Gene editing-mediated enhancement of carotenoid compounds accumulation in common wheat grains

Gene editing-mediated enhancement of carotenoid compounds accumulation in common wheat grains | 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 Gene editing-mediated enhancement of carotenoid compounds accumulation in common wheat grains Yajie Guo, Mengtian Liu, Mengyao Li, Dan Wang, Huiyun Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8056178/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 Common wheat ( Triticum aestivum L.) is a staple food crop for humans, yet it primarily accumulates the non-provitamin A carotenoid lutein and exhibits limited natural variation in provitamin A β-carotene among its various accessions. This characteristic necessitates the development of alternative strategies for provitamin A biofortification. To address this challenge, we targeted key control points in the carotenoid biosynthetic pathway using the CRISPR-Cas9 system in a wheat cultivar Fielder. Specifically, we knocked out the gene encoding lycopene ε-cyclase (LCYE), an enzyme that acts as a gatekeeper opposing the production of β-branch carotenoids. This genetic modification resulted in a significant increase in β-carotene levels in the endosperms at 30 DPA of triple homozygous transgene-free mutant lines revealed by biochemical profiling, an approximate 34.5% enhancement for β-carotene, 125.4% for zeaxanthin, 73.8% for violaxanthin, and 186.5% for antheraxanthin compared to the wild-type control. Despite the drastic reduction in lutein levels, the TaLcye mutations did not significantly impair wheat yield and the mutant lines exhibited elevated levels of amylose and soluble sugar. Additionally, the seed coats and endosperms of the triple homozygous transgene-free mutant lines exhibited an orange-yellow hue. In conclusion, we have successfully developed novel carotenoids biofortified wheat lines through gene-editing approach. Our findings demonstrate the potential of gene editing to significantly enhance the nutritional profile of commercial wheat by increasing carotenoid content, thereby addressing micronutrient deficiencies in modern diets. wheat grain carotenoids provitamin A β-carotene biofortification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key message Targeted knockout of the gene TaLcye , using the CRISPR-Cas9 system, led to significantly elevated levels of β-carotene, zeaxanthin, violaxanthin, and antheraxanthin in the grains of common wheat. Introduction Carotenoids serve as precursors for the synthesis of vitamin A, a nutrient that humans cannot synthesize de novo. Consequently, vitamin A must be obtained through dietary intake. Vitamin A deficiency (VAD) can make serious health consequences, with mild cases manifesting as night blindness and severe cases potentially leading to permanent blindness or even death (Grune et al., 2010 ; Sajovic et al., 2022 ; Carazo et al., 2021 ). Among carotenoids, β-carotene is the principal plant-derived precursor of retinol (vitamin A), essential for a wide range of physiological function (Anand et al., 2022 ). These functions include night vision, cell proliferation, reproduction, embryonic development, brain activity, and immune system regulation (Ahmad and Ahmed 2019 ; Trono 2019 ). Other carotenoids, such as zeaxanthin, antheraxanthin, and violaxanthin, also play crucial roles as antioxidants, neutralizing harmful reactive oxygen species (ROS; Sajovic et al., 2022 ). However, these vital compounds are often underrepresented in modern diets. Carotenoids constitute a diverse group of naturally occurring lipophilic compounds that are extensively distributed in green plants, algae, fungi, and bacteria. These compounds belong to the C40 isoprenoid class and typically consist of eight isoprenoid units (Cunningham et al., 1998). The biosynthetic pathway of carotenoids begins with geranylgeranyl pyrophosphate (GGPP) as the precursor (Lichtenthaler et al., 1999). The first committed step in this pathway is that GGPP is catalyzed by phytoene synthase (PSY), which facilitates the condensation of two GGPP molecules to produce 15-cis-phytoene. Subsequently, phytoene undergoes a successive of desaturation reactions to form lycopene (Zhai et al., 2016 ). These desaturation steps are mediated by phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), ZISO (ζ-carotene isomerase), and CRTISO (carotenoid isomerase) enzymes, respectively (Tanaka et al., 2008 ). The cyclization of lycopene is a critical branching point in carotenoid biosynthesis, diverging into two distinct pathways. One pathway leads to the formation of α-carotene, and the other results in the production of γ-carotene and β-carotene. Plants contain two types of cyclase enzymes which mediate the process of ε-cyclase (LCYE) and β-cyclase (LCYB), respectively (Cunningham et al., 1996 ; Cunningham et al., 2001). The LCYE and LCYB catalyzes the cyclization of one end of lycopene to form an ε-ring, yielding α-carotene. In contrast, LCYB cyclizes both ends of lycopene to form β-rings, thereby producing γ-carotene and β-carotene (Cunningham et al., 1996 ; Bai et al. 2009 ; Zhai et al., 2016 ). Further downstream in the plant carotenoid biosynthetic pathway, α-carotene and β-carotene undergo additional modifications to generate more complex oxygenated derivatives. These modifications are catalyzed by carotenoid hydroxylase (HYD). Specifically, α-carotene is predominantly converted into α-cryptoxanthin and lutein (Bai et al. 2009 ). Meanwhile, β-carotene is transformed into a variety of xanthophylls, including β-cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin, and neoxanthin (Fig. 1 ) (Giuliano 2017 ). The gene Lcye encoding ε-cyclase is a key determinant in the carotenoid biosynthetic pathway, exerting significant influence over the content and compositional ratios of carotenoids in plants. Subsequent studies had demonstrated its pivotal role in modulating carotenoid profiles across various plant species. For instances, the deficiency of LCYE enzyme in Arabidopsis results in the failure to synthesize lutein and a concomitant increase in β-carotene levels (Pogson et al., 2000); in the tomato plants with silenced Lcye , total carotenoids and β-carotene levels were elevated, while lutein content was reduced (Ma et al., 2010); downregulation of the Lcye gene in rapeseed (Brassica napus) showed markedly elevate carotenoid levels in seeds (Yu et al., 2008 ). Similarly, targeted editing of the Lcye gene in bananas led to a six-fold increase of β-carotene content in fruits, albeit with concurrent reductions of α-carotene and lutein levels (Kaur et al., 2020 ). In potatoes, downregulation of Lcye led to a 14-fold increase in β-carotene content and a 2.5-fold rise in total carotenoid content, with the potato callus tissue exhibiting a deep yellow hue (Diretto et al., 2006 ; Ke et al., 2019 ). Moreover, biochemical analyses of durum wheat mutant lines revealed a 75% increase of β-carotene content in grain in the complete mutant line compared to the control (Sestili et al., 2019 ). Collectively, these findings underscored the potential of Lcye as a valuable genetic tool for enhancing β-carotene content in plants, thereby holding significant promise for breeding programs aimed at improving nutritional quality. Common wheat serves as a crucial source of plant-based protein, minerals, and vitamins for human nutrition. The quality of wheat can be broadly categorized into processing quality and nutritional quality (Shewry et al., 2009). With the continuous improvement of living standards and the deepening awareness of healthy eating, the cultivation of wheat varieties with enhanced nutritional quality has gained increasing attention. Among these nutrients, vitamin A content is one of the key indicators for assessing the nutritional quality of wheat, playing a critical role in human health. However, predominantly non-provitamin A carotenoids such as lutein are typically accumulated in wheat seeds, while provitamin A carotenoids such as β-carotene and the downstream metabolite zeaxanthin, antheraxanthin, and violaxanthin of β-carotene are relatively low in content (Cong et al., 2009 ; Yu and Tian, 2018 ). It is necessary to develop the wheat germplasm with enhanced carotenoids by gene editing technology. In this research, we employed the CRISPR gene editing system to target the TaLcye gene in wheat, aiming to enhance the nutritional profile controlled by this gene. Results showed that the carotenoid contents—β-carotene, zeaxanthin, antheraxanthin and violaxanthin—were substantially elevated in the grains at 30 DPA. Notably, these genetic modifications did not significantly affect wheat yield. Our results highlight the potential of gene editing as an effective approach for biofortification, providing a viable solution to address micronutrient deficiencies in major crop. Materials and methods Plant materials and cultivation conditions A wheat cultivar Fielder was acquired from the Crop Germplasm Bank of China. Wheat seeds were grown in pots (20×30 cm) in a growth chamber maintained at 24°C, with 16/8 h light/dark cycle, 300 µmol m − 2 s − 1 light intensity, and 45% humidity. At 15–17 days post-anthesis (DPA), immature wheat grains were collected for Agrobacterium -mediated transformation, following the method described by Wang et al. ( 2017 ). Construction of the vectors The expression vector pWMB110-Cas9 for gene editing was constructed in our previous work by inserting the Cas9 gene into the multiple cloning site (MCS) of pWMB110, which contains the bar gene as a selection marker for generating transgenic plants and the maize ubiquitin (UBI) promoter for driving Cas9 gene (Liu et al. 2020 ). Moreover, a guide RNA sequence 5’-gtatgggaggacgaattcaa-3’ was designed to target the three homologous TaLcye genes based on the sequences of TaLcye-3A (GeneBank accession ACF42349.1), TaLcye-3B (GeneBank accession ACF42350.1), and TaLcye-3D (GeneBank accession ACF42351.1) (Sestili et al., 2019 ). This guide RNA was driven by the TaU3 promoter, and inserted into the multiple cloning site (MCS) of the pWMB110-Cas9 plasmid through homologous recombination, resulting in the final construct named TaLcye -CRISPR (Fig. S1A-S1B). Detection analysis of the TaLcye -edited mutations Genomic DNA was extracted from the candidate T0 transgenic wheat mutants, in which the targeted genes, TaLcye-3A (5’-gtctggtagcatccttcaagtag-3’ and 5’-tatcatgctgcctgatgcaactg-3’), TaLcye-3B (5’-gtctggtagcatccttcaagtag-3’ and 5’-ctgaattccaagatcgaatgtacc-3’), and TaLcye-3D (5’-gtctggtagcatccttcaagtag-3’ and 5’-gaactggtgcacaaacaacatatg-3’), were amplified using their respective specific primers. The bar gene was screened using the primer pairs 5’-ACCATCGTCAACCACTACATCG-3’ and 5’-GCTGCCAGAAACCACGTCATG-3’. The PCR products were digested with the corresponding restriction enzyme (1 U) in a 10 µl reaction buffer containing 3 µl PCR product for 2 h at 37°C. The resultant products were separated on a 2% agarose gel and visualized using a GelDoc XR System (BioRad). The presence of edited mutations was inferred from the altered banding patterns compared to the wildtype controls. Quantitative reverse-transcriptase PCR (qRT-PCR) Samples were collected from the leaves and immature grains at 15 DPA of the TaLcye -edited wheat plants and their wild-type Fielder, which were grown in a semi-controlled greenhouse environment. Total RNA was extracted using Trizol reagent (Invitrogen) following the manufacturer’s protocol. Reverse transcription was performed using HiScript III RT SuperMix (Vazyme Biotech Co., Ltd) according to the manufacturer’s instructions. The qRT-PCR was performed using SevenFast SYBR Green Mix (Seven, Beijing) in a 7500 Fast Real-Time PCR system (Applied Biosystems). Each reaction was carried out in a final volume of 20 µl, with 10 µl SevenFast SYBR Green Mix, 0.4 µM of each primer, and 1 µl cDNA. The primers for the key genes involved in the carotenoid biosynthetic pathway were listed in Table S1. Transcript abundance was expressed relative to that of TaActin using the 2 –2ΔΔCT method, which allows for the quantification of gene expression levels relative to a reference gene (Livak et al. 2001). Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) Leaves and grains at 30 DPA were collected from the TaLcye -edited plants and wild type, and grounded into a fine powder under liquid nitrogen. Subsequently, a solvent mixture composed of n-hexane, acetone, and ethanol (1:1:1, v/v/v), as well as 0.01% butylated hydroxytoluene (BHT, w/v), was added into the powdered samples. Following the extraction process, the samples were centrifuged to eliminate insoluble debris. The resulting supernatant was filtered through a syringe filter to yield a clear extract. This extract was subsequently injected into an ultra-performance liquid chromatography (UPLC) system. Separation was achieved on a C30 column using a gradient elution program. The mobile phase consisted of water and methanol, both of which contained a small amount of formic acid. The separated compounds were then detected via tandem mass spectrometry. The data were processed using Analyst 1.6.3 and MultiQuant 3.0.3 software. The chromatographic peaks corresponding to the analytes in different samples were integrated and corrected based on the retention times and peak shape information of the reference standards. Ultimately, the concentrations of the target compounds were determined using calibration curves derived from standard solutions. Grain protein, wet gluten, and starch content analysis Grain protein and wet gluten content in the TaLcye -edited lines and wild type were measured using the DA7200 near-infrared instrument (Perten, Sweden). Starch and amylose in the whole grain flour were extracted and analyzed using the Solarbio Starch Content Assay Kit and the Amylose Content Assay Kit (Solarbio, Beijing). In each analysis, three biological replicates were prepared for both each TaLcye -edited line and the wild-type. Grain soluble sugar analysis About 0.1 g of fine sample powder from each TaLcye -edited line and the wild-type was weighed into a 15 ml centrifuge tube, and then 4 ml of 80% (v/v) ethanol was added. The centrifuge tube was then placed in a water bath at 80°C for 40 min. After cooling, the sample was centrifuged at 4500 rpm for 10 min. The supernatant was collected and filtered through a 0.45 µm membrane disc filter (Sangon, China). Next, 1 mL of the filtered supernatant was taken, mixed with 5 mL of anthrone reagent, and then placed in a boiling water bath for 10 min. After cooling to room temperature, the absorbance (OD) value at 625 nm was measured using a spectrophotometer, and the soluble sugar content was calculated based on the standard curve prepared with known concentrations of soluble sugar standards. Each sample was subjected to three technical replicates. Scanning electron microscopy analysis of wheat grains Mature wheat grains from the TaLcye -edited lines and wild-type were fixed in 2.5% glutaraldehyde solution for 2–4 h. Then the grains underwent dehydration in a graded ethanol series (30%, 50%, 70%, 90%, and 100% ethanol), maintaining 15–30 min in each concentration. Subsequently, critical point drying was performed using carbon dioxide as the displacement medium to remove ethanol while preserving sample integrity. The dried grains were sectioned to expose internal structures. The sections were then mounted on a sample holder and sputter-coated with a thin gold layer using an ion sputtering device to enhance conductivity. Finally, the samples were placed in the scanning electron microscopy (SEM) chamber for observing the surface and internal structures of the wheat grains. Examination of the main agronomic traits and statistical analysis At wheat maturity stage, ten plants were randomly picked from each replicate plot for evaluating main agronomic traits including tiller number per plant, plant height, spike length, spikelet number per spike, grains per spike, grain length, grain width, and 1000-kernel weight (TKW). Data analysis was performed using SPSS, with t-tests comparing means between the TaLcye -edited lines and wild-type. Significance levels were set at * P < 0.05 (significant) and ** P < 0.01 (highly significant). Results Identification of wheat TaLcye loss-of-function mutants To assess the roles of TaLcye homoeologs in carotenoid metabolism and plant growth in wheat, we edited TaLcye homoeologs in a wheat cultivar Fielder and obtained candidate mutant plants. In the offsprings of TaLcye -edited plants, three transgenic-free homozygous mutant lines (1–4, 2–8, and 5 − 1) were selected after detailed molecular analysis (Fig. S1C). Sanger sequencing revealed that line 5 − 1 exhibited a 2 bp, 4 bp, and 11 bp deletions in exon 2 of the TaLcye-A , TaLcye-B and TaLcye-D homoeologs, in order. Line 2–8 had a 1 bp and 2 bp deletions at the 3 bp upstream of the PAM sites of TaLcye-B and TaLcye-D homoeologs, respectively. Line 1–4 contained a 2 bp deletion at position 469–470 of the TaLcye-A ORF, and a single T insertion at position 476 of the TaLcye-B ORF, and a 15 bp deletion in the TaLcye-D homoeologs (Fig. S2). Expression profiling of the key genes involved in the carotenoid biosynthetic pathway in the TaLcye mutants To investigate the effects of TaLcye -edited homeoalleles on carotenoid biosynthesis in wheat, we assessed the expression levels of other key genes including TaPsy, TaPds, TaZds, TaLcyb, TaHyd1 , and TaHyd2 involved in carotenoid biosynthesis pathway in TaLcye mutant lines using qRT-PCR and compared them with those in the wild-type. In leaves, the relative expression levels of TaPsy and TaPds showed no significant differences among the mutant lines 1–4, 2–8, and 5 − 1. In contrast, the transcript levels of TaLcyb and TaHyd2 were downregulated, while TaZds and TaHyd1 were upregulated in mutant line 5 − 1 (Fig. S3). In grainsa, none of the six genes ( TaPsy , TaPds , TaZds , TaLcyb , TaHyd1 , TaHyd2 ) differed in expression between wild-type and mutants 1–4 or 2–8; only the triple homozygous mutant line 5 − 1 showed significant repression expression of TaPsy and TaHyd2 and induction of TaPds , TaZds , TaLcyb and TaHyd1 (Fig. 2). Carotenoid profiles in the leaves and grains of the wheat TaLcye mutants Vitamin A is an essential nutrient for humans. Provitamin A carotenoids, such as β-carotene, can be converted into vitamin A in the human body, with β-carotene being the most effective (Van Loo-Bouwman et al., 2014). Lutein, another type of carotenoid, cannot be converted into vitamin A (Kirpichenkova et a., 2018). To elucidate the role of TaLcye- edited in carotenoid biosynthesis in the leaves and grains of the mutant lines, we analyzed and compared the carotenoid content in the mutant lines and their wild-type using LC-MS/MS (Fig. 3; Fig. S4-S5). A total of nineteen metabolites were measured in the leaves (Table S2). Compared with wild-type, the mutant line 2–8 exhibited a significant decrease in total carotenoid content. And the mutant lines 1–4 and 5 − 1 experienced a substantial reduction in lutein content (Fig. S6F; Table S2), which is a major compound in the downstream branch of the TaLcye gene. While in another branch, antheraxanthin, violaxanthin, and zeaxanthin levels were significantly elevated relative to the wild-type (Fig. S6C-6E; Table S2). Additionally, the mutant lines 1–4 and 5 − 1 also showed a slight increase in the content of γ-carotene, β-citraurin, phytoene, and β-cryptoxanthin (Table S2). The carotenoid profiles of fourteen metabolites totally were measured in the grains at 30 DPA of the mutant lines, and results revealed lower average values than those observed in their leaves (Fig. 4; Table S3). The content of lutein exhibited a significant decrease in the mutant lines 1–4 and 5 − 1 compared with the wild-type (Fig. 4E). However, the total carotenoid content in these mutant lines did not significantly differ from that in the wild-type (Table S3). This lack of difference could be attributed to the substantial increases in β-carotene (33.2–34.5%), zeaxanthin (112.1-125.4%), violaxanthin (73.5–73.8%), and antheraxanthin (164.1-186.5%) in the mutant lines 1–4 and 5 − 1 (Fig. 4A-4D). Notably, the mutant line 2–8 showed no significant difference in lutein content in comparison with wild-type (Fig. 4E). However, β-carotene content in the mutant line 2–8 was increased by 26.1%, and the total carotenoid content in this mutant line was enhanced to 59.71 µg g⁻¹ (Fig. 4A; Table S3). Comparison of several main agronomic traits between TaLcye mutants and their wild-type To elucidate the impact of TaLcye mutations on the physiology and productivity of wheat plants, we evaluated some growth and yield related traits of mutant lines and their wild-type, which were cultivated in a semi-controlled greenhouse setting. Our findings revealed that, despite reductions in plant height, spike length, spikelet number per spike, and grains per spike in the mutant lines 1–4, 2–8, and 5 − 1 relative to the wild-type, these mutants exhibited significant increases in grain length, and the thousand-kernel weight had no significant difference between wild-type and the mutant lines (Fig. 5A-5D; Table 1). Consequently, the TaLcye mutations did not exert a substantial detrimental effect on wheat yield. Furthermore, the seed coat and endosperm of the triple homozygous mutant lines 1–4 and 5 − 1 displayed an orange-yellow hue (Fig. 5E-5F). Table 1 Main agronomic traits of wild-type and TaLcye -edited plants Line Tiller number Plant height (cm) Spike length (cm) Spikelet number Grains per spike Grain length (cm) Grain width (cm) TKW (g) Fielder 15.6 ± 1.06 102.9 ± 1.02 10.9 ± 0.22 18.1 ± 0.22 56.0 ± 0.51 6.75 ± 0.01 3.52 ± 0.01 45.48 ± 0.25 1–4 15.7 ± 0.93 81.5 ± 0.45** 9.2 ± 0.13** 16.5 ± 0.43* 51.9 ± 1.05* 7.26 ± 0.07** 3.29 ± 0.04** 43.51 ± 0.57 2–8 16.0 ± 0.53 84.2 ± 0.20** 9.6 ± 0.22* 16.8 ± 0.45* 52.4 ± 1.00* 7.16 ± 0.01** 3.35 ± 0.03* 43.52 ± 0.69 5 − 1 13.9 ± 0.27 81.7 ± 0.28** 9.4 ± 0.21** 16.3 ± 0.41* 51.3 ± 1.00* 7.27 ± 0.07** 3.34 ± 0.02* 43.36 ± 1.17 Comparison between the lines 1–4, 2–8, 5 − 1, and Fielder. “*” Represent the P < 0.05, and “**” represent the P < 0.01. Comprehensive analysis of grain quality in wheat TaLcye mutants To systematically evaluate the grain quality of TaLcye -edited plants, we conducted a comprehensive analysis of several key parameters, including protein and wet gluten content, grain hardness, starch and amylose content, as well as soluble sugar content. The results revealed that the mutant lines (1–4, 2–8, and 5 − 1) exhibited no significant differences in protein, wet gluten content, and grain hardness compared to their wild-type counterparts. However, significant improvements were observed in the contents of soluble sugar and amylose in lines 1–4 and 5 − 1. Conversely, the total starch content was significantly reduced in these mutant lines (Table 2). Additionally, scanning electron microscopy (SEM) analysis indicated an increase in A-type starch granules in the mutant lines, which may be attributed to the genetic modifications introduced by the TaLcye editing (Fig. 6). Table 2 Analysis of the major quality traits of wild type and TaLcye -edited grains Line Starch (mg/g) Amylose (mg/g) Soluble sugar (mg/g) Protein (%) Wet gluten (%) Hardness (%) WT 549.312 ± 2.453 138.736 ± 2.253 50.242 ± 0.222 11.27 ± 0.05 25.3 ± 0.08 60.7 ± 0.18 1–4 439.223 ± 1.727** 180.69 ± 0.624** 66.114 ± 0.726** 11.5 ± 0.09 25.44 ± 0.07 60.24 ± 0.07 2–8 517.222 ± 2.025* 148.452 ± 0.25* 62.004 ± 0.796* 11.63 ± 0.08 25.48 ± 0.09 60.01 ± 0.16 5 − 1 474.519 ± 1.556** 178.137 ± 1.556** 65.61 ± 0.869** 11.61 ± 0.1 25.44 ± 0.13 60.05 ± 0.18 Comparison between the lines 1–4, 2–8, 5 − 1, and Fielder. “*” Represent the P < 0.05, and “**” represent the P < 0.01. Discussion Given the crucial role of β-carotene in human nutrition, significant efforts have been devoted to enhance its levels in staple crops. For instance, researchers successfully biofortified rice with improved β-carotene by reconstructing its entire biosynthetic pathway in the endosperm, called as "Golden Rice". This was achieved through the introduction of the several key genes from the carotenoid biosynthetic pathway, thereby significantly increasing the β-carotene content in rice grains (Amna et al., 2020 ; Ye et al., 2000 ). In another study, the durum wheat cultivar Kronos was subjected