Genome variation and LTR-RT analyses of an ancient peach landrace reveal mechanism of blood-flesh fruit color formation and fruit maturity date advancement.

Peach (Prunus persica) landrace has typical regional characteristics, strong environmental adaptability, and contains many valuable genes that provide the foundation for breeding excellent varieties. Therefore, it is necessary to assemble the genomes of specific landraces to facilitate the localization and utilization of these genes. Here, we de novo assembled a high-quality genome from an ancient blood-fleshed Chinese landrace Tianjin ShuiMi (TJSM) that originated from the China North Plain. The assembled genome size was 243.5 Mb with a contig N50 of 23.7 Mb and a scaffold N50 of 28.6 Mb. Compared with the reported peach genomes, our assembled TJSM genome had the largest number of specific structural variants (SVs) and long terminal repeat-retrotransposons (LTR-RTs). Among the LTR-RTs with the potential to regulate their host genes, we identified a 6688 bp LTR-RT (named it blood TE) in the promoter of NAC transcription factor-encoding PpBL, a gene regulating peach blood-flesh formation. The blood TE was not only co-separated with the blood-flesh phenotype but also associated with fruit maturity date advancement and different intensities of blood-flesh color formation. Our findings provide new insights into the mechanism underlying the development of the blood-flesh color and determination of fruit maturity date and highlight the potential of the TJSM genome to mine more variations related to agronomic traits in peach fruit.

Discovery of a key gene associated with fruit maturity date and analysis of its regulatory pathway in peach.

Fruit maturity is an important agronomic trait of fruit crops. Although in previous studies, several molecular markers are developed for the trait, the knowledge about its candidate genes is particularly limited. In this study, a total of 357 peach accessions were re-sequenced to obtain 949,638 SNPs. Combing with 3-year fruit maturity dates, a genome-wide association analysis was performed, and 5, 8, and 9 association loci were identified. To screen the candidate genes for those year-stable loci on chromosomes 4 and 5, two maturity date mutants were used for transcriptome sequencing. Gene expression analysis indicated that Prupe.4G186800 and Prupe.4G187100 on chromosome 4 were essential to fruit ripening in peaches. However, the expression analysis of different tissues showed that the first gene has no tissue-specific character, but transgenic studies showed that the latter is more likely to be a key candidate gene than the first for the maturity date in peach. The yeast two-hybrid assay showed that the proteins encoded by the two genes interacted and then regulated fruit ripening. Moreover, the previously identified 9 bp insertion in Prupe.4G186800 may affect their interaction ability. This research is of great significance for understanding the molecular mechanism of peach fruit ripening and developing practical molecular markers in a breeding program.

Betulin, Synthesized by PpCYP716A1, Is a Key Endogenous Defensive Metabolite of Peach against Aphids.

Wild pest-resistant germplasms employ secondary metabolites to withstand insect attacks. A close wild relative of the cultivated peach, Prunus davidiana, displays strong resistance to green peach aphids by utilizing metabolites to cope with aphid infestation; however, the underlying mechanism of aphid resistance remains mostly unknown. Here, metabolomic analysis was performed to explore the changes in metabolite levels in P. davidiana after aphid infestation. The data revealed that betulin is a key defensive metabolite in peaches that protects against aphids and possesses potent aphidicidal activity. Further toxicity tests demonstrated that betulin was toxic to pests but not to beneficial insects. Additionally, transcriptomic and phylogenetic analyses revealed that the cytochrome P450 gene PpCYP716A1 was responsible for betulin synthesis─this finding was confirmed by the heterologous expression of this gene. This study revealed a strategy whereby plants harness defense metabolites to develop resistance to pests. These findings may facilitate controlling such pests.

Combined nature and human selections reshaped peach fruit metabolome.

