Preferentially expressed endosperm genes reveal unique activities in wheat endosperm during grain filling

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The endosperm-specific expressed genes can precisely provide targets in the endosperm for improving wheat grain quality and nutrition using modern bioengineering technologies. However, the genes specifically expressed in developing endosperms remain largely unknown. Results In this study, 315 preferentially expressed endosperm genes (PEEGs) in the spring wheat cultivar, Chinese Spring, were screened using data obtained from an open bioinformatics database, which reveals a unique grain reserve deposition process and special signal transduction in a developing wheat endosperm. Furthermore, transcription and accumulation of storage proteins in the wheat cultivar, XC26 were evaluated. The results revealed that PEEG plays a critical role in storage protein fragment deposition and is a potential candidate for modifying grain quality and nutrition. Conclusion These results provide new insights into endosperm development and candidate genes and promoters for improving wheat grain quality through genetic engineering and plant breeding techniques. wheat endosperm storage protein developmental transcription Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Bread wheat ( Triticum aestivum L., 2n = 6 × = 42, AABBDD) is one of the essential cereals in the human diet. Wheat endosperm is a major organ for starch and protein accumulation [ 1 ], which determine wheat grain yield and end-use quality [ 2 ]. Endosperm development begins with double fertilization, which produces the embryo and endosperm. The embryo matures approximately 10 days post-anthesis (DPA), while the endosperm undergoes cell division, expansion, and differentiation to form the endosperm structure [ 3 ]. The endosperm then focuses on reserve deposition, also known as the filling stage, which begins approximately 10 DPA and peaks at approximately 20 DPA [ 4 ]. Subsequently, the deposition of storage reserves decreases and the grain starts to desiccate at approximately 30 DPA [ 2 ]. The wheat endosperm undergoes programmed cell death during desiccation, which marks the end of its development [ 5 ]. However, our understanding of the dynamic expression of associated genes and signaling pathways involved in the physiological functions at the different phases of endosperm development remains limited. The endosperm rapidly mobilizes storage reserves required by the embryo to support its germination [ 6 ]. As a triploid organ, the endosperm dies and serves as a temporary nutrient reserve [ 7 ]. Generally, cytokinins, auxins, and gibberellins regulate the development of the embryo and endosperm [ 8 ], while abscisic acid and ethylene promote senescence by regulating programmed cell death in wheat endosperm during grain filling [ 9 ]. However, whether a particular signal transduction system synchronizes the deposition of reserves and cell death during endosperm growth remains unknown. In human beings, starch and storage proteins deposited in the endosperm are the primary sources of energy, and have therefore become the subjects of interest among researchers. Most storage proteins are specifically expressed in the endosperm [ 10 ]. Protein content constitutes 20% of the wheat grain and provides essential nutrients for human beings, which is a key standard for modern wheat breeding program [ 11 ]. The composition of grain protein is a key factor influencing dough nutrition and baking quality of wheat flour [ 12 ]. Osborne [ 13 ] (1924) classified storage proteins into various groups based on their extraction and solubility in dilute alcohol (gliadins), dilute saline (globulins), water (albumins), and dilute acid or alkali (glutenins). Studying the expression of genes associated with storage proteins will provide a key reference for improving wheat grain quality and nutrients using genomic approaches because different storage proteins contribute differently to bread quality and human nutritional requirements [ 14 ]. In this study, we identified 315 genes that were preferentially expressed in wheat endosperm, which contains seed storage proteins, and is associated with defense response and signaling that promote the unique activity of grain filling in wheat endosperm. The results of this study could provide crucial information for improving wheat grain quality through genetic engineering. Methods Screening of preferentially expressed endosperm genes in wheat Transcriptional data of 269,428 genes from 850 wheat samples at different developmental stages and tissues were downloaded [ 15 ] ( https://urgi.versailles.inra.fr/download/iwgsc/IWGSC_RefSeq_Annotations/v1.1/iwgsc_refseqv1.1_rnaseq_mapping_2017July20.zip ) and represented as transcripts per million (TPM). Afterward, 68 wheat samples were selected for further analysis. The preferentially expressed endosperm genes were assigned based on the criterion of a TPM value of more than 10 for the endosperm samples and less than 5 for the other samples. The TPM difference multiple between endosperm and other samples was greater than 100-fold. Plant materials Wheat plants ( T. aestivum cultivar, Chinese Spring, CS) were grown in a greenhouse maintained at day and night temperatures of 24°C and 17°C, respectively, and a 14-h light/10-h dark cycle. The plants were labeled when they began flowering. Root samples were collected from wheat seedlings at the two-leaf stage, while the starchy endosperm, stem, and leaf samples were obtained from wheat seedlings at the grain filling stage (20 DPA), and immediately frozen in liquid nitrogen for RNA isolation (five biological replicates per tissue). For transcriptomic verification and analysis of storage protein deposition, seeds of the spring wheat variety, Xinchun 26 (XC26) were sown in the field at the Junhu Wheat Breeding Experimental Station, Changji, Xinjiang, China. Developing grains in the central spikelet were collected at 7, 14, and 21 DPA, and stored immediately in liquid nitrogen for subsequent RNA-sequencing on an Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA, USA) and storage protein quantification. Three biological replicates were used for each stage. Quantification of gene expression Raw reads were trimmed to remove sequencing adapters using Trimmomatic (v0.33) and reads with low-quality bases were culled. Clean reads were aligned to high and low confidence transcripts (IWGSC RefSeq v1.1 annotation) with the default parameters in Kallisto (v0.46.2). Transcript expression values (TPM) were summarized to gene expression levels using the Tximport package in R (v1.14.0) with the “lengthScaledTPM” option. Protein separation and quantification The grain samples of XC26 were oven-dried at 105°C for 30 min and then at 80°C to a constant mass for grain protein content (GPC) measurements. The continuous shaking extraction method [ 16 ] was used to determine the contents of each protein component (albumins, globulins, gliadins, and glutenins). Three biological replicates were used for the test. qRT-PCR analysis Total RNA was extracted from the starchy endosperm, roots, stems, and leaves of wheat using RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China). Poly(dT) cDNA was prepared from 1 mg of total RNA using PrimeScript II Reverse Transcriptase Kit (TaKaRa Bio, Kusatsu, Japan), according to the manufacturer’s instructions. qRT-PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus, Code No. RR820A; TaKaRa Bio, Kusatsu, Japan) with three technical replicates. The wheat actin