to ethyl methanesulfonate (EMS) mutagenesis to create a TaLcye -silenced mutant, in which the metabolic pathway responsible for the synthesis of α-carotene and lutein was effectively blocked, while the β-carotene content in grains was increased by 75% compared to the wild-type control (Sestili et al., 2019 ). Similarly, knocking out the Lcye gene in lettuce led to a 2.7-fold increase in β-carotene levels (Livneh et al., 2025 ). Wheat, a staple food source for a significant portion of the global population, is characterized by its low β-carotene content in the grains, which limits the bioavailability of essential retinol (vitamin A) (Shewry et al., 2009). In this study, we generated homozygous mutants of the TaLcye gene by utilizing CRISPR-Cas9 gene editing system in wheat. Analysis using UPLC-MS/MS revealed that although the β-carotene content in the mutant leaves was reduced, it was increased in all the grains at 30 DPA of mutant lines (Fig. 3 ; Table S2-S3). Specifically, line 5 − 1 exhibited a 34.5% increase in β-carotene content compared to the wild-type (Fig. 4 A; Table S3). Additionally, line 2–8 demonstrated a slight increase of total carotenoid content in the grains at 30 DPA (Table S3). Zeaxanthin, as a downstream metabolite of β-carotene, is highly concentrated in the macula of the eye's retina, where it functions as a shield against blue light and oxidative stress, thereby reducing the risk of age-related macular degeneration (AMD) (Sajilata et al., 2008 ). In addition, antheraxanthin and violaxanthin was the metabolite of zeaxanthin, have been shown to possess specific anti-cancer properties and may contribute to slow the progression of atherosclerosis (Shimode et al., 2018 ). In plants, antheraxanthin and violaxanthin participate in the xanthophyll cycle and protect the photosynthetic system from light-induced damage under high light conditions (Zhang et al., 2023 ; D'Ambrosio et al., 2023 ). Our present results by UPLC-MS/MS analysis revealed significant increases in the levels of these carotenoids in the leaves of line 5 − 1, with zeaxanthin increasing 2.6-fold, antheraxanthin increasing 2.8-fold, and violaxanthin increasing 1.5-fold compared to the wild type (Fig. S6; Table S2). In the grains of line 1–4, the levels of zeaxanthin, antheraxanthin, and violaxanthin were elevated to 2.1 µg g⁻¹, 13.6 µg g⁻¹, and 29.9 µg g⁻¹, in order (Fig. 4 ; Table S3). Lutein is a yellow carotenoid that typically imparts a pale-yellow hue to cereal grains. In the endosperm of mature tetraploid wheat grains, lutein is the predominant carotenoid component, contributing the seeds with a significantly yellow appearance (Howitt et al., 2009 ). In contrast, carotenes, particularly β-carotene, generally impart an orange color to grains. For instance, the accumulation of β-carotene in grains results in an orange or orange-yellow phenotype (Kim et al., 2022 ). In our current study, the seed coat and endosperm of wheat triple homozygous mutant lines 1–4 and 5 − 1 exhibited an orange-yellow hue, likely due to the elevated levels of β-carotene in the grains (Fig. 5 E- 5 F). Homozygous TaLcye mutants displayed reductions in plant height, spike length, spikelet number, and grains per spike. Interestingly, grain length was increased in the mutants, while thousand-kernel weight remained statistically unchanged (Fig. 5 A- 5 D; Table 1 ), indicating that TaLcye mutations did not adversely affect overall grain yield. Moreover, the mutant lines exhibited significantly higher levels of soluble sugars and amylose (Table 2 ), traits that may enhance their utility in bread, pastry, and other food-processing applications. These findings underscore the potential of TaLcye mutants for dual-purpose improvement of both nutritional quality and processing characteristics, warranting further investigation. Carotenoids are the precursors of ABA biosynthesis (Zhou et al., 2021 ). The 9'-cis-neoxanthin or 9'-cis-violaxanthin was cleaved by the enzyme 9-cis-epoxycarotenoid dioxygenase to produce xanthoxin, the committed precursor of ABA (Finkelstein et al., 2013) (Fig. 1 ). The phytohormone abscisic acid (ABA) plays a central role in the induction and maintenance of seed dormancy. Seed dormancy is an adaptive trait that enables seeds to remain quiescent until conditions become favorable for germination and mainly determines the pre-harvest sprouting tolerance (Nee et al., 2017 ). Our results revealed a significant increase in violaxanthin content in the mutant lines 1–4 and 5 − 1 (Fig. 4 C; Fig. S6D), leading us to hypothesize that ABA levels may differ between the mutants and the wild type, potentially affecting seed dormancy. This possibility will be the focus of our future research. The advent of CRISPR-Cas9 gene editing system has revolutionized the potential for targeted genetic modifications in crop species (Livneh et al., 2025 ). In wheat, this technology enables the precise knockout of the key genes like TaLcye to enhance β-carotene content, a crucial provitamin A precursor, in the grains at 30 DPA (Fig. 4 A; Table S3). Importantly, our field experiments and quality analysis demonstrated that such genetic modifications do not significantly compromise main agronomic traits of the mutant lines (Fig. 5 ; Table 1 – 2 ), thereby maintaining their overall performance and yield potential. These results indicated that the mutants with satisfactory agronomic performance can be seamlessly integrated into breeding programs, paving the way for the development of wheat cultivars with elevated β-carotene levels. Collectively, this study lays a robust foundation for leveraging gene editing technologies to achieve provitamin A biofortification in commercial wheat varieties, thereby providing a valuable reference for improving nutritional outcomes in staple crops. Declarations Authors’ contribution Huiyun Liu conceived the research and designed the experiments; Yajie Guo conducted most of the experiments; Yajie Guo and Mengtian Liu performed the vector construction, wheat transformation, and examination the mutants; Dan Wang and Mengyao Li investigated the agronomic traits; Yajie Guo and Huiyun Liu drafted and revised the manuscript. Acknowledgements This study was financially supported by the Major Science and Technology Projects in Henan Province (231100110300), the Ministry of Science and Technology (China) of China (no. 2021YFF1000203), the Natural Science Foundation of Henan Province, China (no. 232300420189), the Tackling Key Problems in Science and Technology of Henan Province, China (no. 242102111117). Declaration of Interest Statement The authors declare no competing interests. References Ahmad A, Ahmed Z (2019) Fortification in beverages, production and management of beverages, volume1: the science of beverages, pp, 85–122 Amna QS, Tantray AY, Bashir SS, Zaid A, Wani SH (2020) Golden rice: genetic engineering, promises, present status and future prospects, in rice research for quality improvement: genomics and genetic engineering, pp, 581–604 Anand R, Mohan L, Bharadvaja N (2022) Disease prevention and treatment using β-carotene: the ultimate provitamin A. Rev Bras Farmacogn 32:491–501 Bai L, Kin EH, DellaPenna D, Brutnell TP (2009) Novel lycopene epsilon cyclase activities in maize revealed through perturbation of carotenoid biosynthesis. Plant J 59:588–599 Carazo A, Macakova K, Matousova K, Krcmova LK, Protti M, Mladenka P (2021) Vitamin A update: forms, sources, kinetics, detection, function, deficiency, therapeutic use and toxicity. Nutrients 13:703 Cong L, Wang C, Chen L, Liu H, Yang G, He G (2009) Expression of phytoene synthase1 and carotene desaturase crtI genes result in an increase in the total carotenoids content in transgenic elite wheat ( Triticum aestivum L). J Agric Food Chem 57:8652–8660 Cunningham FX, Pogson B, Sun Z, McDonald KA, DellaPenna D, Gantt E (1996) Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8:1613–1626 Cunningham FX, Gantt E (1998) Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 49:557–583 D'Ambrosio C, Stiglianim AL, Rambla JL, Frusciante S, Diretto G, Enfissi EMA, Granell A, Fraser PD, Giorio G (2023) A xanthophyll-derived apocarotenoid regulates carotenogenesis in tomato chromoplasts. Plant Sci 328:111575 Diretto G, Tavazza R, Welsch R, Pizzichini D, Mourgues F, Papacchioli V, Beyer P, Giuliano G (2006) Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biol 6:13 Finkelstein R (2013) Abscisic acid synthesis and response. Arabidopsis Book 11:e0166 Giuliano G (2017) Provitamin a biofortification of crop plants: a gold rush with many miners. Curr Opin Biotechnol 44:169–180 Grune T, Lietz G, Palou A, Ross AC, Stahl W, Tang G, Thurnham D, Yin SA, Biesalski HK (2010) β-Carotene as an important vitamin a source for humans. J Nutr 140:2268–2285 Howitt C, Cavanagh C, Bowerman A, Cazzonelli C, Rampling L, Mimica J, Pogson B (2009) Alternative splicing, activation of cryptic exons and amino acid substitutions in carotenoid biosynthetic genes are associated with lutein accumulation in wheat endosperm. Funct Integr Genomics 9:363–376 Kaur N, Alok A, Shivani Kumar P, Kaur N, Awasthi P, Chaturvedi S, Pandey P, Pandey A, Pandey AK, Tiwari S (2020) CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit. Metab Eng 59:76–86 Ke Q, Kang L, Kim HS, Xie T, Liu C, Ji CY, Kim SH, Park WS, Ahn MJ, Wang S, Li H, Deng X, Kwak SS (2019) Down-regulation of lycopene ɛ-cyclase expression in transgenic sweetpotato plants increases the carotenoid content and tolerance to abiotic stress. Plant Sci 281:52–60 Kim HK, Kim JY, Kim JH, Go JY, Jung YS, Lee HJ, Ahn MJ, Yu J, Bae S, Kim HS, Kwak SS, Kim MS, Cho YG, Jung YJ, Kang KK (2022) Biochemical characterization of orange-colored rice calli induced by target mutagenesis of OsOr Gene. Plants (Basel) 12: 56 Kirpichenkova EV, Korolev AA, Onishchenko GG, Nikitenko EI, Lipatov DV, Kuz'min AG, Dyskin YA, Denisova EL, Fetisov RN (2018) Study of lutein and zeaxanthin content in the diet with the assessment of the relationship between the level of alimentary intake of non-vitamin carotenoids and the density of the macular region of the retina at a young age. Vopr Pitan 87:20–26 Kusama Y, Inoue S, Jimbo H, Takaichi S, Sonoike K, Hihara Y, Nishiyama Y (2015) Zeaxanthin and echinenone protect the repair of photosystem II from Inhibition by singlet oxygen in synechocystis sp. PCC 6803. Plant Cell Physiol 56:906–916 Lichtenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 50:47–65 Liu H, Wang K, Jia Z, Gong Q, Lin Z, Du L, Pei X, Ye X (2020) Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system. J Exp Bot 71:1337–1349 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 –∆∆CT method. Methods 25:402–408 Livneh Y, Leor Librach E, Agmon D, Makov Bouaniche T, Tiwari V, Shor E, Yeselson Y, Masci T, Schaffer A, Charuvi D, Hirschberg J, Vainstein A (2025) Combined enhancement of ascorbic acid, β-carotene and zeaxanthin in gene-edited lettuce. Plant Biotechnol J 23:1954–1967 Ma L, Lin XM (2010) Effects of lutein and zeaxanthin on aspects of eye health. J Sci Food Agr 90:2–12 Nee G, Xiang Y, Soppe WJ (2017) The release of dormancy, a wake-up call for seeds to germinate. Curr Opin Plant Biol 35:8–14 Pogson BJ, Rissler HM (2000) Genetic manipulation of carotenoid biosynthesis and photoprotection. Philos T Roy Soc B 355:1395–1403 Sajilata MG, Singhal RS, Kamat MY (2008) The carotenoid pigment zeaxanthin-a review. Proc. Comp. Rev. Food Sci. Food Safety 7: 29–49 Sajovic J, Meglic A, Glavac D, Markelj S, Hawlina M, Fakin A (2022) The role of vitamin a in retinal diseases. Int J Mol Sci 23:1–27 Sestili F, Garcia-Molina MD, Gambacorta G, Beleggia R, Botticella E, De Vita P, Savatin DV, Masci S, Lafiandra D (2019) Provitamin A biofortification of durum wheat through a TILLING approach. Int J Mol Sci 20:5703 Shewry PR (2009) Wheat. J Exp Bo 60:1537–1553 Shimode S, Miyata K, Araki M, Shindo K (2018) Antioxidant activities of the antheraxanthin-related carotenoids, antheraxanthin, 9-cis-antheraxanthin, and mutatoxanthins. J Oleo Sci 67:977–981 Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54:733–749 Trono D (2019) Carotenoids in cereal food crops: composition and retention throughout grain storage and food processing. Plants 8:551 Van Loo Bouwman CA, Naber TH, Schaafsma G (2014) A review of vitamin A equivalency of β-carotene in various food matrices for human consumption. Br J Nutr 111:2153–2166 Wang K, Liu H, Du L, Ye X (2017) Generation of marker-free transgenic hexaploid wheat via an Agrobacterium -mediated co-transformation strategy in commercial Chinese wheat varieties. Plant Biotechnol J 15:614–623 Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin a (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303–305 Yu B, Lydiate DJ, Young LW, Schäfer UA, Hannoufa A (2008) Enhancing the carotenoid content of brassica napus seeds by downregulating lycopene epsilon cyclase. Transgenic Res 17:573–585 Yu S, Tian L (2018) Breeding major cereal grains through the lens of nutrition sensitivity. Mol Plant 11:23–30 Zhai S, Xia X, He Z (2016) Carotenoids in staple cereals: metabolism, regulation, and genetic manipulation. Front Plant Sci 7:1197 Zhang Z, Zhou D, Li S, Pan J, Liang J, Wu X, Wu XN, Krall L, Zhu G (2023) Multiomics analysis reveals the chemical and genetic bases of pigmented potato tuber. J Agric Food Chem 71:16402–16416 Zhou H, Yang M, Zhao L, Zhu Z, Liu F, Sun H, Sun C, Tan L (2021) HIGH-TILLERING AND DWARF 12 modulates photosynthesis and plant architecture by affecting carotenoid biosynthesis in rice. J Exp Bot 72:1212–1224 Supplementary Files 202511supplementaryfigures.docx Supplementarytables11.