is the causative gene for \u003cem\u003ej175\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGenome sequencing revealed that the protein encoded by J175 consists of 467 amino acids and has a molecular weight of 57.67 kDa. In MaizeGDB, the Zm00001d026669 gene is annotated as a cell division protein FtsZ homolog (Jiao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, Zm00001d026669 is referred to as J175. Protein sequence analysis indicated that Zm00001d026669 encodes FtsZ2-2 (J175), a homologous protein of the FtsZ family, which is highly conserved in evolution. Maize contains only three FtsZ proteins: FtsZ1, FtsZ2-1, and FtsZ2-2. FtsZ2-2 shares a high degree of sequence similarity (77.8%) with FtsZ2-1, indicating similar functions. J175 exhibits high sequence similarity (79.80%) to AtFtsZ2-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; S8). AtFtsZ2-2 is involved in chloroplast division, indicating that J175 may play a role in the process of chloroplast division in maize (Mcandrew et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Additionally, conservative domain analysis indicated that the missense mutation site(Gly to Arg)of \u003cem\u003eJ175\u003c/em\u003e is located at a highly conserved site in the FtsZ protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-E), with the mutated SNP occurring in the second motif. The glycine at this site is highly conserved. We speculate that the mutation at this site affects the function of the protein, leading to the inhibition of chloroplast division and potentially resulting in a complete loss of division ability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Constitutive expression and subcellular localization of \u003cem\u003ej175\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe expression of the \u003cem\u003eJ175\u003c/em\u003e gene in different tissues of wild-type RP125 at the V13 stage (the time when the plant rapidly transitions from vegetative to reproductive growth) was examined using qRT-PCR. \u003cem\u003eJ175\u003c/em\u003e was expressed across various tissues, with the highest expression levels in the bracts and female tassels and the lowest in the male tassels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Analysis of \u003cem\u003ej175\u003c/em\u003e expression in seeds at different days after pollination reveals a trend characterized by an initial increase followed by a subsequent decrease, with the peak expression level observed at 10 DAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the subcellular localization of J175, we fused J175 with GFP and expressed the fusion protein in maize leaf protoplast. Furthermore, as a control, signals from 35: GFP were detected in the nucleus and cytoplasm. In contrast, the signal of 35S: J175 GFP overlaps with the plastid marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), suggesting that J175 functions within the plastid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.8 \u003cem\u003ej175\u003c/em\u003e had abnormal development of amyloplasts and starch granules\u003c/h2\u003e \u003cp\u003eAmyloplasts and chloroplasts are types of plastids that share similarities in their division processes (Mcandrew et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Endosperm cells were selected for observation using fluorescence microscopy. The wild-type endosperm amyloplasts at 12 DAP displayed a smooth spherical shape, whereas the mutant endosperm amyloplasts exhibited a typical bead-on-a-string shape during the powder-making division. However, some of the amyloplasts displayed relatively regular polygon shapes with smooth surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). At 20 DAP, the \u003cem\u003ej175\u003c/em\u003e endosperm amyloplasts developed further into irregular polygons with multiple starch particles clustered together, significantly reducing the number of amyloplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eScanning electron microscopy was utilized to observe the endosperm starch in mature grains of wild-type and \u003cem\u003ej175\u003c/em\u003e. The wild-type starch granules exhibited smooth, spherical shapes arranged in a regular and orderly manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-I). In contrast, starch granules of \u003cem\u003ej175\u003c/em\u003e existed in the form of oligomers, with a smooth surface and compact arrangement, and their number was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ-L). These results indicate that the mutation of the J175 gene affects amyloplast division in the grain, ultimately forming a complex of oligomers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Transcriptome profiling of \u003cem\u003ej175\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo analyze the gene expression differences between \u003cem\u003ej175\u003c/em\u003e and WT, RNA-seq was performed on ear leaves and kernel. Overall, 35,375 genes were