gene was used as an internal control to determine the relative expression levels of preferentially expressed endosperm genes. The gene-specific primers for qRT-PCR are listed in Table S2 . Gene annotation and protein classification Gene annotations were obtained from our previous study [ 17 ] ( https://zenodo.org/record/2541477/files/Genes_transcripts_FPKM.zip ), and computed using eggNOG-mapper based on eggNOG (v5.0) orthology data. Proteins were classified based on the same section of the two annotations. Statistical analysis All statistical analyses were performed using R v3.6.1. The function “chisq.test” with the argument “correct = FALSE” was used for Pearson’s Chi-squared test. Gene expression profiles at different developmental stages of wheat grain were clustered using the R Bioconductor package “Mfuzz” [ 18 ]. Results Screening of preferentially expressed endosperm genes in wheat According to the results of transcriptomic analysis, 315 out of 269,428 genes were identified as preferentially expressed endosperm genes in wheat (Fig. 1 A and Table S1 ) based on their TPM during the grain filling stage (20 DPA). The 315 genes identified were further categorized into four classes (Fig. 1 B) based on their peak transcription during the early (2–10 DPA), middle (20 DPA), and late filling stages (30 DPA). Eighteen randomly selected genes were subjected to qRT-PCR and the results revealed that the genes were highly expressed in the wheat endosperm, but hardly detected in the roots, stems or leaves (Figs. 1 C and S1 ). The results indicate that the genes were preferentially expressed in the wheat endosperm. Composition of preferentially expressed endosperm genes The 315 genes showed increased transcript abundance, which peaked at 20 DPA, accounting for 35.3% of the total mRNA in the endosperm of CS wheat (Fig. 2 A). The high mRNA proportion and transcript abundance were also observed in the developing grain of the wheat variety, XC26 (Fig. 2 A). The results indicate a common transcriptional pattern of preferentially expressed endosperm genes in different wheat varieties. The identified genes were functionally annotated into eight main groups (Fig. 2 B), including storage proteins (37.3%), transcription genes (11.4%), defense genes (8.9%), redox-related genes (4.7%), proteinase inhibitor genes (4.4%) among others, which highlights the importance of storage protein deposition in developing endosperms. However, genes associated with starch metabolism accounted for only 1.3% of the genes, suggesting that storage protein deposition is a unique process in wheat endosperm, whereas starch synthesis may be a common biosynthesis process across wheat tissues, despite being a major activity associated with grain filling in the endosperm. A total of 180 storage genes and 91 defense-related genes in the wheat genome database (IWGSC v1.1) were identified by homologous comparison. Among them, 30% and 36% genes were not classified as preferentially expressed endosperm genes because they were simultaneously expressed in wheat grains and other tissues (Fig. 2 C), suggesting that manipulation of these genes may cause unexpected results beyond the endosperm. Transcription of defense genes involved in endosperm filling Plants have developed complex defense mechanisms to protect themselves against attack by pathogens and pests [ 19 ]. In this study, 28 defense genes, mostly defensin genes (Table S1 ) and their expression levels in CS remained relatively stable from 10 to 20 DPA (Fig. 3 A). However, the defense genes in XC26 exhibited increased transcript abundance (Fig. 3 B), indicating that the synthesis of defensins and defense capability vary among wheat varieties. Fifteen redox-regulating genes were identified as preferentially expressed endosperm genes, and their expression increased with endosperm development. Furthermore, 14 proteinase inhibitors, which are activated in plants in response to injury or attack by insects or pathogens [ 20 ], were identified in the wheat plants and their expression was upregulated during grain filling. The number and high transcript levels of redox-related genes and proteinase inhibitors defined a distinct feature of the developing endosperm. Special signaling and transcriptional factors involved in endosperm filling Plant growth and development require the integration of a variety of environmental and endogenous signals. Six receptors were identified to be associated with the preferentially expressed endosperm genes, including two unique hormone receptors, a phytoestrogen receptor (ER), and an insulin-like growth factor 1 receptor (IGF1R). The ER and IGF1R were first discovered in wheat (Table S1 ), and their expression levels increased sharply from early to middle grain filling stages, but significantly decreased thereafter in CS and XC26 wheat plants (Fig. 4 A). In addition, ER and IGF1R had transcript profiles similar to that of the PLATZ transcription factor (Fig. 4 B), which is known for its role in controlling grain length by promoting cell proliferation [ 21 ]. The results suggest that ER and IGF1R are involved in early endosperm development. Abscisic acid (ABA) is a major plant hormone that influences the process of seed maturation [ 8 ]. Two ABA receptors were identified as preferentially expressed endosperm genes. Notably, two glutamate receptors, which regulate ABA biosynthesis [ 22 ], were identified and their expression profiles were similar to those of ABA receptors in CS and XC26 wheat plants (Figs. 4 A and 4 B). Similarly, NAC transcription factors and ABA-responsive genes [ 23 ] exhibited similar expression profiles, suggesting that the endosperm has its own ABA signaling system to enhance its development. Ethylene response factors were also identified and their expression levels increased gradually with grain maturation (Figs. 4 A and 4 B), suggesting that ethylene is crucial during the late stage of wheat grain development. Storage proteins and genes expressed during grain filling The transcript profiles of storage proteins exhibited an increasing trend during the filling stage in CS and XC26 wheat plants (Fig. 5 A). Among the identified storage proteins, gliadins were the most abundant (57, Table S1 ), with peak transcript expression being observed at approximately 14–20 DPA in CS and XC26 wheat plants. Globulins also exhibited a similar trend (Fig. 5 B). The quantification of storage proteins in XC26 showed that both gliadin and globulin contents increased with the progression of endosperm development, suggesting that the two types of proteins constantly accumulated during endosperm development. In contrast, the expression of albumin and glutelin transcripts peaked at approximately 7–10 DPA, and decreased thereafter. The abundance of corresponding proteins in XC26 was maximum during the middle grain filling stage (approximately 14 DPA). These results suggest that storage proteins exhibited varying accumulation profiles during wheat grain filling. An evaluation of the transcription of storage protein genes in XC26 revealed similar transcript abundance and profiles (Table S2 ). The results revealed that several genes substantially contributed to the transcript abundance in CS and XC26 wheat plants (Fig. 5 C and Table S2 ) and exhibited typical storage protein gene profiles, which could be potential candidate genes and promoters of grain protein quality improvement in wheat or for the development of gene products through genetic engineering. Discussion In this study, 315 preferentially expressed endosperm genes were identified in wheat. Their