BACKGROUND: Plant metabolites reshaped by nature and human beings are crucial for both their lives and human health. However, which metabolites respond most strongly to selection pressure at different evolutionary stages and what roles they undertake on perennial fruit crops such as peach remain unclear. RESULTS: Here, we report 18,052 significant locus-trait associations, 12,691 expression-metabolite correlations, and 294,676 expression quantitative trait loci (eQTLs) for peach. Our results indicate that amino acids accumulated in landraces may be involved in the environmental adaptation of peaches by responding to low temperature and drought. Moreover, the contents of flavonoids, the major nutrients in fruits, have kept decreasing accompanied by the reduced bitter flavor during both domestication and improvement stages. However, citric acid, under the selection of breeders' and consumers' preference for flavor, shows significantly different levels between eastern and western varieties. This correlates with differences in activity against cancer cells in vitro in fruit from these two regions. Based on the identified key genes regulating flavonoid and acid contents, we propose that more precise and targeted breeding technologies should be designed to improve peach varieties with rich functional contents because of the linkage of genes related to bitterness and acid taste, antioxidant and potential anti-cancer activity that are all located at the top of chromosome 5. CONCLUSIONS: This study provides powerful data for future improvement of peach flavor, nutrition, and resistance in future and expands our understanding of the effects of natural and artificial selection on metabolites.

Multi-omics approaches identify a key gene, PpTST1, for organic acid accumulation in peach.

Organic acid content in fruit is an important determinant of peach organoleptic quality, which undergoes considerable variations during development and maturation. However, its molecular mechanism remains largely unclear. In this study, an integrative approach of genome-wide association studies and comparative transcriptome analysis were applied to identify candidate genes involved in organic acid accumulation in peach. A key gene PpTST1, encoding tonoplast sugar transporter, was identified and the genotype of PpTST1 with a single-base transversion (G1584T) in the third exon which leads to a single amino acid substitution (Q528H) was associated with low level of organic acid content in peach. Overexpression of PpTST1His resulted in reduced organic acid content along with increased sugar content both in peach and tomato fruits, suggesting its dual function in sugar accumulation and organic acid content reduction. Two V-type proton ATPases interact with PpTST1 in yeast two-hybridization assay. In addition, the G1584T transversion appeared and gradually accumulated during domestication and improvement, which indicated that PpTST1 was under selection. The identification and characterization of PpTST1 would facilitate the improvement of peach fruit quality.

Comparative transcriptome analysis reveals distinct gene expression profiles in Brachypodium distachyon infected by two fungal pathogens.

BACKGROUND: The production of cereal crops is frequently affected by diseases caused by Fusarium graminearum and Magnaporthe oryzae, two devastating fungal pathogens. To improve crop resistance, many studies have focused on understanding the mechanisms of host defense against these two fungi individually. However, our knowledge of the common and different host defenses against these pathogens is very limited. RESULTS: In this study, we employed Brachypodium distachyon as a model for cereal crops and performed comparative transcriptomics to study the dynamics of host gene expression at different infection stages. We found that infection with either F. graminearum or M. oryzae triggered massive transcriptomic reprogramming in the diseased tissues. Numerous defense-related genes were induced with dynamic changes during the time course of infection, including genes that function in pattern detection, MAPK cascade, phytohormone signaling, transcription, protein degradation, and secondary metabolism. In particular, the expression of jasmonic acid signaling genes and proteasome component genes were likely specifically inhibited or manipulated upon infection by F. graminearum. CONCLUSIONS: Our analysis showed that, although the affected host pathways are similar, their expression programs and regulations are distinct during infection by F. graminearum and M. oryzae. The results provide valuable insight into the interactions between B. distachyon and two important cereal pathogens.

New high-quality peach (Prunus persica L. Batsch) genome assembly to analyze the molecular evolutionary mechanism of volatile compounds in peach fruits.

Peach (Prunus persica L. Batsch) is an economically important fruit crop worldwide. Although a high-quality peach genome has previously been published, Sanger sequencing was used for its assembly, which generated short contigs. Here, we report a chromosome-level genome assembly and sequence analysis of Chinese Cling, an important founder cultivar for peach breeding programs worldwide. The assembled genome contained 247.33 Mb with a contig N50 of 4.13 Mb and a scaffold N50 of 29.68 Mb, representing 99.8% of the estimated genome. Comparisons between this genome and the recently published one (Lovell peach) uncovered 685 407 single nucleotide polymorphisms, 162 655 insertions and deletions, and 16 248 structural variants. Gene family analysis highlighted the contraction of the gene families involved in flavone, flavonol, flavonoid, and monoterpenoid biosynthesis. Subsequently, the volatile compounds of 256 peach varieties were quantitated in mature fruits in 2015 and 2016 to perform a genome-wide association analysis. A comparison with the identified domestication genomic regions allowed us to identify 25 quantitative trait loci, associated with seven volatile compounds, in the domestication region, which is consistent with the differences in volatile compounds between wild and cultivated peaches. Finally, a gene encoding terpene synthase, located within a previously reported quantitative trait loci region, was identified to be associated with linalool synthesis. Such findings highlight the importance of this new assembly for the analysis of evolutionary mechanisms and gene identification in peach species. Furthermore, this high-quality peach genome provides valuable information for future fruit improvement.