detected. In the comparison of j175-green-leaf with WT-leaf, 769 genes showed differential expression, with 389 upregulated and 380 downregulated genes. In the \u003cem\u003ej\u003c/em\u003e175-white-leaf vs. WT-leaf comparison, 2,850 genes were differentially expressed, with 2,178 upregulated and 672 downregulated genes. Between \u003cem\u003ej175\u003c/em\u003e-green-leaf and \u003cem\u003ej175\u003c/em\u003e-white-leaf, 3,529 genes showed differential expression, with 2,552 upregulated and 977 downregulated genes. For WT-kernel vs. \u003cem\u003ej\u003c/em\u003e175-kernel, 528 genes were differentially expressed, with 172 upregulated and 356 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e-A). A clustering heatmap analysis of the differential genes revealed that replicates from each sample are grouped closely, indicating similar expression patterns among replicates (Fig. S9).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe gene ontology (GO) pathway enrichment analysis of differentially expressed genes in leaves revealed that the main enriched biological processes included \"carbohydrate metabolism processes\" and \"photosynthesis.\" Functional categories of GO molecules, including \"iron ion binding,\" \"oxidoreductase activity,\" and \"UDP glycosyltransferase activity,\" alongside the component categories of GO cells, such as \"photosystem I\" and \"photosystem II,\" were significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA; Appendix G). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that differentially expressed genes were mainly concentrated in metabolic and photosynthetic pathways, including \"sucrose and starch metabolism,\" \"carbon metabolism,\" and \"photosynthesis,\" with downregulation being more prevalent than upregulation. The upregulated genes were primarily concentrated in pathways such as plant-pathogen interactions, plant hormone signal transduction, and the MAPK signaling pathway plant. The different leaves of J175 will have varying effects on plant metabolism, hormone signal transduction, and MAPK plant disease resistance signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, S6). The GO pathway enrichment analysis of differentially expressed genes in kernels indicated that the main biological processes enriched were \"immune response\" and \"Protein morphology.\" The functional categories of GO molecules, such as \"defense response,\" \"protein stabilization,\" and \"response to heat,\" along with the component categories of GO cells, such as \"protein folding chaperone complex\" and \"protein aggregate center,\" were significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Similar to the leaf transcriptome, KEGG analysis of the kernel transcriptome showed that differentially expressed genes are in the pathway sets of \"plant hormone signal transduction\" and \"plant pathway interaction.\" However, the difference is that the kernel differential genes are largely enriched in \"protein processing in endoplasmic reticulum\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression of plastid division-related genes in \u003cem\u003ej175\u003c/em\u003e green and white leaves was inconsistent. Most plastid division-related genes, including \u003cem\u003ej175\u003c/em\u003e, were downregulated in \u003cem\u003ej175\u003c/em\u003e green leaves and kernel, while the upregulation of \u003cem\u003eFtsZ2-1\u003c/em\u003e may partially complement the J175 gene mutation. Additionally, the expression levels of \u003cem\u003eARC3\u003c/em\u003e and \u003cem\u003eGC1\u003c/em\u003e, which are negative regulators of chloroplast division, were significantly increased in the mutant. To further verify the reliability of the transcriptome data, we conducted qPCR validation of several genes, and the results were consistent with the transcriptome data (Appendix H). Many essential genes involved in kernel development were also significantly differentially expressed between the WT kernels and \u003cem\u003ej175\u003c/em\u003e kernels. Among these, genes such as \u003cem\u003esmk9\u003c/em\u003e (small kernel opaque endosperm2), \u003cem\u003eo2\u003c/em\u003e (opaque endosperm2), \u003cem\u003efl2\u003c/em\u003e (floury endosperm2), \u003cem\u003efl3, fl4\u003c/em\u003e, \u003cem\u003ebt2\u003c/em\u003e (brittle endosperm) were downregulated in \u003cem\u003ej175\u003c/em\u003e kernel. However, genes such as \u003cem\u003ebetl3\u003c/em\u003e (basal endosperm transfer layer3), \u003cem\u003ebetl4, betl9, betl10\u003c/em\u003e, and \u003cem\u003ebap2\u003c/em\u003e (basal layer antifungal protein 2) were upregulated in \u003cem\u003ej175\u003c/em\u003e kernel (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). To explore the significant differences in chlorophyll content between the WT and \u003cem\u003ej175\u003c/em\u003e, we analyzed the expression levels of key enzyme genes involved in the chlorophyll synthesis pathway. In Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eD, the expression of the \u003cem\u003eCHLH\u003c/em\u003e gene, which encodes the H subunit of magnesium chelatase, was significantly downregulated in J175 white leaves. Together, CHLH, CHLI, and CHLD form a magnesium-chelating enzyme (MgCh) that catalyzes the transformation of protoporphyrin IX into magnesium protoporphyrin IX. This enzyme is crucial in chlorophyll synthesis, and the mutation in the J\u003cem\u003e175\u003c/em\u003e gene also inhibits this process.