composition and expression patterns revealed a unique activity in wheat endosperm at different developmental stages, which enhances our understanding of the wheat grain filling process and provides valuable insights for manipulating wheat grain products through genetic engineering. As a key component of wheat grain, the endosperm determines the final yield and nutritional value of the grain and it is the primary focus of wheat quality enhancement. With the rapid development of biotechnology, improving wheat grain quality using transgenic technology has become an important field. Some strong constitutive promoters, such as the cauliflower mosaic virus (CaMV) 35S and maize ubiquitin (PZmUbi) promoters, have been widely used in plant genetic engineering [ 24 ]. However, their activities frequently fail to meet the seed storage protein gene expression requirements [ 25 ] and nontargeted expression of foreign genes may interfere with plant growth and development. Thus, strong endosperm-specific promoters could be better candidates for biotechnological applications in cereals [ 26 ]. Plant endosperm-specific promoters are typically acquired from cereal storage protein genes. In this study, novel preferentially expressed endosperm genes other than the typical protein storage genes were characterized. The genes could serve as candidate promoters for the genetic engineering of cereals. The various transcription profiles of the screened genes could offer researchers more options for the selection of appropriate promoters to drive target genes during appropriate periods and quantities to manipulate wheat grain contents. Combined genome sequencing of different cultivars [ 27 ] could facilitate the identification of single nucleotide polymorphisms associated with endosperm-specific promoters and crucial information for manipulating the expression levels of the candidate genes at different developmental stages of wheat grain in the future. Moreover, preferentially expressed endosperm genes, particularly the storage protein genes, could be key candidates for current wheat grain development and mapping of quality quantitative trait loci. Several ABA receptors and regulatory genes were identified in this study. The preferential expression of these genes in the wheat endosperm indicates that the endosperm has a specific ABA signaling system that is divergent from other wheat tissues, which may provide more clues for the role of ABA in maintaining the balance between dehydration response and starch accumulation [ 28 ]. A significant outcome was the identification of two novel development-related hormone receptors, ER and IGF1R. Both receptors are closely associated with the development of human cells [ 29 – 30 ], but their roles in plant species remain unknown. Our results showed that both hormone receptors are predominantly expressed at approximately 10 DPA when the deposition of starch and storage proteins is initiated, which demonstrates its role during the early developmental stage of wheat endosperm. These findings indicate intact signaling in plants, which form a basis for further research on the regulation of wheat endosperm development to improve grain yield and quality. Conclusion A total of 315 preferentially expressed endosperm genes were identified in CS wheat. The analysis of the expression profiles of the genes provided a global perspective on the unique activity of wheat grain reserve deposition, and revealed key candidate genes and promoters for enhancing the nutritional quality of wheat grains through genetic engineering and plant breeding. Abbreviations DPA：Days post-anthesis TPM：Transcripts Per Kilobase of exon model per Million mapped reads qRT-PCR：Quantitative real-time PCR HMW-GSs：High molecular weight glutenin subunits LMW-GSs：Low molecular weight glutenin subunits Declarations Data availability The raw RNA-Seq data were deposited in the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession numbers PRJNA1021180. Authors’ contributions Laboratory procedures, data analysis and writing the first draft of the manuscript were done by JS, YQZ and PZ. HMY, CSW and JQX commented on previous versions of the manuscript. JS and PZ were involved in genome analysis. ZZ, ZLW and ZYY assisted in data analysis and visualization. YQZ performed RT-qPCR. SBX and YQZ conceptualized the experiment, supervised the project and finalized the manuscript. All authors read and approved the final manuscript. Funding This work was supported by Xinjiang Academy of Agricultural Sciences young science and technology backbone innovation ability training project (xjnkq-2022006); “Two Zones” Science and Technology Development Project (2023LQJ04); Silk Road Economic Belt Innovation-driven Development Pilot Zone, Wuchangshi National Independent Innovation Demonstration Zone Science and Technology Development Plan (2022LQ03017); Supported by the earmarked fund for XJARS (XJARS-01). Ethics approval and consent to participate The experimental studies conducted on plants were in compliance with the relevant institutional, national, and international guidelines and legislation. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Institute of Nuclear and Biological Technologies, Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Crop Biotechnology/Key Laboratory of Oasis-Desert Crop Physiology Ecology and Cultivation of Ministry of Agricultural and Rural Affairs/Crop Chemical Regulation Engineering Technology Research Center in Xinjiang, Urumqi 830091, China 2 State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling, Shaanxi 712100, China 3 Institute of Applied Microbiology, Xinjiang Academy of Agricultural Sciences/Xinjiang Laboratory of Special Environmental Microbiology, Urumqi, Xinjiang 830091, PR China References Tasleem-Tahir A, Nadaud I, Chambon C, Branlard G. Expression profiling of starchy endosperm metabolic proteins at 21 stages of wheat grain development. J Proteome Res. 2012;11:2754–73. 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Additional Declarations No competing interests reported. Supplementary Files FigS1.jpg Fig. S1. Detection of 14 candidate preferentially expressed endosperm genes by quantitative PCR. The x-axis represents different wheat plant parts and the y-axis represents the relative gene expression levels. Error bars represent standard deviations of five replicates (Chinese Spring, CS at 20 DPA). Abbreviation: DPA, days post-anthesis. TableS1.xls Additional files Table S1. Identification of preferentially expressed endosperm genes and their functional classification TableS2.xlsx Table S2. TPM of different storage protein genes in grains TableS3.xls Table S3. The gene-specific primers used for quantitative PCR Cite Share Download PDF Status: Published Journal Publication published 22 Aug, 2024 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 27 Feb, 2024 Reviews received at journal 19 Feb, 2024 Reviewers agreed at journal 02 Feb, 2024 Reviewers invited by journal 02 Feb, 2024 Editor assigned by journal 29 Jan, 2024 Editor invited by journal 29 Jan, 2024 Submission checks completed at journal 29 Jan, 2024 First submitted to journal 24 Jan, 2024 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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Crop Biotechnology/Key Laboratory of Oasis-Desert Crop Physiology Ecology and Cul","correspondingAuthor":false,"prefix":"","firstName":"Jianqiang","middleName":"","lastName":"Xia","suffix":""},{"id":269974726,"identity":"9308ada9-4dc0-4b06-93ec-4c18991d0c5e","order_by":6,"name":"Zhun Zhao","email":"","orcid":"","institution":"Institute