Selection and validation reference genes for qRT-PCR normalization in different cultivars during fruit ripening and softening of peach (Prunus persica).

Quantitative real-time PCR (qRT-PCR) has been emerged as an effective method to explore the gene function and regulatory mechanisms. However, selecting appropriate reference gene (s) is a prerequisite for obtaining accurate qRT-PCR results. Peach is one of important fruit in Rosaceae and is widely cultivated worldwide. In this study, to explore reliable reference gene (s) in peach with different types during fruit ripening and softening (S1-S4), nine candidate reference genes (EF-1α, GAPDH, TBP, UBC, eIF-4α, TUB-A, TUB-B, ACTIN, and HIS) were selected from the whole-genome data. Then, the expression levels of the nine selected genes were detected using qRT-PCR in three peach types, including 'Hakuho' (melting type), 'Xiacui' (stony hard type), 'Fantasia' and 'NJC108' (non-melting type) cultivars were detected using qRT-PCR. Four software (geNorm, NormFinder, BestKeeper and RefFinder) were applied to evaluate the expression stability of these candidate reference genes. Gene expression was characterized in different peach types during fruit ripening and softening stages. The overall performance of each candidate in all samples was evaluated. The Actin gene (ACTIN) was a suitable reference gene and displayed excellent stability in 'Total' set, 'Hakuho' samples, S3 and S4 fruit developmental stages. Ubiquitin C gene (UBC) showed the best stability in most independent samples, including 'Fantasia', 'NJC108', S2 sets. Elongation factor-1α gene (EF-1α) was the most unstable gene across the set of all samples, 'NJC108' and S2 sets, while showed the highest stability in 'Xiacui' samples. The stability of candidate reference genes was further verified by analyzing the relative expression level of ethylene synthase gene of Prunus persica (PpACS1) in fruit ripening and softening periods of 'Hakuho'. Taken together, the results from this study provide a basis for future research on the mining of important functional genes, expression patterns and regulatory mechanisms in peach.

Genomic analyses provide insights into peach local adaptation and responses to climate change.

The environment has constantly shaped plant genomes, but the genetic bases underlying how plants adapt to environmental influences remain largely unknown. We constructed a high-density genomic variation map of 263 geographically representative peach landraces and wild relatives. A combination of whole-genome selection scans and genome-wide environmental association studies (GWEAS) was performed to reveal the genomic bases of peach adaptation to diverse climates. A total of 2092 selective sweeps that underlie local adaptation to both mild and extreme climates were identified, including 339 sweeps conferring genomic pattern of adaptation to high altitudes. Using genome-wide environmental association studies (GWEAS), a total of 2755 genomic loci strongly associated with 51 specific environmental variables were detected. The molecular mechanism underlying adaptive evolution of high drought, strong UVB, cold hardiness, sugar content, flesh color, and bloom date were revealed. Finally, based on 30 yr of observation, a candidate gene associated with bloom date advance, representing peach responses to global warming, was identified. Collectively, our study provides insights into molecular bases of how environments have shaped peach genomes by natural selection and adds candidate genes for future studies on evolutionary genetics, adaptation to climate changes, and breeding.

Reduced expression of a subunit gene of sucrose non-fermenting 1 related kinase, PpSnRK1ßγ, confers flat fruit abortion in peach by regulating sugar and starch metabolism.

BACKGROUND: Fruit abortion is a major limiting factor for fruit production. In flat peach, fruit abortion is present in the whole tree of some accessions during early fruit development. However, the physiological factors and genetic mechanism underlying flat fruit abortion remain largely elusive. RESULTS: In this study, we have revealed that the fertilization process was accomplished and the reduction of sucrose and starch contents might result in flat fruit abortion. By combining association and gene expression analysis, a key candidate gene, PpSnRK1ßγ, was identified. A 1.67-Mb inversion co-segregated with flat fruit shape altered the promoter activity of PpSnRK1ßγ, resulting in much lower expression in aborting flat peach. Ectopic transformation in tomato and transient overexpression in peach fruit have shown that PpSnRK1ßγ could increase sugar and starch contents. Comparative transcriptome analysis further confirmed that PpSnRK1ßγ participated in carbohydrate metabolism. Subcellular localization found that PpSnRK1ßγ was located in nucleus. CONCLUSIONS: This study provides a possible reason for flat fruit abortion and identified a critical candidate gene, PpSnRK1ßγ, that might be responsible for flat fruit abortion in peach. The results will provide great help in peach breeding and facilitate gene identification for fruit abortion in other plant species.