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe leaf is the site where photosynthetic products are synthesized, determining the production of assimilates and acting as the \"source\" of yield formation (Hofius and B\u0026ouml;rnke \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). As an important C4 plant, 90% of corn yield is derived from leaf photosynthesis (Khanna-Chopra \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Zhu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Grain serves as the \"reservoir\" for materials stored in plants, with starch being the most important storage material in grains, accounting for approximately 70% of the total content (Luchese et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We discovered that a SNP mutation in the exon of \u003cem\u003ej175\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) prevented chloroplast division and inhibited chlorophyll synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Thus, this reduced the photosynthetic efficiency in \u003cem\u003ej175\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). Simultaneously, this mutation limited the division of the amyloplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F), altering the shape of starch granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-L). Therefore, the \u003cem\u003ej175\u003c/em\u003e mutation affects the \"source\" and \"sink\" of yield composition, resulting in a significant reduction in most agronomic traits of this material (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).As the allelic mutants of \u003cem\u003ej175\u003c/em\u003e, \u003cem\u003ej175-1\u003c/em\u003e and \u003cem\u003ej175-2\u003c/em\u003e, due to the higher position of the \u003cem\u003ej175-1\u003c/em\u003e mutation site, more structural domain functions are lost, resulting in a more severe reduction in agronomic traits for \u003cem\u003ej175-2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This result further proves that \u003cem\u003ej175\u003c/em\u003e mutation will have a certain impact on the agronomic traits of maize.\u003c/p\u003e \u003cp\u003eChloroplast division mutants are essential genetic materials for studying the underlying mechanisms of chloroplast division in plants (Glynn et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Vitha and S. 2001). Partial genes regulating chloroplast division have been identified, and their regulatory mechanisms have been elucidated through the studies on Arabidopsis mutants deficient in chloroplast division (Okazaki \u003cem\u003eet al.\u003c/em\u003e 2009, Pyke et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Robertson et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Yoder et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This includes FtsZ1, ARC5, ARC6, and PDV1 (plastid division 1). In Arabidopsis, AtFtsZ directly or indirectly affects the localization and assembly of other proteins in the division complex, thereby inhibiting chloroplast division (Miyagishima et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Osteryoung \u003cem\u003eet al.\u003c/em\u003e 2014). An increase in AtFtsZ1-1 protein levels inhibits chloroplast division, with higher AtFtsZ1-1 protein levels resulting in more severe phenotypes (Stokes \u003cem\u003eet al.\u003c/em\u003e 2000). Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant (Schmitz \u003cem\u003eet al.\u003c/em\u003e 2009), we found through evolutionary tree analysis that AtFtsZ2-1 and AtFtsZ2-2 have the highest similarity of 83.55%, while ZmFtsZ2-2 and ZmFtsZ2-1 have only 63.11% similarity. ZmFtsZ2-1 has the highest sequence similarity with SbFtsZ2-2 in sorghum. Does this imply that maize ZmFtsZ2 has different functions compared to Arabidopsis AtFtsZ2. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The \u003cem\u003ej175\u003c/em\u003e mutant gene found in maize not only affects the chloroplast division as observed in other related genes, but also causes the yellowing of the \u003cem\u003ej175\u003c/em\u003e seedlings, weak leaf growth, and the formation of white stripes on the leaves, affecting plant growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, F; S1,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This suggests that the \u003cem\u003ej175\u003c/em\u003e gene may serve different functions across various species. The high expression of j175 gene in green tissues such as the ears, ligules, and bracts further indicates its potential role in regulating plant growth and development (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-A). In the mesophyll cells of \u003cem\u003ej175\u003c/em\u003e, the chloroplasts are larger and fewer compared to those of the wild-type chloroplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G), suggesting that mutations in the FtsZ 2\u0026ndash;2 protein encoded by j175 causes damage to the Z-ring structure during maize chloroplast division, thereby hindering the division process. Furthermore, the RNA-seq and qPCR results demonstrated that most genes related to chloroplast division are inhibited to varying degrees in the \u003cem\u003ej175\u003c/em\u003e green leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eD; S11), supporting this hypothesis. The interference with chloroplast development may affect the chlorophyll synthesis, and genes involved in chlorophyll biosynthesis (including \u003cem\u003eZmHEMD, ZmCHLH, ZmCHLD, and ZmPORD\u003c/em\u003e) are inhibited in the mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). As the production sites of salicylic and jasmonic acid, which are important mediators of plant immunity, chloroplasts also participate in PAMP-induced defense gene expression, playing a vital role in plant immunity (Nomura et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, in the GO and KEGG pathway analyses, several genes related to hormone signal transduction and plant-pathogen interactions were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, C; S10). Additionally, the significant reduction in peroxidase activity observed in the enzyme activity assay could also indicate that the stress resistance of \u003cem\u003ej175\u003c/em\u003e was weakened (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Thus, mutations in the genes related to chloroplast division may reduce plant immunity.Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant,\u003c/p\u003e \u003cp\u003eUnlike the compound starch granules found in the rice endosperm, maize endosperm contains simple starch granules, with only one starch granule per amyloplast (Kawagoe \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Shannon et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Changes in the expression levels of amyloplast division proteins can alter starch granule synthesis (Yun MinSoo et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, the membrane structure of amyloplasts is also affected by the levels of plastid division proteins (Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the \u003cem\u003ej175\u003c/em\u003e mutant, amyloplast division is inhibited during growth and maturity, and amyloplasts in the mature grain endosperm existed in the form of oligomers, with a significant decrease in quantity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-L). An increase in StFtsZ1 protein levels in potatoes results in a significant reduction in the number and size of starch granules in tubers (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Sack \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1991\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Similarly, in Arabidopsis FtsZ deficient mutants, amyloplasts did not proliferate (Fujiwara et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Osteryoung and Pyke \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), while the \u003cem\u003ej175\u003c/em\u003e mutant in maize exhibited a change in amyloplast shape, implying that the regulation of the maize \u003cem\u003ej175\u003c/em\u003e gene on amyloplasts differs from that of Arabidopsis and potato. Normal amyloplast division, such as during gravity sensing, is essential for plant growth and development (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Sack \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1991\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Amyloplasts in Arabidopsis are essential for root gravity sensing and for repolarizing LAZY proteins through sedimentation (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e, Kiss and Sack \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1989\u003c/span\u003e, Nishimura et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Whether the \u003cem\u003ej175\u003c/em\u003e gene in maize regulates gravity sensing in plants still requires further study. The formation of vitreous/opaque endosperm depends on the close interactions between proteosomes (storage gliadin) and amyloplasts (Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Most opaque and floury genes in maize regulate zein synthesis by influencing the regulatory or structural genes of zein (Gillikin et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Kim et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Schmidt et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, Wang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The