of Nuclear and Biological Technologies, Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Crop Biotechnology/Key Laboratory of Oasis-Desert Crop Physiology Ecology and Cul","correspondingAuthor":false,"prefix":"","firstName":"Zhun","middleName":"","lastName":"Zhao","suffix":""},{"id":269974728,"identity":"f6ef22cc-ffe1-4fb6-a529-ca16b4b7b2eb","order_by":7,"name":"Zhenlong Wang","email":"","orcid":"","institution":"Institute of Nuclear and Biological Technologies, Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Crop Biotechnology/Key Laboratory of Oasis-Desert Crop Physiology Ecology and 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Cul","correspondingAuthor":false,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Wang","suffix":""},{"id":269974734,"identity":"79d6af32-dedb-4a57-b491-bdde9bd42d0f","order_by":10,"name":"Shengbao Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYHACNmaGCgjrAwMDM7FazoAZjDOI18LYRooWgxvpzx4XzruTOH9G8sMGhgrrxAb2swfwapGckWNuPHPbs8QNN9IMGxjOpCc28OQl4NXCL5HDJs277XDuBokE8weMbYcTGyR4DPB7RCL9mTTvnMO582ekf2xg/EeEFn6JBDNp3obDuQ03cgwbGBuI0CLZ88ZMesaxw/UbzrwpbEg4lm7cxpODX4vBcaDDCmoOG8u3p29s+FBjLdvPfga/FlSQAPIdCepHwSgYBaNgFOAAAMfJRrQi4ZbLAAAAAElFTkSuQmCC","orcid":"","institution":"State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Shengbao","middleName":"","lastName":"Xu","suffix":""},{"id":269974735,"identity":"e6eb07fa-6834-4e0c-bd22-8e962d09c670","order_by":11,"name":"Yueqiang Zhang","email":"","orcid":"","institution":"Institute of Nuclear and Biological Technologies, Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Crop Biotechnology/Key Laboratory of Oasis-Desert Crop Physiology Ecology and Cul","correspondingAuthor":false,"prefix":"","firstName":"Yueqiang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-01-25 03:29:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3895854/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3895854/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-024-10713-4","type":"published","date":"2024-08-22T15:57:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50407281,"identity":"98ff7af6-fce5-496d-9383-090a7f05341c","added_by":"auto","created_at":"2024-01-31 05:38:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2217879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of endosperm-specific genes in Chinese Spring (CS) wheat. \u003c/strong\u003e(A) Expression patterns of endosperm-specific genes. Each row indicates an organization. Abbreviations: DPA, days post-anthesis; WE, whole endosperm; SE, starchy endosperm. (B) Expression pattern classification of endosperm-specific genes. The x-axis represents days after flowering and the y-axis represents relative gene expression levels. Yellow or green lines indicate genes with low numbers, red and purple lines indicate genes with high numbers. (C) Detection of four genes with the highest expression level by quantitative real-time PCR. The x-axis represents different wheat plant parts and the y-axis represents the relative gene expression levels. Error bars represent standard deviations of five replicates (Chinese Spring, CS of 20 DPA).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/57070d3b18bce6467ffb55f2.jpg"},{"id":50407287,"identity":"be6f70fc-1a5a-463e-8375-7af683666477","added_by":"auto","created_at":"2024-01-31 05:38:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1683858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComposition and distribution of preferentially expressed endosperm genes in Chinese spring and Xinchun 26.\u003c/strong\u003e(A) The abundance ratios of 315 gene transcripts to total transcripts from Chinese pring and Xinchun 26 wheat grains. (B) The composition of preferentially expressed endosperm genes. Different parts of the pie chart represent various proteins encoded by endosperm-specific genes, including other genes that are divided into four categories. (C) Expression patterns of storage protein and defense genes in the wheat genome. Each row represents a wheat plant part. Abbreviations: EPSG, endosperm-preferred storage genes; NEPSG, non-endosperm-preferred storage genes; EPDG endosperm-preferred defense genes; NEPDG, non-endosperm-preferred defense genes.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/bc75c5efb5c75a84216e6fcb.jpg"},{"id":50407921,"identity":"bc912ec6-44c8-482d-bda9-affad910ff96","added_by":"auto","created_at":"2024-01-31 05:54:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":372158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscript abundance ratios of defense-related genes in Chinese Spring and Xinchun 26. \u003c/strong\u003e(A) Transcript abundance ratios of defense-related genes in Chinese Spring wheat. The x-axis represents the grain filling stage and the y-axis represents the ratio of each protein transcript to the total endosperm transcripts. Blue, orange, and red represent the transcript abundance of defense-related genes, redox-related genes, and proteinase inhibitors, respectively. (B) Transcript abundance ratios of defense-related genes in Xinchun 26 wheat. The x-axis represents the distinct stages of grain filling and the y-axis represents the ratio of each storage protein transcript to the total endosperm transcripts. Blue, orange, and red represent the transcript abundance of defense-related genes, redox-related genes, and proteinase inhibitors, respectively.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/afd38995af4246b2aaa0891c.jpg"},{"id":50407290,"identity":"5c9255a9-2933-4d07-ac85-2de6a975aafc","added_by":"auto","created_at":"2024-01-31 05:38:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1258772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of special signaling genes and transcription factors in Chinese Spring and Xinchun 26. \u003c/strong\u003e(A) The expression levels of special signaling genes during the developmental stages of Chinese Spring (left) and Xinchun 26 (right) endosperms. The x-axis represents the different stages of grain filling and the y-axis represents the expression levels of signaling genes. Blue, red, and green represent the expression levels of glutamate receptors, ABA receptors, and development-related hormone receptors, respectively. (B) Expression levels of transcription factors in Chinese Spring (left) and Xinchun 26 (right) wheat. The x-axis represents the different stages of grain filling and the y-axis represents the expression levels of transcription factors. Purple, green, and orange represent the expression levels of NAC TFs, ERFs, and PLATZ TFs, respectively. Abbreviations: ABA, abscisic acid; TFs, transcription factors; ERFs, estrogen receptors.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/46869d80a890d6e133811bcf.jpg"},{"id":50407567,"identity":"f7e74663-3859-4b5e-8e6a-8aad4981016f","added_by":"auto","created_at":"2024-01-31 05:46:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2107142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscript abundances and expression of storage proteins. \u003c/strong\u003e(A) Transcript abundance ratios of storage proteins in Chinese Spring and Xinchun 26, and storage protein content in Xinchun 26. (B) Transcript abundance ratios of four types of storage proteins (albumin, globulin, gliadin, and glutenin) in Chinese Spring and Xinchun 26, and the storage protein content of Xinchun 26. (C) A heatmap representing the expression levels (in transcripts per million, TPM) in samples obtained at different grain filling stages. Early, early grain filling stage; expression of storage proteins in Chinese Spring at 2 DPA (left) and in Xinchun 26 at 7 DPA (right). Medium, middle grain filling stage; the average expression of storage proteins in Chinese Spring at 10 DPA and 20 DPA (left); the average expression of storage proteins in Xinchun 26 at 7 DPA and 14 DPA (right). Late, late grain filling stage; expression of storage proteins in Chinese Spring at 30 DPA. Abbreviations: DPA, days post-anthesis.