An integrated peach genome structural variation map uncovers genes associated with fruit traits.

BACKGROUND: Genome structural variations (SVs) have been associated with key traits in a wide range of agronomically important species; however, SV profiles of peach and their functional impacts remain largely unexplored. RESULTS: Here, we present an integrated map of 202,273 SVs from 336 peach genomes. A substantial number of SVs have been selected during peach domestication and improvement, which together affect 2268 genes. Genome-wide association studies of 26 agronomic traits using these SVs identify a number of candidate causal variants. A 9-bp insertion in Prupe.4G186800, which encodes a NAC transcription factor, is shown to be associated with early fruit maturity, and a 487-bp deletion in the promoter of PpMYB10.1 is associated with flesh color around the stone. In addition, a 1.67 Mb inversion is highly associated with fruit shape, and a gene adjacent to the inversion breakpoint, PpOFP1, regulates flat shape formation. CONCLUSIONS: The integrated peach SV map and the identified candidate genes and variants represent valuable resources for future genomic research and breeding in peach.

Dynamic proteome changes of wheat developing grains in response to water deficit and high-nitrogen fertilizer conditions.

This study investigated grain proteomic profiles in response to water deficit, high nitrogen (N) fertilizer, and their combined treatments in elite Chinese bread wheat cultivar Jingdong 17, using a two-dimensional difference gel electrophoresis (2D-DIGE)-based approach. Water deficit negatively affected the main agronomic traits of wheat and grain yield, while high-N fertilizer had the opposite effects. The application of a high-N fertilizer under water deficit conditions moderately improved kernel development and grain yield. 2D-DIGE led to the identification of 124 differentially accumulated protein (DAP) spots during five different grain developmental stages, corresponding to 97 unique proteins. The more significant changes of DAPs occurred at 10-20 days after flowering. DAPs were involved in carbohydrate metabolism, protein turnover, protein folding, cell cycle control, stress response, nitrogen metabolism, photosynthesis, and energy metabolism. In particular, water deficit caused a significant downregulation of proteins involved in starch biosynthesis, whereas high-N fertilizer led to a significant upregulation of proteins involved in nitrogen metabolism, carbohydrate metabolism, and starch biosynthesis. The combined treatment resulted in a moderate upregulation of DAPs related to carbohydrate metabolism, starch biosynthesis, and nitrogen metabolism. Our results indicated that high-N fertilization could alleviate yield loss caused by water deficit by promoting the accumulation of proteins involved in nitrogen and carbohydrate metabolism.

An orphan protein of Fusarium graminearum modulates host immunity by mediating proteasomal degradation of TaSnRK1α.

Fusarium graminearum is a causal agent of Fusarium head blight (FHB) and a deoxynivalenol (DON) producer. In this study, OSP24 is identified as an important virulence factor in systematic characterization of the 50 orphan secreted protein (OSP) genes of F. graminearum. Although dispensable for growth and initial penetration, OSP24 is important for infectious growth in wheat rachis tissues. OSP24 is specifically expressed during pathogenesis and its transient expression suppresses BAX- or INF1-induced cell death. Osp24 is translocated into plant cells and two of its 8 cysteine-residues are required for its function. Wheat SNF1-related kinase TaSnRK1α is identified as an Osp24-interacting protein and shows to be important for FHB resistance in TaSnRK1α-overexpressing or silencing transgenic plants. Osp24 accelerates the degradation of TaSnRK1α by facilitating its association with the ubiquitin-26S proteasome. Interestingly, TaSnRK1α also interacts with TaFROG, an orphan wheat protein induced by DON. TaFROG competes against Osp24 for binding with the same region of TaSnRKα and protects it from degradation. Overexpression of TaFROG stabilizes TaSnRK1α and increases FHB resistance. Taken together, Osp24 functions as a cytoplasmic effector by competing against TaFROG for binding with TaSnRK1α, demonstrating the counteracting roles of orphan proteins of both host and fungal pathogens during their interactions.