upregulation of \u003cem\u003eo2, fl2, fl4, de30\u003c/em\u003e, and other genes in the kernels of the \u003cem\u003ej175\u003c/em\u003e mutant may serve as compensation for the deletion of the \u003cem\u003ej175\u003c/em\u003e gene, allowing stable synthesis of gliadin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). However, this specific interaction requires further investigation. The expression of all basal endosperm transfer layer genes and basal antifungal protein genes in maize was downregulated in the j175-kernel (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). This finding implies that the \u003cem\u003ej175\u003c/em\u003e gene plays a critical role in regulating the development and immunity of the maize endosperm transfer layer, which may contribute to the smaller kernel size observed in j175 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe mutation of maize white stripe is caused by a single nucleotide substitution in the exon of the filamentous temperature-sensitive Z protein gene, \u003cem\u003ej175\u003c/em\u003e. This mutation affects the division of chloroplasts and amyloplasts, thereby affecting agronomic traits.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (32272184).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuayang Lv: Writing – original draft, Data curation.Xuewu He: Methodology. Hongyu Zhang: Investigation. Zeting Mou: Investigation. Xuerui He: Investigation. Hanmei Liu: Software, Visualization. Yangping Li: Investigation. Yinghong Liu: Investigation. Yufeng Hu: Investigation. Zhiming Zhang: Resources. Yubi Huang: Conceptualization. Junjie Zhang: Conceptualization, Supervision, Funding acquisition.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAaron J. Schmitza b, c, Jonathan M. Glynna,d, Bradley J.S.C. Olsona,e,f, Kevin D. Stokesa,g, Osteryounga K W. 2009. Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant, but FtsZ-based plastid division is not essential for chloroplast partitioning or plant growth and development. \u003cem\u003eMolecular plant,\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 1211-1222.\u003c/li\u003e\n \u003cli\u003eBailey T L, Johnson J, Grant C E, Noble W S. 2015. The MEME suite. \u003cem\u003eNucleic acids research,\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, W39-W49.\u003c/li\u003e\n \u003cli\u003eBalmer Y, Vensel W, Cai N, Manieri W, Schurmann P, Hurkman W, Buchanan B. 2006. A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts. \u003cem\u003eProceedings of the National Academy of Sciences,\u003c/em\u003e \u003cstrong\u003e103\u003c/strong\u003e, 2988-2993.\u003c/li\u003e\n \u003cli\u003eChen C, MacCready J S, Ducat D C, Osteryoung K W. 2018a. The molecular machinery of chloroplast division. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e176\u003c/strong\u003e, 138-151.\u003c/li\u003e\n \u003cli\u003eChen C, Maccready J S, Ducat D C, Osteryoung K W. 2018b. The Molecular Machinery of Chloroplast Division. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e176\u003c/strong\u003e, 138.\u003c/li\u003e\n \u003cli\u003eChen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, Liu Y, Feng J, Chen H, He Y. 2023a. TBtools-II: A \u0026ldquo;one for all, all for one\u0026rdquo; bioinformatics platform for biological big-data mining. \u003cem\u003eMolecular plant,\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1733-1742.\u003c/li\u003e\n \u003cli\u003eChen J, Yu R, Li N, ZhaoguoZhang, XinxinZhao, YaranQu, ChengfuYuan, YanfangPan, ZhexianZhou, YangyangLi, KunlunWang, JiajunChen, ZhirenWang, XiaoyiWang, XiaolianHe, Shu-NanDong, JuanDeng, Xing WangChen, Haodong. 2023b. Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants. \u003cem\u003eCell,\u003c/em\u003e \u003cstrong\u003e186\u003c/strong\u003e, 4788-+.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ede Pater S, Caspers M, Kottenhagen M, Meima H, Ter Stege R, de Vetten N. 2006.\u0026nbsp;Manipulation of starch granule size distribution in potato tubers by modulation of plastid division. \u003cem\u003ePlant Biotechnology Journal,\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 123-134.\u003c/li\u003e\n \u003cli\u003eDenyer K, Pike M. 2008. Isolation of amyloplasts. \u003cem\u003eCurrent protocols in cell biology,\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 3.28. 21-23.28. 15.\u003c/li\u003e\n \u003cli\u003eEcheverria E, Boyer C, Liu K-C, Shannon J. 1985. Isolation of amyloplasts from developing maize endosperm. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 513-519.\u003c/li\u003e\n \u003cli\u003eEsch L, Ngai Q Y, Barclay J E, Mcnelly R, Hayta S, Smedley M, Smith A M, Seung D. 2023. Increasing amyloplast size in wheat endosperm through mutation of PARC6 affects starch granule morphology. \u003cem\u003eThe New phytologist\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eFujiwara M T, Yoshioka Y, Kazama Y, Hirano T, Niwa Y, Moriyama T, Sato N, Abe T, Yoshida S, Itoh R D. 2024. Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana. \u003cem\u003ePlant physiology\u003c/em\u003e, kiae314.\u003c/li\u003e\n \u003cli\u003eGillikin J W, Zhang F, Coleman C E, Bass H W, Larkins B A, Boston R S. 1997. A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, 345-352.\u003c/li\u003e\n \u003cli\u003eGlynn J M, Miyagishima S y, Yoder D W, Osteryoung K W, Vitha S. 2007. Chloroplast division. \u003cem\u003eTraffic,\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 451-461.\u003c/li\u003e\n \u003cli\u003eHofius D, B\u0026ouml;rnke F A. 2007. Photosynthesis, carbohydrate metabolism and source\u0026ndash;sink relations. In: \u003cem\u003ePotato biology and biotechnology\u003c/em\u003e. Elsevier. pp. 257-285.\u003c/li\u003e\n \u003cli\u003eHu B, Jin J, Guo A-Y, Zhang H, Luo J, Gao G. 2015. GSDS 2.0: an upgraded gene feature visualization server. \u003cem\u003eBioinformatics,\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 1296-1297.\u003c/li\u003e\n \u003cli\u003eJiao Y, Peluso P, Shi J, Liang T, Stitzer M C, Wang B, Campbell M S, Stein J C, Wei X, Chin C-S. 2017. Improved maize reference genome with single-molecule technologies. \u003cem\u003eNature,\u003c/em\u003e \u003cstrong\u003e546\u003c/strong\u003e, 524-527.\u003c/li\u003e\n \u003cli\u003eKawagoe Y. 2013. The characteristic polyhedral, sharp-edged shape of compound-type starch granules in rice endosperm is achieved via the septum-like structure of the amyloplast. \u003cem\u003eJournal of Applied Glycoscience,\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 29-36.\u003c/li\u003e\n \u003cli\u003eKawagoe Y Y. 2009. Amyloplast Division Progresses Simultaneously at Multiple Sites in the Endosperm of Rice. \u003cem\u003ePlant \u0026amp; Cell Physiology,\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 1617.\u003c/li\u003e\n \u003cli\u003eKhanna-Chopra R. 2000. Photosynthesis in relation to crop productivity. \u003cem\u003eProbing Photosynthesis: Mechanisms, Regulation and Adaptations\u003c/em\u003e, 263-280.\u003c/li\u003e\n \u003cli\u003eKim C S, Hunter B G, Kraft J, Boston R S, Yans S, Jung R, Larkins B A. 2004. A defective signal peptide in a 19-kD \u0026alpha;-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e, 380-387.\u003c/li\u003e\n \u003cli\u003eKiss J Z, Sack H F D. 1989. Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana. \u003cem\u003ePlanta,\u003c/em\u003e \u003cstrong\u003e177\u003c/strong\u003e, 198-206.\u003c/li\u003e\n \u003cli\u003eLi, Han-Hai, Xue, Shan, Le, Jie, Zou, Jun-Jie, Wang, Yu-Ren. 2016. The role of Arabidopsis Actin-Related Protein 3 in amyloplast sedimentation and polar auxin transport in root gravitropism. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eLiu M, Yu J, Yang M, Cao L, Chen C. 2024. Adaptive evolution of chloroplast division mechanisms during plant terrestrialization. \u003cem\u003eCell Reports,\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eLuchese C L, Spada J C, Tessaro I C. 2017. Starch content affects physicochemical properties of corn and cassava starch-based films. \u003cem\u003eIndustrial Crops and Products,\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 619-626.\u003c/li\u003e\n \u003cli\u003eMatsushima R, Hisano H. 2019. Imaging amyloplasts in the developing endosperm of barley and rice. \u003cem\u003eScientific reports,\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 3745.\u003c/li\u003e\n \u003cli\u003eMcandrew R S, Froehlich J E, Vitha S, Osteryoung S K W. 2001. Colocalization of Plastid Division Proteins in the Chloroplast Stromal Compartment Establishes a New Functional Relationship between FtsZ1 and FtsZ2 in Higher Plants. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 1656-1666.\u003c/li\u003e\n \u003cli\u003eMiyagishima S Y. Mechanism of Plastid Division: From a Bacterium to an Organelle1.\u003c/li\u003e\n \u003cli\u003eMiyagishima S Y. 2005. Origin and evolution of the chloroplast division machinery. \u003cem\u003eJournal of Plant Research,\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, 295-306.\u003c/li\u003e\n \u003cli\u003eMiyagishima S Y. 2011. Mechanism of Plastid Division: From a Bacterium to an Organelle. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e155\u003c/strong\u003e, 1533-1544.\u003c/li\u003e\n \u003cli\u003eMiyagishima S Y, Froehlich J E, Osteryoung K W. 2006. PDV1 and PDV2 Mediate Recruitment of the Dynamin-Related Protein ARC5 to the Plastid Division Site. \u003cem\u003eTHE PLANT CELL ONLINE,\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 2517-2530.\u003c/li\u003e\n \u003cli\u003eMiyagishima S Y, Takahara M, Mori T, Kuroiwa H, Kuroiwa T. 2001. Plastid Division Is Driven by a Complex Mechanism That Involves Differential Transition of the Bacterial and Eukaryotic Division Rings. \u003cem\u003eThe Plant Cell,\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2257-2268.