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/6dd52f34ab0047ca55afeb36.jpg"},{"id":63300149,"identity":"f8ee7f50-14b6-4994-9973-470bdf05fcb7","added_by":"auto","created_at":"2024-08-26 16:11:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8249298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/f9c78351-1f76-42f7-8e01-bf0c417269fa.pdf"},{"id":50407284,"identity":"59502b7b-6fe9-46ea-9bbc-197891abeb75","added_by":"auto","created_at":"2024-01-31 05:38:03","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":947566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1.\u003c/strong\u003e Detection of 14 candidate preferentially expressed endosperm genes by quantitative PCR. The x-axis represents different wheat plant parts and the y-axis represents the relative gene expression levels. Error bars represent standard deviations of five replicates (Chinese Spring, CS at 20 DPA). Abbreviation: DPA, days post-anthesis.\u003c/p\u003e","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/5c85c0eb5912b17541ed3ff3.jpg"},{"id":50407282,"identity":"be4ab367-16e6-4ee2-95e0-810bd1bc7520","added_by":"auto","created_at":"2024-01-31 05:38:03","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":280064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional files\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1. \u003c/strong\u003eIdentification of preferentially expressed endosperm genes and their functional classification\u003c/p\u003e","description":"","filename":"TableS1.xls","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/25f8d0d89a581cd6d59d3868.xls"},{"id":50407286,"identity":"56cfc72e-2d7d-4fcb-b5f4-d692d574b8a4","added_by":"auto","created_at":"2024-01-31 05:38:03","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2.\u003c/strong\u003e TPM of different storage protein genes in grains\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/740264f4edca08425662766a.xlsx"},{"id":50407288,"identity":"e9ca4861-b6ba-4463-b22e-28ece5634bb4","added_by":"auto","created_at":"2024-01-31 05:38:03","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":33792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3. \u003c/strong\u003eThe gene-specific primers used for quantitative PCR\u003c/p\u003e","description":"","filename":"TableS3.xls","url":"https://assets-eu.researchsquare.com/files/rs-3895854/v1/459362411e7a7b5c2aae1d2b.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preferentially expressed endosperm genes reveal unique activities in wheat endosperm during grain filling","fulltext":[{"header":"Background","content":"\u003cp\u003eBread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L., 2n\u0026thinsp;=\u0026thinsp;6 \u0026times; = 42, AABBDD) is one of the essential cereals in the human diet. Wheat endosperm is a major organ for starch and protein accumulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which determine wheat grain yield and end-use quality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEndosperm development begins with double fertilization, which produces the embryo and endosperm. The embryo matures approximately 10 days post-anthesis (DPA), while the endosperm undergoes cell division, expansion, and differentiation to form the endosperm structure [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The endosperm then focuses on reserve deposition, also known as the filling stage, which begins approximately 10 DPA and peaks at approximately 20 DPA [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Subsequently, the deposition of storage reserves decreases and the grain starts to desiccate at approximately 30 DPA [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The wheat endosperm undergoes programmed cell death during desiccation, which marks the end of its development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, our understanding of the dynamic expression of associated genes and signaling pathways involved in the physiological functions at the different phases of endosperm development remains limited.\u003c/p\u003e \u003cp\u003eThe endosperm rapidly mobilizes storage reserves required by the embryo to support its germination [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As a triploid organ, the endosperm dies and serves as a temporary nutrient reserve [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Generally, cytokinins, auxins, and gibberellins regulate the development of the embryo and endosperm [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], while abscisic acid and ethylene promote senescence by regulating programmed cell death in wheat endosperm during grain filling [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, whether a particular signal transduction system synchronizes the deposition of reserves and cell death during endosperm growth remains unknown.\u003c/p\u003e \u003cp\u003eIn human beings, starch and storage proteins deposited in the endosperm are the primary sources of energy, and have therefore become the subjects of interest among researchers. Most storage proteins are specifically expressed in the endosperm [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Protein content constitutes 20% of the wheat grain and provides essential nutrients for human beings, which is a key standard for modern wheat breeding program [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The composition of grain protein is a key factor influencing dough nutrition and baking quality of wheat flour [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOsborne [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] (1924) classified storage proteins into various groups based on their extraction and solubility in dilute alcohol (gliadins), dilute saline (globulins), water (albumins), and dilute acid or alkali (glutenins). Studying the expression of genes associated with storage proteins will provide a key reference for improving wheat grain quality and nutrients using genomic approaches because different storage proteins contribute differently to bread quality and human nutritional requirements [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we identified 315 genes that were preferentially expressed in wheat endosperm, which contains seed storage proteins, and is associated with defense response and signaling that promote the unique activity of grain filling in wheat endosperm. The results of this study could provide crucial information for improving wheat grain quality through genetic engineering.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eScreening of preferentially expressed endosperm genes in wheat\u003c/h2\u003e \u003cp\u003eTranscriptional data of 269,428 genes from 850 wheat samples at different developmental stages and tissues were downloaded [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://urgi.versailles.inra.fr/download/iwgsc/IWGSC_RefSeq_Annotations/v1.1/iwgsc_refseqv1.1_rnaseq_mapping_2017July20.zip\u003c/span\u003e\u003cspan address=\"https://urgi.versailles.inra.fr/download/iwgsc/IWGSC_RefSeq_Annotations/v1.1/iwgsc_refseqv1.1_rnaseq_mapping_2017July20.zip\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and represented as transcripts per million (TPM). Afterward, 68 wheat samples were selected for further analysis. The preferentially expressed endosperm genes were assigned based on the criterion of a TPM value of more than 10 for the endosperm samples and less than 5 for the other samples. The TPM difference multiple between endosperm and other samples was greater than 100-fold.