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. 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08:41:11","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50825,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/522320cfcb6498daa25c45be.png"},{"id":96976990,"identity":"cef7f8c7-d847-46aa-8ce4-c83e134aeefc","added_by":"auto","created_at":"2025-11-28 08:41:11","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146930,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED25013460structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/0d344b76a2d7e1663af7f4ed.xml"},{"id":96976988,"identity":"ad398156-1de1-4fd8-b02d-63bb3c182c41","added_by":"auto","created_at":"2025-11-28 08:41:11","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154870,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/814bda3e499d26d4a3327056.html"},{"id":96976901,"identity":"db3a64a2-8a5b-4204-b97e-ade1fc42ad08","added_by":"auto","created_at":"2025-11-28 08:41:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":103787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA simplified scheme of carotenoid biosynthetic pathway in higher plants\u003c/strong\u003e. Dashed arrows denote multiple reaction steps. GGPP, geranylgeranyl pyrophosphate; PSY, phytoene synthase; PDS, phytoene desaturase; ZISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; LCYB, lycopene β-cyclase; LCYE, lycopene ε-cyclase; HYD, β-carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NXS, neoxanthin synthase; CRTISO, carotenoid isomerase; ABA, abscisic acid.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/f8e256e6b93ab30d369c54e2.png"},{"id":96976919,"identity":"476f2282-03cb-45a2-8681-c78c9bcfc403","added_by":"auto","created_at":"2025-11-28 08:41:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":198238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression pattern of genes involved in the carotenoid biosynthesis in grains. \u003c/strong\u003eA:\u003cem\u003e \u003c/em\u003eThe expression level of\u003cem\u003e TaPsy\u003c/em\u003e; B:\u003cem\u003e \u003c/em\u003ethe expression level of\u003cem\u003e TaPds\u003c/em\u003e; C:\u003cem\u003e \u003c/em\u003ethe expression level of\u003cem\u003e TaZds\u003c/em\u003e; D:\u003cem\u003e \u003c/em\u003ethe expression level of\u003cem\u003e TaLcyb\u003c/em\u003e; E:\u003cem\u003e \u003c/em\u003ethe expression level of\u003cem\u003e TaHyd1\u003c/em\u003e; F: the expression level of\u003cem\u003e TaHyd2\u003c/em\u003e; the levels of gene expression are normalized using wheat \u003cem\u003eTaActin \u003c/em\u003egene; values are means \u003cem\u003e± \u003c/em\u003esd of three biological replicates; standard error is indicated above each bar; two-tailed student’s \u003cem\u003et-tests\u003c/em\u003e showed relative expression values with significant (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) and highly significant (**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) differences compared to the control are designated by the asterisk.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/2724b4802be7a3e17b28564d.png"},{"id":96976927,"identity":"2ec4a5bf-9868-48c3-bcd3-cbe42b73f753","added_by":"auto","created_at":"2025-11-28 08:41:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap analysis of metabolite profiles in the wild-type and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTaLcye\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutants. \u003c/strong\u003eA: Leaves at 30 DPA; B: grains at 30 DPA; color scale, standardized metabolite abundance (red = high, green = low); group, samples are clustered by similarity in metabolite composition.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/74b9a9d6d502e836065175e3.png"},{"id":96976938,"identity":"d6531d58-0c7b-4be9-acae-dde3f870fe92","added_by":"auto","created_at":"2025-11-28 08:41:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":169914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCarotenoid content in the grains at 30 DPA of wild-type and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTaLcye \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutant lines\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eValues are reported as micrograms pergram of fresh weight (F.W.). Standard errors are shown above each bar, along with significant (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) and highly significant (**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) differences compared to the control are designated by the asterisk.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/e3f2415424f218326906caa9.png"},{"id":96976921,"identity":"3e4ff582-0e8b-4ae6-9adb-69b9b72ac00d","added_by":"auto","created_at":"2025-11-28 08:41:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the main agronomic traits in wild-type and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTaLcye-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eedited\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003elines. \u003c/strong\u003eA: Whole-plants phenotype of wild-type (1) and mutant lines 1-4 (2), 2-8 (3), and 5-1 (4); B: spike morphology of wild-type (1) and mutant lines 1-4 (2), 2-8 (3), and 5-1 (4); C: grains length measurements for wild-type (1) and mutant lines 1-4 (2), 2-8 (3), and 5-1 (4); D: grains width measurements for wild-type (1) and mutant lines 1-4 (2), 2-8 (3), and 5-1 (4); E: the seed coat color comparison between wild-type (1) and mutant lines 1-4 (2), 2-8 (3), and 5-1 (4); F: the endosperms color variation in wild-type (1) relative to mutant lines 1-4 (2), 2-8 (3), and 5-1 (4).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/018dd004cf5d8076e423e409.png"},{"id":96976951,"identity":"ae170bbb-30e5-4298-91c4-947371c52803","added_by":"auto","created_at":"2025-11-28 08:41:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":276182,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM analysis of endosperm starch granules in wild-type and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTaLcye\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant grains. \u003c/strong\u003eA: Wild-type Fielder; B: mutant line 1-4; C: mutant line 2-8; D: mutant line 5-1; scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/58d6a3c28f64b6e8657d8cf2.png"},{"id":98451989,"identity":"715e72c2-2dcb-44dd-9f59-0a9615dff1c6","added_by":"auto","created_at":"2025-12-17 17:34:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2300156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/174bec30-5a83-4ba4-a444-e3de76989797.pdf"},{"id":96976925,"identity":"11297ea3-df7b-43f4-bf1e-c3866f650cd4","added_by":"auto","created_at":"2025-11-28 08:41:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1072740,"visible":true,"origin":"","legend":"","description":"","filename":"202511supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/f98642d87a24ce35d6a7d987.docx"},{"id":97138743,"identity":"0615ec2a-1b81-4623-8d0e-d0170a2095f1","added_by":"auto","created_at":"2025-12-01 09:59:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29090,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytables11.docx","url":"https://assets-eu.researchsquare.com/files/rs-8056178/v1/4b589d57e6775774dee93085.docx"}],"financialInterests":"","formattedTitle":"Gene editing-mediated enhancement of carotenoid compounds accumulation in common wheat grains","fulltext":[{"header":"Key message ","content":"\u003cp\u003eTargeted knockout of the gene \u003cem\u003eTaLcye\u003c/em\u003e, using the CRISPR-Cas9 system, led to significantly elevated levels of \u0026beta;-carotene, zeaxanthin, violaxanthin, and antheraxanthin in the grains of common wheat.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCarotenoids serve as precursors for the synthesis of vitamin A, a nutrient that humans cannot synthesize de novo. Consequently, vitamin A must be obtained through dietary intake. Vitamin A deficiency (VAD) can make serious health consequences, with mild cases manifesting as night blindness and severe cases potentially leading to permanent blindness or even death (Grune et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sajovic et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Carazo et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among carotenoids, β-carotene is the principal plant-derived precursor of retinol (vitamin A), essential for a wide range of physiological function (Anand et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These functions include night vision, cell proliferation, reproduction, embryonic development, brain activity, and immune system regulation (Ahmad and Ahmed \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Trono \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Other carotenoids, such as zeaxanthin, antheraxanthin, and violaxanthin, also play crucial roles as antioxidants, neutralizing harmful reactive oxygen species (ROS; Sajovic et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, these vital compounds are often underrepresented in modern diets.