\u003c/li\u003e\n \u003cli\u003eNishimura T, Mori S, Shikata H, Nakamura M, Hashiguchi Y, Abe Y, Hagihara T, Yoshikawa H Y, Toyota M, Higaki T. 2023. Cell polarity linked to gravity sensing is generated by LZY translocation from statoliths to the plasma membrane. \u003cem\u003eScience,\u003c/em\u003e \u003cstrong\u003e381\u003c/strong\u003e, 1006-1010.\u003c/li\u003e\n \u003cli\u003eNomura H, Komori T, Uemura S, Kanda Y, Shimotani K, Nakai K, Furuichi T, Takebayashi K, Sugimoto T, Sano S. 2012. Chloroplast-mediated activation of plant immune signalling in Arabidopsis. \u003cem\u003eNature communications,\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 926.\u003c/li\u003e\n \u003cli\u003eOkazaki, Kumiko, Kabeya, Yukihiro, Suzuki, Kenji, Mori, Toshiyuki, Ichikawa, Takanari. 2009. The PLASTID DIVISION1 and 2 Components of the Chloroplast Division Machinery Determine the Rate of Chloroplast Division in Land Plant Cell Differentiation. \u003cem\u003ePlant Cell\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eOsteryoung, Katherine, W., Pyke, Kevin, A. 2014. Division and Dynamic Morphology of Plastids. \u003cem\u003eAnnual review of plant biology,\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 443-472.\u003c/li\u003e\n \u003cli\u003eOsteryoung K W, Mcandrew R S. 2001. THE PLASTID DIVISION MACHINE. \u003cem\u003eAnnual Review of Plant Physiology \u0026amp; Plant Molecular Biology,\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 315.\u003c/li\u003e\n \u003cli\u003eOsteryoung K W, Pyke K A. 2014. Division and dynamic morphology of plastids. \u003cem\u003eAnnual review of plant biology,\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 443-472.\u003c/li\u003e\n \u003cli\u003eOsteryoung K W, Stokes K D, Rutherford S M, Lee P W Y. 1998. Chloroplast Division in Higher Plants Requires Members of Two Functionally Divergent Gene Families with Homology to Bacterial ftsZ. \u003cem\u003eTHE PLANT CELL ONLINE,\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1991-2004.\u003c/li\u003e\n \u003cli\u003ePyke K A, Rutherford S M, Robertson E J, Leech R M. 1994. arc6, A Fertile Arabidopsis Mutant with Only Two Mesophyll Cell Chloroplasts. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 1169-1177.\u003c/li\u003e\n \u003cli\u003eRobertson E J, Rutherford S M, Leech R M. 1996. Characterization of Chloroplast Division Using the Arabidopsis Mutant arc5. \u003cem\u003ePlant physiology\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eSack F D. 1991. Plant Gravity Sensing. \u003cem\u003eInternational Review of Cytology,\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 193-252.\u003c/li\u003e\n \u003cli\u003eSack F D. 1997. Plastids and gravitropic sensing. \u003cem\u003ePlanta,\u003c/em\u003e \u003cstrong\u003e203\u003c/strong\u003e, S63-S68.\u003c/li\u003e\n \u003cli\u003eSchmidt R J, Burr F A, Burr B. 1987. Transposon tagging and molecular analysis of the maize regulatory locus opaque-2. \u003cem\u003eScience,\u003c/em\u003e \u003cstrong\u003e238\u003c/strong\u003e, 960-963.\u003c/li\u003e\n \u003cli\u003eShannon J C, Echeverria E, Boyer C. 1987. [22] Isolation of amyloplasts from developing endosperm of maize (Zea mays L.). In: \u003cem\u003eMethods in enzymology\u003c/em\u003e. Elsevier. pp. 226-234.\u003c/li\u003e\n \u003cli\u003eStokes, McAndrew, Figueroa, Vitha. 2000. Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. \u003cem\u003ePlant physiology\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eSun Q, Cao X, Liu Z, An C, Hu J, Wang Y, Qiao M, Gao T, Cheng W, Zhang Y. 2023. Structural and functional insights into the chloroplast division site regulators PARC6 and PDV1 in the intermembrane space. \u003cem\u003eProceedings of the National Academy of Sciences,\u003c/em\u003e \u003cstrong\u003e120\u003c/strong\u003e, e2215575120.\u003c/li\u003e\n \u003cli\u003eTamura K, Stecher G, Kumar S. 2021. MEGA11: molecular evolutionary genetics analysis version 11. \u003cem\u003eMolecular biology and evolution,\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 3022-3027.\u003c/li\u003e\n \u003cli\u003eVitha, S. 2001. Ftsz Ring Formation at the Chloroplast Division Site in Plants. \u003cem\u003eJournal of Cell Biology,\u003c/em\u003e \u003cstrong\u003e153\u003c/strong\u003e, 111-120.\u003c/li\u003e\n \u003cli\u003eWang G, Qi W, Wu Q, Yao D, Zhang J, Zhu J, Wang G, Wang G, Tang Y, Song R. 2014. Identification and characterization of maize floury4 as a novel semidominant opaque mutant that disrupts protein body assembly. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e165\u003c/strong\u003e, 582-594.\u003c/li\u003e\n \u003cli\u003eWang H, Huang Y, Xiao Q, Huang X, Li C, Gao X, Wang Q, Xiang X, Zhu Y, Wang J. 2020. Carotenoids modulate kernel texture in maize by influencing amyloplast envelope integrity. \u003cem\u003eNature communications,\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 5346.