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eWheat plants (\u003cem\u003eT. aestivum\u003c/em\u003e cultivar, Chinese Spring, CS) were grown in a greenhouse maintained at day and night temperatures of 24\u0026deg;C and 17\u0026deg;C, respectively, and a 14-h light/10-h dark cycle. The plants were labeled when they began flowering. Root samples were collected from wheat seedlings at the two-leaf stage, while the starchy endosperm, stem, and leaf samples were obtained from wheat seedlings at the grain filling stage (20 DPA), and immediately frozen in liquid nitrogen for RNA isolation (five biological replicates per tissue).\u003c/p\u003e \u003cp\u003eFor transcriptomic verification and analysis of storage protein deposition, seeds of the spring wheat variety, Xinchun 26 (XC26) were sown in the field at the Junhu Wheat Breeding Experimental Station, Changji, Xinjiang, China. Developing grains in the central spikelet were collected at 7, 14, and 21 DPA, and stored immediately in liquid nitrogen for subsequent RNA-sequencing on an Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA, USA) and storage protein quantification. Three biological replicates were used for each stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of gene expression\u003c/h2\u003e \u003cp\u003eRaw reads were trimmed to remove sequencing adapters using Trimmomatic (v0.33) and reads with low-quality bases were culled. Clean reads were aligned to high and low confidence transcripts (IWGSC RefSeq v1.1 annotation) with the default parameters in Kallisto (v0.46.2). Transcript expression values (TPM) were summarized to gene expression levels using the Tximport package in R (v1.14.0) with the \u0026ldquo;lengthScaledTPM\u0026rdquo; option.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eProtein separation and quantification\u003c/h2\u003e \u003cp\u003eThe grain samples of XC26 were oven-dried at 105\u0026deg;C for 30 min and then at 80\u0026deg;C to a constant mass for grain protein content (GPC) measurements. The continuous shaking extraction method [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] was used to determine the contents of each protein component (albumins, globulins, gliadins, and glutenins). Three biological replicates were used for the test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the starchy endosperm, roots, stems, and leaves of wheat using RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China). Poly(dT) cDNA was prepared from 1 mg of total RNA using PrimeScript II Reverse Transcriptase Kit (TaKaRa Bio, Kusatsu, Japan), according to the manufacturer\u0026rsquo;s instructions. qRT-PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus, Code No. RR820A; TaKaRa Bio, Kusatsu, Japan) with three technical replicates. The wheat actin gene was used as an internal control to determine the relative expression levels of preferentially expressed endosperm genes. The gene-specific primers for qRT-PCR are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGene annotation and protein classification\u003c/h2\u003e \u003cp\u003eGene annotations were obtained from our previous study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zenodo.org/record/2541477/files/Genes_transcripts_FPKM.zip\u003c/span\u003e\u003cspan address=\"https://zenodo.org/record/2541477/files/Genes_transcripts_FPKM.zip\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and computed using eggNOG-mapper based on eggNOG (v5.0) orthology data. Proteins were classified based on the same section of the two annotations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using R v3.6.1. The function \u0026ldquo;chisq.test\u0026rdquo; with the argument \u0026ldquo;correct\u0026thinsp;=\u0026thinsp;FALSE\u0026rdquo; was used for Pearson\u0026rsquo;s Chi-squared test. Gene expression profiles at different developmental stages of wheat grain were clustered using the R Bioconductor package \u0026ldquo;Mfuzz\u0026rdquo; [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eScreening of preferentially expressed endosperm genes in wheat\u003c/h2\u003e \u003cp\u003eAccording to the results of transcriptomic analysis, 315 out of 269,428 genes were identified as preferentially expressed endosperm genes in wheat (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) based on their TPM during the grain filling stage (20 DPA). The 315 genes identified were further categorized into four classes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) based on their peak transcription during the early (2\u0026ndash;10 DPA), middle (20 DPA), and late filling stages (30 DPA).\u003c/p\u003e \u003cp\u003eEighteen randomly selected genes were subjected to qRT-PCR and the results revealed that the genes were highly expressed in the wheat endosperm, but hardly detected in the roots, stems or leaves (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The results indicate that the genes were preferentially expressed in the wheat endosperm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComposition of preferentially expressed endosperm genes\u003c/h2\u003e \u003cp\u003eThe 315 genes showed increased transcript abundance, which peaked at 20 DPA, accounting for 35.3% of the total mRNA in the endosperm of CS wheat (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The high mRNA proportion and transcript abundance were also observed in the developing grain of the wheat variety, XC26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The results indicate a common transcriptional pattern of preferentially expressed endosperm genes in different wheat varieties.\u003c/p\u003e \u003cp\u003eThe identified genes were functionally annotated into eight main groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), including storage proteins (37.3%), transcription genes (11.4%), defense genes (8.9%), redox-related genes (4.7%), proteinase inhibitor genes (4.4%) among others, which highlights the importance of storage protein deposition in developing endosperms. However, genes associated with starch metabolism accounted for only 1.3% of the genes, suggesting that storage protein deposition is a unique process in wheat endosperm, whereas starch synthesis may be a common biosynthesis process across wheat tissues, despite being a major activity associated with grain filling in the endosperm.\u003c/p\u003e \u003cp\u003eA total of 180 storage genes and 91 defense-related genes in the wheat genome database (IWGSC v1.1) were identified by homologous comparison. Among them, 30% and 36% genes were not classified as preferentially expressed endosperm genes because they were simultaneously expressed in wheat grains and other tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting that manipulation of these genes may cause unexpected results beyond the endosperm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTranscription of defense genes involved in endosperm filling\u003c/h2\u003e \u003cp\u003ePlants have developed complex defense mechanisms to protect themselves against attack by pathogens and pests [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this study, 28 defense genes, mostly defensin genes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and their expression levels in CS remained relatively stable from 10 to 20 DPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, the defense genes in XC26 exhibited increased transcript abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating that the synthesis of defensins and defense capability vary among wheat varieties.