Effects of Independent and Combined Water-Deficit and High-Nitrogen Treatments on Flag Leaf Proteomes during Wheat Grain Development.

We present the first comprehensive proteome analysis of wheat flag leaves under water-deficit, high-nitrogen (N) fertilization, and combined treatments during grain development in the field. Physiological and agronomic trait analyses showed that leaf relative water content, total chlorophyll content, photosynthetic efficiency, and grain weight and yield were significantly reduced under water-deficit conditions, but dramatically enhanced under high-N fertilization and moderately promoted under the combined treatment. Two-dimensional electrophoresis detected 72 differentially accumulated protein (DAP) spots representing 65 unique proteins, primarily involved in photosynthesis, signal transduction, carbohydrate metabolism, redox homeostasis, stress defense, and energy metabolism. DAPs associated with photosynthesis and protein folding showed significant downregulation and upregulation in response to water-deficit and high-N treatments, respectively. The combined treatment caused a moderate upregulation of DAPs related to photosynthesis and energy and carbohydrate metabolism, suggesting that high-N fertilization can alleviate losses in yield caused by water-deficit conditions by enhancing leaf photosynthesis and grain storage compound synthesis.

Comparative population genomics identified genomic regions and candidate genes associated with fruit domestication traits in peach.

Crop evolution is a long-term process involving selection by natural evolutionary forces and anthropogenic influences; however, the genetic mechanisms underlying the domestication and improvement of fruit crops have not been well studied to date. Here, we performed a population structure analysis in peach (Prunus persica) based on the genome-wide resequencing of 418 accessions and confirmed the presence of an obvious domestication event during evolution. We identified 132 and 106 selective sweeps associated with domestication and improvement, respectively. Analysis of their tissue-specific expression patterns indicated that the up-regulation of selection genes during domestication occurred mostly in fruit and seeds as opposed to other organs. However, during the improvement stage, more up-regulated selection genes were identified in leaves and seeds than in the other organs. Genome-wide association studies (GWAS) using 4.24 million single nucleotide polymorphisms (SNPs) revealed 171 loci associated with 26 fruit domestication traits. Among these loci, three candidate genes were highly associated with fruit weight and the sorbitol and catechin content in fruit. We demonstrated that as the allele frequency of the SNPs associated with high polyphenol composition decreased during peach evolution, alleles associated with high sugar content increased significantly. This indicates that there is genetic potential for the breeding of more nutritious fruit with enhanced bioactive polyphenols without disturbing a harmonious sugar and acid balance by crossing with wild species. This study also describes the development of the genomic resources necessary for evolutionary research in peach and provides the large-scale characterization of key agronomic traits in this crop species.

Genomic analyses of an extensive collection of wild and cultivated accessions provide new insights into peach breeding history.

BACKGROUND: Human selection has a long history of transforming crop genomes. Peach (Prunus persica) has undergone more than 5000 years of domestication that led to remarkable changes in a series of agronomically important traits, but genetic bases underlying these changes and the effects of artificial selection on genomic diversity are not well understood. RESULTS: Here, we report a comprehensive analysis of peach evolution based on genome sequences of 480 wild and cultivated accessions. By focusing on a set of quantitative trait loci (QTLs), we provide evidence supporting that distinct phases of domestication and improvement have led to an increase in fruit size and taste and extended its geographic distribution. Fruit size was predominantly selected during domestication, and selection for large fruits has led to the loss of genetic diversity in several fruit weight QTLs. In contrast, fruit taste-related QTLs were successively selected for by domestication and improvement, with more QTLs selected for during improvement. Genome-wide association studies of 11 agronomic traits suggest a set of candidate genes controlling these traits and potential markers for molecular breeding. Candidate loci for genes that contributed to the adaption to low-chill regions were identified. Furthermore, the genomic bases of divergent selection for fruit texture and local breeding for different flavors between Asian and European/North American cultivars were also determined. CONCLUSIONS: Our results elucidate the genetic basis of peach evolution and provide new resources for future genomics-guided peach breeding.

Lysine acetylproteome profiling under water deficit reveals key acetylated proteins involved in wheat grain development and starch biosynthesis.