\u003c/p\u003e\u003cp\u003eCarotenoids constitute a diverse group of naturally occurring lipophilic compounds that are extensively distributed in green plants, algae, fungi, and bacteria. These compounds belong to the C40 isoprenoid class and typically consist of eight isoprenoid units (Cunningham et al., 1998). The biosynthetic pathway of carotenoids begins with geranylgeranyl pyrophosphate (GGPP) as the precursor (Lichtenthaler et al., 1999). The first committed step in this pathway is that GGPP is catalyzed by phytoene synthase (PSY), which facilitates the condensation of two GGPP molecules to produce 15-cis-phytoene. Subsequently, phytoene undergoes a successive of desaturation reactions to form lycopene (Zhai et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These desaturation steps are mediated by phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), ZISO (ζ-carotene isomerase), and CRTISO (carotenoid isomerase) enzymes, respectively (Tanaka et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe cyclization of lycopene is a critical branching point in carotenoid biosynthesis, diverging into two distinct pathways. One pathway leads to the formation of α-carotene, and the other results in the production of γ-carotene and β-carotene. Plants contain two types of cyclase enzymes which mediate the process of ε-cyclase (LCYE) and β-cyclase (LCYB), respectively (Cunningham et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Cunningham et al., 2001). The LCYE and LCYB catalyzes the cyclization of one end of lycopene to form an ε-ring, yielding α-carotene. In contrast, LCYB cyclizes both ends of lycopene to form β-rings, thereby producing γ-carotene and β-carotene (Cunningham et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Bai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zhai et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurther downstream in the plant carotenoid biosynthetic pathway, α-carotene and β-carotene undergo additional modifications to generate more complex oxygenated derivatives. These modifications are catalyzed by carotenoid hydroxylase (HYD). Specifically, α-carotene is predominantly converted into α-cryptoxanthin and lutein (Bai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Meanwhile, β-carotene is transformed into a variety of xanthophylls, including β-cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin, and neoxanthin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Giuliano \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe gene \u003cem\u003eLcye\u003c/em\u003e encoding ε-cyclase is a key determinant in the carotenoid biosynthetic pathway, exerting significant influence over the content and compositional ratios of carotenoids in plants. Subsequent studies had demonstrated its pivotal role in modulating carotenoid profiles across various plant species. For instances, the deficiency of LCYE enzyme in \u003cem\u003eArabidopsis\u003c/em\u003e results in the failure to synthesize lutein and a concomitant increase in β-carotene levels (Pogson et al., 2000); in the tomato plants with silenced \u003cem\u003eLcye\u003c/em\u003e, total carotenoids and β-carotene levels were elevated, while lutein content was reduced (Ma et al., 2010); downregulation of the \u003cem\u003eLcye\u003c/em\u003e gene in rapeseed (Brassica napus) showed markedly elevate carotenoid levels in seeds (Yu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Similarly, targeted editing of the \u003cem\u003eLcye\u003c/em\u003e gene in bananas led to a six-fold increase of β-carotene content in fruits, albeit with concurrent reductions of α-carotene and lutein levels (Kaur et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In potatoes, downregulation of \u003cem\u003eLcye\u003c/em\u003e led to a 14-fold increase in β-carotene content and a 2.5-fold rise in total carotenoid content, with the potato callus tissue exhibiting a deep yellow hue (Diretto et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ke et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, biochemical analyses of durum wheat mutant lines revealed a 75% increase of β-carotene content in grain in the complete mutant line compared to the control (Sestili et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Collectively, these findings underscored the potential of \u003cem\u003eLcye\u003c/em\u003e as a valuable genetic tool for enhancing β-carotene content in plants, thereby holding significant promise for breeding programs aimed at improving nutritional quality.\u003c/p\u003e\u003cp\u003eCommon wheat serves as a crucial source of plant-based protein, minerals, and vitamins for human nutrition. The quality of wheat can be broadly categorized into processing quality and nutritional quality (Shewry et al., 2009). With the continuous improvement of living standards and the deepening awareness of healthy eating, the cultivation of wheat varieties with enhanced nutritional quality has gained increasing attention. Among these nutrients, vitamin A content is one of the key indicators for assessing the nutritional quality of wheat, playing a critical role in human health. However, predominantly non-provitamin A carotenoids such as lutein are typically accumulated in wheat seeds, while provitamin A carotenoids such as β-carotene and the downstream metabolite zeaxanthin, antheraxanthin, and violaxanthin of β-carotene are relatively low in content (Cong et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yu and Tian, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is necessary to develop the wheat germplasm with enhanced carotenoids by gene editing technology.\u003c/p\u003e\u003cp\u003eIn this research, we employed the CRISPR gene editing system to target the \u003cem\u003eTaLcye\u003c/em\u003e gene in wheat, aiming to enhance the nutritional profile controlled by this gene. Results showed that the carotenoid contents\u0026mdash;β-carotene, zeaxanthin, antheraxanthin and violaxanthin\u0026mdash;were substantially elevated in the grains at 30 DPA. Notably, these genetic modifications did not significantly affect wheat yield. Our results highlight the potential of gene editing as an effective approach for biofortification, providing a viable solution to address micronutrient deficiencies in major crop.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials and cultivation conditions\u003c/h2\u003e\u003cp\u003eA wheat cultivar Fielder was acquired from the Crop Germplasm Bank of China. Wheat seeds were grown in pots (20\u0026times;30 cm) in a growth chamber maintained at 24\u0026deg;C, with 16/8 h light/dark cycle, 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity, and 45% humidity. At 15\u0026ndash;17 days post-anthesis (DPA), immature wheat grains were collected for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation, following the method described by Wang et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eConstruction of the vectors\u003c/h3\u003e\n\u003cp\u003eThe expression vector pWMB110-Cas9 for gene editing was constructed in our previous work by inserting the \u003cem\u003eCas9\u003c/em\u003e gene into the multiple cloning site (MCS) of pWMB110, which contains the \u003cem\u003ebar\u003c/em\u003e gene as a selection marker for generating transgenic plants and the maize \u003cem\u003eubiquitin (UBI)\u003c/em\u003e promoter for driving \u003cem\u003eCas9\u003c/em\u003e gene (Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, a guide RNA sequence 5\u0026rsquo;-gtatgggaggacgaattcaa-3\u0026rsquo; was designed to target the three homologous \u003cem\u003eTaLcye\u003c/em\u003e genes based on the sequences of \u003cem\u003eTaLcye-3A\u003c/em\u003e (GeneBank accession ACF42349.1), \u003cem\u003eTaLcye-3B\u003c/em\u003e (GeneBank accession ACF42350.1), and \u003cem\u003eTaLcye-3D\u003c/em\u003e (GeneBank accession ACF42351.1) (Sestili et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This guide RNA was driven by the \u003cem\u003eTaU3\u003c/em\u003e promoter, and inserted into the multiple cloning site (MCS) of the pWMB110-Cas9 plasmid through homologous recombination, resulting in the final construct named \u003cem\u003eTaLcye\u003c/em\u003e-CRISPR (Fig. S1A-S1B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection analysis of the\u003c/b\u003e \u003cb\u003eTaLcye\u003c/b\u003e\u003cb\u003e-edited mutations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from the candidate T0 transgenic wheat mutants, in which the targeted genes, \u003cem\u003eTaLcye-3A\u003c/em\u003e (5\u0026rsquo;-gtctggtagcatccttcaagtag-3\u0026rsquo; and 5\u0026rsquo;-tatcatgctgcctgatgcaactg-3\u0026rsquo;), \u003cem\u003eTaLcye-3B\u003c/em\u003e (5\u0026rsquo;-gtctggtagcatccttcaagtag-3\u0026rsquo; and 5\u0026rsquo;-ctgaattccaagatcgaatgtacc-3\u0026rsquo;), and \u003cem\u003eTaLcye-3D\u003c/em\u003e (5\u0026rsquo;-gtctggtagcatccttcaagtag-3\u0026rsquo; and 5\u0026rsquo;-gaactggtgcacaaacaacatatg-3\u0026rsquo;), were amplified using their respective specific primers. The \u003cem\u003ebar\u003c/em\u003e gene was screened using the primer pairs 5\u0026rsquo;-ACCATCGTCAACCACTACATCG-3\u0026rsquo; and 5\u0026rsquo;-GCTGCCAGAAACCACGTCATG-3\u0026rsquo;. The PCR products were digested with the corresponding restriction enzyme (1 U) in a 10 \u0026micro;l reaction buffer containing 3 \u0026micro;l PCR product for 2 h at 37\u0026deg;C. The resultant products were separated on a 2% agarose gel and visualized using a GelDoc XR System (BioRad). The presence of edited mutations was inferred from the altered banding patterns compared to the wildtype controls.\u003c/p\u003e\n\u003ch3\u003eQuantitative reverse-transcriptase PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eSamples were collected from the leaves and immature grains at 15 DPA of the \u003cem\u003eTaLcye\u003c/em\u003e-edited wheat plants and their wild-type Fielder, which were grown in a semi-controlled greenhouse environment. Total RNA was extracted using Trizol reagent (Invitrogen) following the manufacturer\u0026rsquo;s protocol. Reverse transcription was performed using HiScript III RT SuperMix (Vazyme Biotech Co., Ltd) according to the manufacturer\u0026rsquo;s instructions. The qRT-PCR was performed using SevenFast SYBR Green Mix (Seven, Beijing) in a 7500 Fast Real-Time PCR system (Applied Biosystems). Each reaction was carried out in a final volume of 20 \u0026micro;l, with 10 \u0026micro;l SevenFast SYBR Green Mix, 0.4 \u0026micro;M of each primer, and 1 \u0026micro;l cDNA. The primers for the key genes involved in the carotenoid biosynthetic pathway were listed in Table S1. Transcript abundance was expressed relative to that of \u003cem\u003eTaActin\u003c/em\u003e using the 2\u003csup\u003e\u0026ndash;2ΔΔCT\u003c/sup\u003e method, which allows for the quantification of gene expression levels relative to a reference gene (Livak et al. 2001).\u003c/p\u003e\n\u003ch3\u003eUltra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)\u003c/h3\u003e\n\u003cp\u003eLeaves and grains at 30 DPA were collected from the \u003cem\u003eTaLcye\u003c/em\u003e-edited plants and wild type, and grounded into a fine powder under liquid nitrogen. Subsequently, a solvent mixture composed of n-hexane, acetone, and ethanol (1:1:1, v/v/v), as well as 0.01% butylated hydroxytoluene (BHT, w/v), was added into the powdered samples. Following the extraction process, the samples were centrifuged to eliminate insoluble debris. The resulting supernatant was filtered through a syringe filter to yield a clear extract. This extract was subsequently injected into an ultra-performance liquid chromatography (UPLC) system. Separation was achieved on a C30 column using a gradient elution program. The mobile phase consisted of water and methanol, both of which contained a small amount of formic acid. The separated compounds were then detected via tandem mass spectrometry.\u003c/p\u003e\u003cp\u003eThe data were processed using Analyst 1.6.3 and MultiQuant 3.0.3 software. The chromatographic peaks corresponding to the analytes in different samples were integrated and corrected based on the retention times and peak shape information of the reference standards. Ultimately, the concentrations of the target compounds were determined using calibration curves derived from standard solutions.\u003c/p\u003e\n\u003ch3\u003eGrain protein, wet gluten, and starch content analysis\u003c/h3\u003e\n\u003cp\u003eGrain protein and wet gluten content in the \u003cem\u003eTaLcye\u003c/em\u003e-edited lines and wild type were measured using the DA7200 near-infrared instrument (Perten, Sweden). Starch and amylose in the whole grain flour were extracted and analyzed using the Solarbio Starch Content Assay Kit and the Amylose Content Assay Kit (Solarbio, Beijing). In each analysis, three biological replicates were prepared for both each \u003cem\u003eTaLcye\u003c/em\u003e-edited line and the wild-type.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGrain soluble sugar analysis\u003c/h2\u003e\u003cp\u003eAbout 0.1 g of fine sample powder from each \u003cem\u003eTaLcye\u003c/em\u003e-edited line and the wild-type was weighed into a 15 ml centrifuge tube, and then 4 ml of 80% (v/v) ethanol was added. The centrifuge tube was then placed in a water bath at 80\u0026deg;C for 40 min. After cooling, the sample was centrifuged at 4500 rpm for 10 min. The supernatant was collected and filtered through a 0.45 \u0026micro;m membrane disc filter (Sangon, China).\u003c/p\u003e\u003cp\u003eNext, 1 mL of the filtered supernatant was taken, mixed with 5 mL of anthrone reagent, and then placed in a boiling water bath for 10 min. After cooling to room temperature, the absorbance (OD) value at 625 nm was measured using a spectrophotometer, and the soluble sugar content was calculated based on the standard curve prepared with known concentrations of soluble sugar standards. Each sample was subjected to three technical replicates.