\u003c/p\u003e \u003cp\u003eFifteen redox-regulating genes were identified as preferentially expressed endosperm genes, and their expression increased with endosperm development. Furthermore, 14 proteinase inhibitors, which are activated in plants in response to injury or attack by insects or pathogens [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], were identified in the wheat plants and their expression was upregulated during grain filling. The number and high transcript levels of redox-related genes and proteinase inhibitors defined a distinct feature of the developing endosperm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSpecial signaling and transcriptional factors involved in endosperm filling\u003c/h2\u003e \u003cp\u003ePlant growth and development require the integration of a variety of environmental and endogenous signals. Six receptors were identified to be associated with the preferentially expressed endosperm genes, including two unique hormone receptors, a phytoestrogen receptor (ER), and an insulin-like growth factor 1 receptor (IGF1R). The ER and IGF1R were first discovered in wheat (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and their expression levels increased sharply from early to middle grain filling stages, but significantly decreased thereafter in CS and XC26 wheat plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, ER and IGF1R had transcript profiles similar to that of the PLATZ transcription factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which is known for its role in controlling grain length by promoting cell proliferation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The results suggest that ER and IGF1R are involved in early endosperm development.\u003c/p\u003e \u003cp\u003eAbscisic acid (ABA) is a major plant hormone that influences the process of seed maturation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Two ABA receptors were identified as preferentially expressed endosperm genes. Notably, two glutamate receptors, which regulate ABA biosynthesis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], were identified and their expression profiles were similar to those of ABA receptors in CS and XC26 wheat plants (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Similarly, NAC transcription factors and ABA-responsive genes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] exhibited similar expression profiles, suggesting that the endosperm has its own ABA signaling system to enhance its development. Ethylene response factors were also identified and their expression levels increased gradually with grain maturation (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting that ethylene is crucial during the late stage of wheat grain development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStorage proteins and genes expressed during grain filling\u003c/h2\u003e \u003cp\u003eThe transcript profiles of storage proteins exhibited an increasing trend during the filling stage in CS and XC26 wheat plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Among the identified storage proteins, gliadins were the most abundant (57, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), with peak transcript expression being observed at approximately 14\u0026ndash;20 DPA in CS and XC26 wheat plants. Globulins also exhibited a similar trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The quantification of storage proteins in XC26 showed that both gliadin and globulin contents increased with the progression of endosperm development, suggesting that the two types of proteins constantly accumulated during endosperm development. In contrast, the expression of albumin and glutelin transcripts peaked at approximately 7\u0026ndash;10 DPA, and decreased thereafter. The abundance of corresponding proteins in XC26 was maximum during the middle grain filling stage (approximately 14 DPA). These results suggest that storage proteins exhibited varying accumulation profiles during wheat grain filling.\u003c/p\u003e \u003cp\u003eAn evaluation of the transcription of storage protein genes in XC26 revealed similar transcript abundance and profiles (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The results revealed that several genes substantially contributed to the transcript abundance in CS and XC26 wheat plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) and exhibited typical storage protein gene profiles, which could be potential candidate genes and promoters of grain protein quality improvement in wheat or for the development of gene products through genetic engineering.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, 315 preferentially expressed endosperm genes were identified in wheat. Their composition and expression patterns revealed a unique activity in wheat endosperm at different developmental stages, which enhances our understanding of the wheat grain filling process and provides valuable insights for manipulating wheat grain products through genetic engineering.\u003c/p\u003e \u003cp\u003eAs a key component of wheat grain, the endosperm determines the final yield and nutritional value of the grain and it is the primary focus of wheat quality enhancement. With the rapid development of biotechnology, improving wheat grain quality using transgenic technology has become an important field.\u003c/p\u003e \u003cp\u003eSome strong constitutive promoters, such as the cauliflower mosaic virus (CaMV) 35S and maize ubiquitin (PZmUbi) promoters, have been widely used in plant genetic engineering [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, their activities frequently fail to meet the seed storage protein gene expression requirements [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and nontargeted expression of foreign genes may interfere with plant growth and development. Thus, strong endosperm-specific promoters could be better candidates for biotechnological applications in cereals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Plant endosperm-specific promoters are typically acquired from cereal storage protein genes. In this study, novel preferentially expressed endosperm genes other than the typical protein storage genes were characterized. The genes could serve as candidate promoters for the genetic engineering of cereals. The various transcription profiles of the screened genes could offer researchers more options for the selection of appropriate promoters to drive target genes during appropriate periods and quantities to manipulate wheat grain contents.\u003c/p\u003e \u003cp\u003eCombined genome sequencing of different cultivars [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] could facilitate the identification of single nucleotide polymorphisms associated with endosperm-specific promoters and crucial information for manipulating the expression levels of the candidate genes at different developmental stages of wheat grain in the future. Moreover, preferentially expressed endosperm genes, particularly the storage protein genes, could be key candidates for current wheat grain development and mapping of quality quantitative trait loci.\u003c/p\u003e \u003cp\u003eSeveral ABA receptors and regulatory genes were identified in this study. The preferential expression of these genes in the wheat endosperm indicates that the endosperm has a specific ABA signaling system that is divergent from other wheat tissues, which may provide more clues for the role of ABA in maintaining the balance between dehydration response and starch accumulation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A significant outcome was the identification of two novel development-related hormone receptors, ER and IGF1R. Both receptors are closely associated with the development of human cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], but their roles in plant species remain unknown. Our results showed that both hormone receptors are predominantly expressed at approximately 10 DPA when the deposition of starch and storage proteins is initiated, which demonstrates its role during the early developmental stage of wheat endosperm. These findings indicate intact signaling in plants, which form a basis for further research on the regulation of wheat endosperm development to improve grain yield and quality.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA total of 315 preferentially expressed endosperm genes were identified in CS wheat. The analysis of the expression profiles of the genes provided a global perspective on the unique activity of wheat grain reserve deposition, and revealed key candidate genes and promoters for enhancing the nutritional quality of wheat grains through genetic engineering and plant breeding.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDPA：Days post-anthesis\u003c/p\u003e\n\u003cp\u003eTPM：Transcripts Per Kilobase of exon model per Million mapped reads\u003c/p\u003e\n\u003cp\u003eqRT-PCR：Quantitative real-time PCR\u003c/p\u003e\n\u003cp\u003eHMW-GSs：High molecular weight glutenin subunits\u003c/p\u003e\n\u003cp\u003eLMW-GSs：Low molecular weight glutenin subunits\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw RNA-Seq data were deposited in the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession numbers PRJNA1021180.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLaboratory procedures, data analysis and writing the first draft of the manuscript were done by JS, YQZ and PZ. HMY, CSW and JQX commented on previous versions of the manuscript. JS and PZ were involved in genome analysis. ZZ, ZLW and ZYY assisted in data analysis and visualization. YQZ performed RT-qPCR. SBX and YQZ conceptualized the experiment, supervised the project and finalized the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Xinjiang Academy of Agricultural Sciences young science and technology backbone innovation ability training project (xjnkq-2022006); \u0026ldquo;Two Zones\u0026rdquo; Science and Technology Development Project (2023LQJ04); Silk Road Economic Belt Innovation-driven Development Pilot Zone, Wuchangshi National Independent Innovation Demonstration Zone Science and Technology Development Plan (2022LQ03017); Supported by the earmarked fund for XJARS (XJARS-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental studies conducted on plants were in compliance with the relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Institute of Nuclear and Biological Technologies, Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Crop Biotechnology/Key Laboratory of Oasis-Desert Crop Physiology Ecology and Cultivation of Ministry of Agricultural and Rural Affairs/Crop Chemical Regulation Engineering Technology Research Center in Xinjiang, Urumqi 830091, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A\u0026amp;F University, Yangling, Shaanxi 712100, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Institute of Applied Microbiology, Xinjiang Academy of Agricultural Sciences/Xinjiang Laboratory of Special Environmental Microbiology, Urumqi, Xinjiang 830091, PR China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTasleem-Tahir A, Nadaud I, Chambon C, Branlard G. 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ABA biosynthesis and degradation contributing to ABA homeostasis during barley seed development under control and terminal drought-stress conditions. J Exp Bot. 2011;62:2615\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan J, Zhou W, Hafner M, Blake RA, Chalouni C, Chen IP, et al. Therapeutic Ligands Antagonize Estrogen Receptor Function by Impairing Its Mobility. Cell. 2019;178:949\u0026ndash;963e18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Y, Kong GK-W, Menting JG, Margetts MB, Delaine CA, Jenkin LM, et al. How ligand binds to the type 1 insulin-like growth factor receptor. Nat Commun. 2018;9:821.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"wheat, endosperm, storage protein, developmental transcription","lastPublishedDoi":"10.21203/rs.3.rs-3895854/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3895854/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) endosperm contains starch and proteins, which determine the final yield, quality, and nutritional value of wheat grain. The endosperm-specific expressed genes can precisely provide targets in the endosperm for improving wheat grain quality and nutrition using modern bioengineering technologies. However, the genes specifically expressed in developing endosperms remain largely unknown.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, 315 preferentially expressed endosperm genes (PEEGs) in the spring wheat cultivar, Chinese Spring, were screened using data obtained from an open bioinformatics database, which reveals a unique grain reserve deposition process and special signal transduction in a developing wheat endosperm. Furthermore, transcription and accumulation of storage proteins in the wheat cultivar, XC26 were evaluated. The results revealed that PEEG plays a critical role in storage protein fragment deposition and is a potential candidate for modifying grain quality and nutrition.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese results provide new insights into endosperm development and candidate genes and promoters for improving wheat grain quality through genetic engineering and plant breeding techniques.\u003c/p\u003e","manuscriptTitle":"Preferentially expressed endosperm genes reveal unique activities in wheat endosperm during grain filling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-31 05:37:58","doi":"10.21203/rs.3.rs-3895854/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-27T10:23:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-20T00:26:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11e38a4b-a3e4-4a50-ae43-319c45db5046","date":"2024-02-02T16:43:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-02T08:29:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-29T09:55:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-29T05:50:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-29T05:49:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-01-25T03:25:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be4500bf-09a9-4365-85a6-940e97196b2d","owner":[],"postedDate":"January 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:00:55+00:00","versionOfRecord":{"articleIdentity":"rs-3895854","link":"https://doi.org/10.1186/s12864-024-10713-4","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2024-08-22 15:57:13","publishedOnDateReadable":"August 22nd, 2024"},"versionCreatedAt":"2024-01-31 05:37:58","video":"","vorDoi":"10.1186/s12864-024-10713-4","vorDoiUrl":"https://doi.org/10.1186/s12864-024-10713-4","workflowStages":[]},"version":"v1","identity":"rs-3895854","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3895854","identity":"rs-3895854","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}