\u003c/li\u003e\n \u003cli\u003eYang Y, Glynn J M, Olson B J, Schmitz A J, Osteryoung K W. 2008. Plastid division: across time and space. \u003cem\u003eCurrent opinion in plant biology,\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 577-584.\u003c/li\u003e\n \u003cli\u003eYoder D W, Kadirjan-Kalbach D, Olson B J S C, Miyagishima S Y, Deblasio S L, Hangarter R P, Osteryoung, W. K. 2007. Effects of Mutations in Arabidopsis FtsZ1 on Plastid Division, FtsZ Ring Formation and Positioning, and FtsZ Filament Morphology in Vivo. \u003cem\u003ePlant \u0026amp; Cell Physiology,\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 775.\u003c/li\u003e\n \u003cli\u003eYun MinSoo Y M, Umemoto T, Kawagoe Y. 2011. Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis.\u003c/li\u003e\n \u003cli\u003eZhang N, Guil S, Wang Y. 2023. LAZing around: The intricate dance of amyloplast sedimentation and gravity sensing in plants. \u003cem\u003eMolecular Plant\u003c/em\u003e, 016.\u003c/li\u003e\n \u003cli\u003eZhang R, Wu Y, Qu X, Yang W, Wu Q, Huang L, Jiang Q, Ma J, Zhang Y, Qi P. 2024. The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function. \u003cem\u003eNature communications,\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 6905.\u003c/li\u003e\n \u003cli\u003eZhao F, Jing L, Wang D, Bao F, Wang G. 2018. Grain and starch granule morphology in superior and inferior kernels of maize in response to nitrogen. \u003cem\u003eScientific Reports,\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 6343.\u003c/li\u003e\n \u003cli\u003eZhu X-G, Long S P, Ort D R. 2010. Improving photosynthetic efficiency for greater yield. \u003cem\u003eAnnual review of plant biology,\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 235-261.\u003c/li\u003e\n \u003cli\u003eZhu Y S, Merkle-Lehman D L, Kung S D. 1984. Light-Induced Transformation of Amyloplasts into Chloroplasts in Potato Tubers. \u003cem\u003ePlant physiology,\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 142-145.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Maize, Chloroplast, Amyloplast, FtsZ","lastPublishedDoi":"10.21203/rs.3.rs-5848477/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5848477/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlastid division regulatory genes play a crucial role in the morphogenesis of chloroplasts and amyloplasts. Chloroplasts are the main sites for photosynthesis and metabolic reactions, while amyloplasts are the organelles responsible for forming and storing starch granules. The proper division of chloroplasts and amyloplasts is essential for plant growth and maintenance yield. Therefore, this study aims to examine the \u003cem\u003eJ175 (FtsZ2-2)\u003c/em\u003e gene, cloned from an ethyl methanesulphonate\u003cem\u003e (\u003c/em\u003eEMS)mutant involved in chloroplast and amyloplast division in maize, through map-based cloning. We found that \u003cem\u003eJ175\u003c/em\u003e encodes a cell division protein, FtsZ (filamentous temperature-sensitive z). The FtsZ family of proteins is widely distributed in plants and may be related to the division of chloroplasts and amyloplasts. J175 is localized in plastids and expressed across various tissues. From the seedling stage, the leaves of the \u003cem\u003ej175\u003c/em\u003e mutant exhibited white stripes, while the division of chloroplasts was inhibited, leading to a significant increase in volume and a reduction in their number. Measurement of the photosynthetic rate showed a significant decrease in the photosynthetic efficiency of \u003cem\u003ej175\u003c/em\u003e. Additionally, the division of amyloplasts in \u003cem\u003ej175\u003c/em\u003e grains at different stages was impeded, resulting in irregular polygonal starch granules. RNA-seq analyses of leaves and kernels also showed that multiple genes affecting plastid division, such as \u003cem\u003eFtsZ1, ARC3, ARC6, PDV1-1, PDV2\u003c/em\u003e, and \u003cem\u003eMinE1\u003c/em\u003e, were significantly downregulated. This study demonstrates that the maize gene \u003cem\u003ej175\u003c/em\u003e is essential for maintaining the division of chloroplasts and amyloplasts, ensuring normal plant growth, and providing an important gene resource for maize molecular breeding.\u003c/p\u003e","manuscriptTitle":"Filamentous temperature-sensitive Z protein J175 regulates maize chloroplasts and amyloplasts division and development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-24 15:15:37","doi":"10.21203/rs.3.rs-5848477/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3015ebdd-50b3-46fc-9b98-4d0fa7eb093f","owner":[],"postedDate":"April 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-09T01:28:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-24 15:15:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5848477","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5848477","identity":"rs-5848477","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}