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eScanning electron microscopy analysis of wheat grains\u003c/h3\u003e\n\u003cp\u003eMature wheat grains from the \u003cem\u003eTaLcye\u003c/em\u003e-edited lines and wild-type were fixed in 2.5% glutaraldehyde solution for 2\u0026ndash;4 h. Then the grains underwent dehydration in a graded ethanol series (30%, 50%, 70%, 90%, and 100% ethanol), maintaining 15\u0026ndash;30 min in each concentration. Subsequently, critical point drying was performed using carbon dioxide as the displacement medium to remove ethanol while preserving sample integrity. The dried grains were sectioned to expose internal structures. The sections were then mounted on a sample holder and sputter-coated with a thin gold layer using an ion sputtering device to enhance conductivity. Finally, the samples were placed in the scanning electron microscopy (SEM) chamber for observing the surface and internal structures of the wheat grains.\u003c/p\u003e\n\u003ch3\u003eExamination of the main agronomic traits and statistical analysis\u003c/h3\u003e\n\u003cp\u003eAt wheat maturity stage, ten plants were randomly picked from each replicate plot for evaluating main agronomic traits including tiller number per plant, plant height, spike length, spikelet number per spike, grains per spike, grain length, grain width, and 1000-kernel weight (TKW). Data analysis was performed using SPSS, with t-tests comparing means between the \u003cem\u003eTaLcye\u003c/em\u003e-edited lines and wild-type. Significance levels were set at *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (significant) and **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (highly significant).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification of wheat\u003c/strong\u003e \u003cstrong\u003eTaLcye\u003c/strong\u003e \u003cstrong\u003eloss-of-function mutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the roles of \u003cem\u003eTaLcye\u003c/em\u003e homoeologs in carotenoid metabolism and plant growth in wheat, we edited \u003cem\u003eTaLcye\u003c/em\u003e homoeologs in a wheat cultivar Fielder and obtained candidate mutant plants. In the offsprings of \u003cem\u003eTaLcye\u003c/em\u003e-edited plants, three transgenic-free homozygous mutant lines (1–4, 2–8, and 5 − 1) were selected after detailed molecular analysis (Fig. S1C). Sanger sequencing revealed that line 5 − 1 exhibited a 2 bp, 4 bp, and 11 bp deletions in exon 2 of the \u003cem\u003eTaLcye-A\u003c/em\u003e, \u003cem\u003eTaLcye-B\u003c/em\u003e and \u003cem\u003eTaLcye-D\u003c/em\u003e homoeologs, in order. Line 2–8 had a 1 bp and 2 bp deletions at the 3 bp upstream of the PAM sites of \u003cem\u003eTaLcye-B\u003c/em\u003e and \u003cem\u003eTaLcye-D\u003c/em\u003e homoeologs, respectively. Line 1–4 contained a 2 bp deletion at position 469–470 of the \u003cem\u003eTaLcye-A\u003c/em\u003e ORF, and a single T insertion at position 476 of the \u003cem\u003eTaLcye-B\u003c/em\u003e ORF, and a 15 bp deletion in the \u003cem\u003eTaLcye-D\u003c/em\u003e homoeologs (Fig. S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression profiling of the key genes involved in the carotenoid biosynthetic pathway in the\u003c/strong\u003e \u003cstrong\u003eTaLcye\u003c/strong\u003e \u003cstrong\u003emutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of \u003cem\u003eTaLcye\u003c/em\u003e-edited homeoalleles on carotenoid biosynthesis in wheat, we assessed the expression levels of other key genes including \u003cem\u003eTaPsy, TaPds, TaZds, TaLcyb, TaHyd1\u003c/em\u003e, and \u003cem\u003eTaHyd2\u003c/em\u003e involved in carotenoid biosynthesis pathway in \u003cem\u003eTaLcye\u003c/em\u003e mutant lines using qRT-PCR and compared them with those in the wild-type.\u003c/p\u003e\n\u003cp\u003eIn leaves, the relative expression levels of \u003cem\u003eTaPsy\u003c/em\u003e and \u003cem\u003eTaPds\u003c/em\u003e showed no significant differences among the mutant lines 1–4, 2–8, and 5 − 1. In contrast, the transcript levels of \u003cem\u003eTaLcyb\u003c/em\u003e and \u003cem\u003eTaHyd2\u003c/em\u003e were downregulated, while \u003cem\u003eTaZds\u003c/em\u003e and \u003cem\u003eTaHyd1\u003c/em\u003e were upregulated in mutant line 5 − 1 (Fig. S3).\u003c/p\u003e\n\u003cp\u003eIn grainsa, none of the six genes (\u003cem\u003eTaPsy\u003c/em\u003e, \u003cem\u003eTaPds\u003c/em\u003e, \u003cem\u003eTaZds\u003c/em\u003e, \u003cem\u003eTaLcyb\u003c/em\u003e, \u003cem\u003eTaHyd1\u003c/em\u003e, \u003cem\u003eTaHyd2\u003c/em\u003e) differed in expression between wild-type and mutants 1–4 or 2–8; only the triple homozygous mutant line 5 − 1 showed significant repression expression of \u003cem\u003eTaPsy\u003c/em\u003e and \u003cem\u003eTaHyd2\u003c/em\u003e and induction of \u003cem\u003eTaPds\u003c/em\u003e, \u003cem\u003eTaZds\u003c/em\u003e, \u003cem\u003eTaLcyb\u003c/em\u003e and \u003cem\u003eTaHyd1\u003c/em\u003e (Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCarotenoid profiles in the leaves and grains of the wheat\u003c/strong\u003e \u003cstrong\u003eTaLcye\u003c/strong\u003e \u003cstrong\u003emutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVitamin A is an essential nutrient for humans. Provitamin A carotenoids, such as β-carotene, can be converted into vitamin A in the human body, with β-carotene being the most effective (Van Loo-Bouwman et al., 2014). Lutein, another type of carotenoid, cannot be converted into vitamin A (Kirpichenkova et a., 2018). To elucidate the role of \u003cem\u003eTaLcye-\u003c/em\u003eedited in carotenoid biosynthesis in the leaves and grains of the mutant lines, we analyzed and compared the carotenoid content in the mutant lines and their wild-type using LC-MS/MS (Fig.\u0026nbsp;3; Fig. S4-S5). A total of nineteen metabolites were measured in the leaves (Table S2). Compared with wild-type, the mutant line 2–8 exhibited a significant decrease in total carotenoid content. And the mutant lines 1–4 and 5 − 1 experienced a substantial reduction in lutein content (Fig. S6F; Table S2), which is a major compound in the downstream branch of the \u003cem\u003eTaLcye\u003c/em\u003e gene. While in another branch, antheraxanthin, violaxanthin, and zeaxanthin levels were significantly elevated relative to the wild-type (Fig. S6C-6E; Table S2). Additionally, the mutant lines 1–4 and 5 − 1 also showed a slight increase in the content of γ-carotene, β-citraurin, phytoene, and β-cryptoxanthin (Table S2).\u003c/p\u003e\n\u003cp\u003eThe carotenoid profiles of fourteen metabolites totally were measured in the grains at 30 DPA of the mutant lines, and results revealed lower average values than those observed in their leaves (Fig.\u0026nbsp;4; Table S3). The content of lutein exhibited a significant decrease in the mutant lines 1–4 and 5 − 1 compared with the wild-type (Fig.\u0026nbsp;4E). However, the total carotenoid content in these mutant lines did not significantly differ from that in the wild-type (Table S3). This lack of difference could be attributed to the substantial increases in β-carotene (33.2–34.5%), zeaxanthin (112.1-125.4%), violaxanthin (73.5–73.8%), and antheraxanthin (164.1-186.5%) in the mutant lines 1–4 and 5 − 1 (Fig.\u0026nbsp;4A-4D). Notably, the mutant line 2–8 showed no significant difference in lutein content in comparison with wild-type (Fig.\u0026nbsp;4E). However, β-carotene content in the mutant line 2–8 was increased by 26.1%, and the total carotenoid content in this mutant line was enhanced to 59.71 µg g⁻¹ (Fig.\u0026nbsp;4A; Table S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of several main agronomic traits between\u003c/strong\u003e \u003cstrong\u003eTaLcye\u003c/strong\u003e \u003cstrong\u003emutants and their wild-type\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the impact of \u003cem\u003eTaLcye\u003c/em\u003e mutations on the physiology and productivity of wheat plants, we evaluated some growth and yield related traits of mutant lines and their wild-type, which were cultivated in a semi-controlled greenhouse setting. Our findings revealed that, despite reductions in plant height, spike length, spikelet number per spike, and grains per spike in the mutant lines 1–4, 2–8, and 5 − 1 relative to the wild-type, these mutants exhibited significant increases in grain length, and the thousand-kernel weight had no significant difference between wild-type and the mutant lines (Fig.\u0026nbsp;5A-5D; Table\u0026nbsp;1). Consequently, the \u003cem\u003eTaLcye\u003c/em\u003e mutations did not exert a substantial detrimental effect on wheat yield. Furthermore, the seed coat and endosperm of the triple homozygous mutant lines 1–4 and 5 − 1 displayed an orange-yellow hue (Fig.\u0026nbsp;5E-5F).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eMain agronomic traits of wild-type and \u003cem\u003eTaLcye\u003c/em\u003e-edited plants\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLine\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTiller number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlant height\u003c/p\u003e\n \u003cp\u003e(cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpike length\u003c/p\u003e\n \u003cp\u003e(cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpikelet number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrains per\u003c/p\u003e\n \u003cp\u003espike\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrain length\u003c/p\u003e\n \u003cp\u003e(cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrain width\u003c/p\u003e\n \u003cp\u003e(cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTKW\u003c/p\u003e\n \u003cp\u003e(g)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFielder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.6 ± 1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e102.9 ± 1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.9 ± 0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.1 ± 0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56.0 ± 0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.75 ± 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.52 ± 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.48 ± 0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1–4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.7 ± 0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81.5 ± 0.45**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.2 ± 0.13**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.5 ± 0.43*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.9 ± 1.05*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.26 ± 0.07**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.29 ± 0.04**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.51 ± 0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2–8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.0 ± 0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e84.2 ± 0.20**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.6 ± 0.22*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.8 ± 0.45*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52.4 ± 1.00*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.16 ± 0.01**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.35 ± 0.03*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.52 ± 0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 − 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.9 ± 0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81.7 ± 0.28**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.4 ± 0.21**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.3 ± 0.41*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.3 ± 1.00*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.27 ± 0.07**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.34 ± 0.02*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.36 ± 1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003eComparison between the lines 1–4, 2–8, 5 − 1, and Fielder. “*” Represent the \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, and “**” represent the \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eComprehensive analysis of grain quality in wheat\u003c/strong\u003e \u003cstrong\u003eTaLcye\u003c/strong\u003e \u003cstrong\u003emutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo systematically evaluate the grain quality of \u003cem\u003eTaLcye\u003c/em\u003e-edited plants, we conducted a comprehensive analysis of several key parameters, including protein and wet gluten content, grain hardness, starch and amylose content, as well as soluble sugar content. The results revealed that the mutant lines (1–4, 2–8, and 5 − 1) exhibited no significant differences in protein, wet gluten content, and grain hardness compared to their wild-type counterparts. However, significant improvements were observed in the contents of soluble sugar and amylose in lines 1–4 and 5 − 1. Conversely, the total starch content was significantly reduced in these mutant lines (Table 2). Additionally, scanning electron microscopy (SEM) analysis indicated an increase in A-type starch granules in the mutant lines, which may be attributed to the genetic modifications introduced by the \u003cem\u003eTaLcye\u003c/em\u003e editing (Fig. 6).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAnalysis of the major quality traits of wild type and \u003cem\u003eTaLcye\u003c/em\u003e-edited grains\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLine\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStarch\u003c/p\u003e\n \u003cp\u003e(mg/g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmylose\u003c/p\u003e\n \u003cp\u003e(mg/g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSoluble sugar (mg/g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWet gluten\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHardness\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e549.312 ± 2.453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e138.736 ± 2.253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50.242 ± 0.222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.27 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.3 ± 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.7 ± 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1–4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e439.223 ± 1.727**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e180.69 ± 0.624**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e66.114 ± 0.726**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.5 ± 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.44 ± 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.24 ± 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2–8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e517.222 ± 2.025*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e148.452 ± 0.25*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e62.004 ± 0.796*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.63 ± 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.48 ± 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.01 ± 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 − 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e474.519 ± 1.556**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e178.137 ± 1.556**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65.61 ± 0.869**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.61 ± 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.44 ± 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.05 ± 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eComparison between the lines 1–4, 2–8, 5 − 1, and Fielder. “*” Represent the \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, and “**” represent the \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGiven the crucial role of β-carotene in human nutrition, significant efforts have been devoted to enhance its levels in staple crops. For instance, researchers successfully biofortified rice with improved β-carotene by reconstructing its entire biosynthetic pathway in the endosperm, called as \"Golden Rice\". This was achieved through the introduction of the several key genes from the carotenoid biosynthetic pathway, thereby significantly increasing the β-carotene content in rice grains (Amna et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ye et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In another study, the durum wheat cultivar Kronos was subjected to ethyl methanesulfonate (EMS) mutagenesis to create a \u003cem\u003eTaLcye\u003c/em\u003e-silenced mutant, in which the metabolic pathway responsible for the synthesis of α-carotene and lutein was effectively blocked, while the β-carotene content in grains was increased by 75% compared to the wild-type control (Sestili et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, knocking out the \u003cem\u003eLcye\u003c/em\u003e gene in lettuce led to a 2.7-fold increase in β-carotene levels (Livneh et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Wheat, a staple food source for a significant portion of the global population, is characterized by its low β-carotene content in the grains, which limits the bioavailability of essential retinol (vitamin A) (Shewry et al., 2009).\u003c/p\u003e\u003cp\u003eIn this study, we generated homozygous mutants of the \u003cem\u003eTaLcye\u003c/em\u003e gene by utilizing CRISPR-Cas9 gene editing system in wheat. Analysis using UPLC-MS/MS revealed that although the β-carotene content in the mutant leaves was reduced, it was increased in all the grains at 30 DPA of mutant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table S2-S3). Specifically, line 5\u0026thinsp;\u0026minus;\u0026thinsp;1 exhibited a 34.5% increase in β-carotene content compared to the wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Table S3). Additionally, line 2\u0026ndash;8 demonstrated a slight increase of total carotenoid content in the grains at 30 DPA (Table S3). Zeaxanthin, as a downstream metabolite of β-carotene, is highly concentrated in the macula of the eye's retina, where it functions as a shield against blue light and oxidative stress, thereby reducing the risk of age-related macular degeneration (AMD) (Sajilata et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In addition, antheraxanthin and violaxanthin was the metabolite of zeaxanthin, have been shown to possess specific anti-cancer properties and may contribute to slow the progression of atherosclerosis (Shimode et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In plants, antheraxanthin and violaxanthin participate in the xanthophyll cycle and protect the photosynthetic system from light-induced damage under high light conditions (Zhang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; D'Ambrosio et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our present results by UPLC-MS/MS analysis revealed significant increases in the levels of these carotenoids in the leaves of line 5\u0026thinsp;\u0026minus;\u0026thinsp;1, with zeaxanthin increasing 2.6-fold, antheraxanthin increasing 2.8-fold, and violaxanthin increasing 1.5-fold compared to the wild type (Fig. S6; Table S2). In the grains of line 1\u0026ndash;4, the levels of zeaxanthin, antheraxanthin, and violaxanthin were elevated to 2.1 \u0026micro;g g⁻\u0026sup1;, 13.6 \u0026micro;g g⁻\u0026sup1;, and 29.9 \u0026micro;g g⁻\u0026sup1;, in order (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Table S3).\u003c/p\u003e\u003cp\u003eLutein is a yellow carotenoid that typically imparts a pale-yellow hue to cereal grains. In the endosperm of mature tetraploid wheat grains, lutein is the predominant carotenoid component, contributing the seeds with a significantly yellow appearance (Howitt et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In contrast, carotenes, particularly β-carotene, generally impart an orange color to grains. For instance, the accumulation of β-carotene in grains results in an orange or orange-yellow phenotype (Kim et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In our current study, the seed coat and endosperm of wheat triple homozygous mutant lines 1\u0026ndash;4 and 5\u0026thinsp;\u0026minus;\u0026thinsp;1 exhibited an orange-yellow hue, likely due to the elevated levels of β-carotene in the grains (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eHomozygous \u003cem\u003eTaLcye\u003c/em\u003e mutants displayed reductions in plant height, spike length, spikelet number, and grains per spike. Interestingly, grain length was increased in the mutants, while thousand-kernel weight remained statistically unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eD; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that \u003cem\u003eTaLcye\u003c/em\u003e mutations did not adversely affect overall grain yield. Moreover, the mutant lines exhibited significantly higher levels of soluble sugars and amylose (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), traits that may enhance their utility in bread, pastry, and other food-processing applications. These findings underscore the potential of \u003cem\u003eTaLcye\u003c/em\u003e mutants for dual-purpose improvement of both nutritional quality and processing characteristics, warranting further investigation.\u003c/p\u003e\u003cp\u003eCarotenoids are the precursors of ABA biosynthesis (Zhou et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The 9'-cis-neoxanthin or 9'-cis-violaxanthin was cleaved by the enzyme 9-cis-epoxycarotenoid dioxygenase to produce xanthoxin, the committed precursor of ABA (Finkelstein et al., 2013) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The phytohormone abscisic acid (ABA) plays a central role in the induction and maintenance of seed dormancy. Seed dormancy is an adaptive trait that enables seeds to remain quiescent until conditions become favorable for germination and mainly determines the pre-harvest sprouting tolerance (Nee et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our results revealed a significant increase in violaxanthin content in the mutant lines 1\u0026ndash;4 and 5\u0026thinsp;\u0026minus;\u0026thinsp;1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; Fig. S6D), leading us to hypothesize that ABA levels may differ between the mutants and the wild type, potentially affecting seed dormancy. This possibility will be the focus of our future research.\u003c/p\u003e\u003cp\u003eThe advent of CRISPR-Cas9 gene editing system has revolutionized the potential for targeted genetic modifications in crop species (Livneh et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In wheat, this technology enables the precise knockout of the key genes like \u003cem\u003eTaLcye\u003c/em\u003e to enhance β-carotene content, a crucial provitamin A precursor, in the grains at 30 DPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Table S3). Importantly, our field experiments and quality analysis demonstrated that such genetic modifications do not significantly compromise main agronomic traits of the mutant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), thereby maintaining their overall performance and yield potential. These results indicated that the mutants with satisfactory agronomic performance can be seamlessly integrated into breeding programs, paving the way for the development of wheat cultivars with elevated β-carotene levels. Collectively, this study lays a robust foundation for leveraging gene editing technologies to achieve provitamin A biofortification in commercial wheat varieties, thereby providing a valuable reference for improving nutritional outcomes in staple crops.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuiyun Liu conceived the research and designed the experiments; Yajie Guo conducted most of the experiments; Yajie Guo and Mengtian Liu performed the vector construction, wheat transformation, and examination the mutants; Dan Wang and Mengyao Li investigated the agronomic traits; Yajie Guo and Huiyun Liu drafted and revised the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the Major Science and Technology Projects in Henan Province (231100110300), the Ministry of Science and Technology (China) of China (no. 2021YFF1000203), the Natural Science Foundation of Henan Province, China (no. 232300420189), the Tackling Key Problems in Science and Technology of Henan Province, China (no. 242102111117). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmad A, Ahmed Z (2019) Fortification in beverages, production and management of beverages, volume1: the science of beverages, pp, 85\u0026ndash;122\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmna QS, Tantray AY, Bashir SS, Zaid A, Wani SH (2020) Golden rice: genetic engineering, promises, present status and future prospects, in rice research for quality improvement: genomics and genetic engineering, pp, 581\u0026ndash;604\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnand R, Mohan L, Bharadvaja N (2022) Disease prevention and treatment using β-carotene: the ultimate provitamin A. Rev Bras Farmacogn 32:491\u0026ndash;501\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBai L, Kin EH, DellaPenna D, Brutnell TP (2009) Novel lycopene epsilon cyclase activities in maize revealed through perturbation of carotenoid biosynthesis. Plant J 59:588\u0026ndash;599\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarazo A, Macakova K, Matousova K, Krcmova LK, Protti M, Mladenka P (2021) Vitamin A update: forms, sources, kinetics, detection, function, deficiency, therapeutic use and toxicity. Nutrients 13:703\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCong L, Wang C, Chen L, Liu H, Yang G, He G (2009) Expression of phytoene synthase1 and carotene desaturase \u003cem\u003ecrtI\u003c/em\u003e genes result in an increase in the total carotenoids content in transgenic elite wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L). J Agric Food Chem 57:8652\u0026ndash;8660\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCunningham FX, Pogson B, Sun Z, McDonald KA, DellaPenna D, Gantt E (1996) Functional analysis of the beta and epsilon lycopene cyclase enzymes of \u003cem\u003eArabidopsis\u003c/em\u003e reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8:1613\u0026ndash;1626\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCunningham FX, Gantt E (1998) Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 49:557\u0026ndash;583\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD'Ambrosio C, Stiglianim AL, Rambla JL, Frusciante S, Diretto G, Enfissi EMA, Granell A, Fraser PD, Giorio G (2023) A xanthophyll-derived apocarotenoid regulates carotenogenesis in tomato chromoplasts. Plant Sci 328:111575\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDiretto G, Tavazza R, Welsch R, Pizzichini D, Mourgues F, Papacchioli V, Beyer P, Giuliano G (2006) Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biol 6:13\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFinkelstein R (2013) Abscisic acid synthesis and response. Arabidopsis Book 11:e0166\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGiuliano G (2017) Provitamin a biofortification of crop plants: a gold rush with many miners. Curr Opin Biotechnol 44:169\u0026ndash;180\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrune T, Lietz G, Palou A, Ross AC, Stahl W, Tang G, Thurnham D, Yin SA, Biesalski HK (2010) β-Carotene as an important vitamin a source for humans. J Nutr 140:2268\u0026ndash;2285\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHowitt C, Cavanagh C, Bowerman A, Cazzonelli C, Rampling L, Mimica J, Pogson B (2009) Alternative splicing, activation of cryptic exons and amino acid substitutions in carotenoid biosynthetic genes are associated with lutein accumulation in wheat endosperm. Funct Integr Genomics 9:363\u0026ndash;376\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaur N, Alok A, Shivani Kumar P, Kaur N, Awasthi P, Chaturvedi S, Pandey P, Pandey A, Pandey AK, Tiwari S (2020) CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit. Metab Eng 59:76\u0026ndash;86\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKe Q, Kang L, Kim HS, Xie T, Liu C, Ji CY, Kim SH, Park WS, Ahn MJ, Wang S, Li H, Deng X, Kwak SS (2019) Down-regulation of lycopene ɛ-cyclase expression in transgenic sweetpotato plants increases the carotenoid content and tolerance to abiotic stress. Plant Sci 281:52\u0026ndash;60\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim HK, Kim JY, Kim JH, Go JY, Jung YS, Lee HJ, Ahn MJ, Yu J, Bae S, Kim HS, Kwak SS, Kim MS, Cho YG, Jung YJ, Kang KK (2022) Biochemical characterization of orange-colored rice calli induced by target mutagenesis of \u003cem\u003eOsOr\u003c/em\u003e Gene. Plants (Basel) 12: 56\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKirpichenkova EV, Korolev AA, Onishchenko GG, Nikitenko EI, Lipatov DV, Kuz'min AG, Dyskin YA, Denisova EL, Fetisov RN (2018) Study of lutein and zeaxanthin content in the diet with the assessment of the relationship between the level of alimentary intake of non-vitamin carotenoids and the density of the macular region of the retina at a young age. Vopr Pitan 87:20\u0026ndash;26\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKusama Y, Inoue S, Jimbo H, Takaichi S, Sonoike K, Hihara Y, Nishiyama Y (2015) Zeaxanthin and echinenone protect the repair of photosystem II from Inhibition by singlet oxygen in synechocystis sp. PCC 6803. Plant Cell Physiol 56:906\u0026ndash;916\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLichtenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 50:47\u0026ndash;65\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu H, Wang K, Jia Z, Gong Q, Lin Z, Du L, Pei X, Ye X (2020) Efficient induction of haploid plants in wheat by editing of \u003cem\u003eTaMTL\u003c/em\u003e using an optimized Agrobacterium-mediated CRISPR system. J Exp Bot 71:1337\u0026ndash;1349\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2\u003csup\u003e\u0026ndash;∆∆CT\u003c/sup\u003e method. Methods 25:402\u0026ndash;408\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLivneh Y, Leor Librach E, Agmon D, Makov Bouaniche T, Tiwari V, Shor E, Yeselson Y, Masci T, Schaffer A, Charuvi D, Hirschberg J, Vainstein A (2025) Combined enhancement of ascorbic acid, β-carotene and zeaxanthin in gene-edited lettuce. Plant Biotechnol J 23:1954\u0026ndash;1967\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa L, Lin XM (2010) Effects of lutein and zeaxanthin on aspects of eye health. J Sci Food Agr 90:2\u0026ndash;12\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNee G, Xiang Y, Soppe WJ (2017) The release of dormancy, a wake-up call for seeds to germinate. Curr Opin Plant Biol 35:8\u0026ndash;14\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePogson BJ, Rissler HM (2000) Genetic manipulation of carotenoid biosynthesis and photoprotection. Philos T Roy Soc B 355:1395\u0026ndash;1403\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSajilata MG, Singhal RS, Kamat MY (2008) The carotenoid pigment zeaxanthin-a review. Proc. Comp. Rev. Food Sci. Food Safety 7: 29\u0026ndash;49\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSajovic J, Meglic A, Glavac D, Markelj S, Hawlina M, Fakin A (2022) The role of vitamin a in retinal diseases. Int J Mol Sci 23:1\u0026ndash;27\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSestili F, Garcia-Molina MD, Gambacorta G, Beleggia R, Botticella E, De Vita P, Savatin DV, Masci S, Lafiandra D (2019) Provitamin A biofortification of durum wheat through a TILLING approach. Int J Mol Sci 20:5703\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShewry PR (2009) Wheat. J Exp Bo 60:1537\u0026ndash;1553\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShimode S, Miyata K, Araki M, Shindo K (2018) Antioxidant activities of the antheraxanthin-related carotenoids, antheraxanthin, 9-cis-antheraxanthin, and mutatoxanthins. J Oleo Sci 67:977\u0026ndash;981\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54:733\u0026ndash;749\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrono D (2019) Carotenoids in cereal food crops: composition and retention throughout grain storage and food processing. Plants 8:551\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Loo Bouwman CA, Naber TH, Schaafsma G (2014) A review of vitamin A equivalency of β-carotene in various food matrices for human consumption. Br J Nutr 111:2153\u0026ndash;2166\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang K, Liu H, Du L, Ye X (2017) Generation of marker-free transgenic hexaploid wheat via an \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated co-transformation strategy in commercial Chinese wheat varieties. Plant Biotechnol J 15:614\u0026ndash;623\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYe X, Al-Babili S, Kl\u0026ouml;ti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin a (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303\u0026ndash;305\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu B, Lydiate DJ, Young LW, Sch\u0026auml;fer UA, Hannoufa A (2008) Enhancing the carotenoid content of brassica napus seeds by downregulating lycopene epsilon cyclase. Transgenic Res 17:573\u0026ndash;585\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu S, Tian L (2018) Breeding major cereal grains through the lens of nutrition sensitivity. Mol Plant 11:23\u0026ndash;30\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhai S, Xia X, He Z (2016) Carotenoids in staple cereals: metabolism, regulation, and genetic manipulation. Front Plant Sci 7:1197\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Z, Zhou D, Li S, Pan J, Liang J, Wu X, Wu XN, Krall L, Zhu G (2023) Multiomics analysis reveals the chemical and genetic bases of pigmented potato tuber. J Agric Food Chem 71:16402\u0026ndash;16416\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou H, Yang M, Zhao L, Zhu Z, Liu F, Sun H, Sun C, Tan L (2021) HIGH-TILLERING AND DWARF 12 modulates photosynthesis and plant architecture by affecting carotenoid biosynthesis in rice. J Exp Bot 72:1212\u0026ndash;1224\u003c/span\u003e\u003c/li\u003e\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":"wheat grain, carotenoids, provitamin A, β-carotene, biofortification","lastPublishedDoi":"10.21203/rs.3.rs-8056178/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8056178/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCommon wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) is a staple food crop for humans, yet it primarily accumulates the non-provitamin A carotenoid lutein and exhibits limited natural variation in provitamin A β-carotene among its various accessions. This characteristic necessitates the development of alternative strategies for provitamin A biofortification. To address this challenge, we targeted key control points in the carotenoid biosynthetic pathway using the CRISPR-Cas9 system in a wheat cultivar Fielder. Specifically, we knocked out the gene encoding lycopene ε-cyclase (LCYE), an enzyme that acts as a gatekeeper opposing the production of β-branch carotenoids. This genetic modification resulted in a significant increase in β-carotene levels in the endosperms at 30 DPA of triple homozygous transgene-free mutant lines revealed by biochemical profiling, an approximate 34.5% enhancement for β-carotene, 125.4% for zeaxanthin, 73.8% for violaxanthin, and 186.5% for antheraxanthin compared to the wild-type control. Despite the drastic reduction in lutein levels, the \u003cem\u003eTaLcye\u003c/em\u003e mutations did not significantly impair wheat yield and the mutant lines exhibited elevated levels of amylose and soluble sugar. Additionally, the seed coats and endosperms of the triple homozygous transgene-free mutant lines exhibited an orange-yellow hue. In conclusion, we have successfully developed novel carotenoids biofortified wheat lines through gene-editing approach. Our findings demonstrate the potential of gene editing to significantly enhance the nutritional profile of commercial wheat by increasing carotenoid content, thereby addressing micronutrient deficiencies in modern diets.\u003c/p\u003e","manuscriptTitle":"Gene editing-mediated enhancement of carotenoid compounds accumulation in common wheat grains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 08:41:04","doi":"10.21203/rs.3.rs-8056178/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":"04c19f8b-6ee3-41f6-bf3a-0c4228643161","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-17T17:33:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-28 08:41:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8056178","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8056178","identity":"rs-8056178","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}