Lysine acetylation is a widespread protein posttranslational modification in all organisms. However, quantitative acetylproteome characterization in response to water deficit during crop grain development remains unknown. In the study, we performed the first large-scale acetylproteome analysis of developing wheat grains under water-deficit using label-free quantitative proteome approach. In total, 716 acetylated sites corresponding to 442 acetylated proteins were identified, of which 106 acetylated sites representing 93 acetylated proteins (including 88 non-histones) showed significant changes under water-deficit. The functional classification showed that 57% and 20% of acetylated proteins were related to metabolic and cellular processes, respectively. Water-deficit caused widespread functional crosstalk between protein acetylation and other PTMs. Particularly, both acetylation and phosphorylation occurred in two key enzymes involved in starch biosynthesis, sucrose synthase (SuSy) and ADP glucose pyrophosphorylase (AGPase). Their crosstalk could play important roles in starch biosynthesis and yield formation under drought conditions. Western blot analysis combined with tandem mass spectrometry identification further verified the reliability of the acetylproteome results. Most of the acetylated proteins showed consistences between transcription and post-translation levels by quantitative real-time PCR. A putative metabolic pathway was proposed to dissect the roles of protein acetylation in regulation of drought response and defense during wheat grain development. SIGNIFICANCE: Lysine acetylation is a widespread modification in all organisms. We performed the first large-scale acetylproteome analysis of developing wheat grains under water-deficit and revealed key acetylated proteins involved in wheat grain development and starch biosynthesis.

Transcriptome analysis reveals novel genes involved in anthocyanin biosynthesis in the flesh of peach.

Peach is an important deciduous fruit tree species. Anthocyanins play an important role in fruit color formation and, through linkage analysis, previous studies have identified and mapped the key genes regulating anthocyanins' accumulation to chromosomes 3 and 5 in two different germplasms. To understand the overall regulatory network of anthocyanins biosynthesis, genes co-expressed with these key genes were identified in the red-fleshed 'Tianjin Shui Mi' and white-fleshed 'Hakuho' germplasms. Analysis of their flesh anthocyanin contents revealed differences 15 days before maturation. Therefore, transcriptome analysis of the flesh of fruits belonging to these two germplasms was performed to search for genes that were up-regulated at the late stage of development of 'Tianjin Shui Mi' but not of 'Hakuho', and identified 183 genes. These genes were also analyzed in the flesh transcriptomes of peach fruits belonging to 30 peach varieties with different anthocyanin contents at maturation, and the Pearson's correlation coefficients between their expression levels and anthocyanin contents were determined. The results showed that 66 genes were significantly correlated to anthocyanin contents, most of which previously reported as regulatory, biosynthetic, and transporter genes involved in anthocyanins' regulatory network. The results of this study enrich the understanding of key genes involved in the biological pathway regulating anthocyanins biosynthesis. The genes mostly associated with anthocyanins biosynthesis presented in this study are of great importance for molecular marker-assisted breeding.

Comparative metabolome analysis of wheat embryo and endosperm reveals the dynamic changes of metabolites during seed germination.

In this study, we performed the first comparative metabolomic analysis of the wheat embryo and endosperm during seed germination using GC-MS/MS. In total, 82 metabolites were identified in the embryo and endosperm. Principal component analysis (PCA), metabolite-metabolite correlation and hierarchical cluster analysis (HCA) revealed distinct dynamic changes in metabolites between the embryo and endosperm during seed germination. Generally, the metabolite changes in the embryo were much greater than those in the endosperm, suggesting that the embryo is more active than the endosperm during seed germination. Most amino acids were upregulated in both embryo and endosperm, while polysaccharides and organic acids associated with sugars were mainly downregulated in the embryo. Most of the sugars showed an upregulated trend in the endosperm, but significant changes in lipids occurred only in the embryo. Our results suggest that the embryo mobilises mainly protein and lipid metabolism, while the endosperm mobilises storage starch and minor protein metabolism during seed germination. The primary energy was generated mainly in the embryo by glycolysis during seed imbibition. The embryo containing most of the genetic information showed increased nucleotides during seed germination process, indicating more active transcription and translation metabolisms.

Comparative Phosphoproteomic Analysis under High-Nitrogen Fertilizer Reveals Central Phosphoproteins Promoting Wheat Grain Starch and Protein Synthesis.

Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation.