Table_1_Impacts of Nitrogen Deficiency on Wheat (Triticum aestivum L.) Grain During the Medium Filling Stage: Transcriptomic and Metabolomic Comparisons.XLSX

Nitrogen (N) supplementation is essential to the yield and quality of bread wheat (Triticum aestivum L.). The impact of N-deficiency on wheat at the seedling stage has been previously reported, but the impact of distinct N regimes applied at the seedling stage with continuous application on filling and maturing wheat grains is lesser known, despite the filling stage being critical for final grain yield and flour quality. Here, we compared phenotype characteristics such as grain yield, grain protein and sugar quality, plant growth, leaf photosynthesis of wheat under N-deficient and N-sufficient conditions imposed prior to sowing (120 kg/hm2) and in the jointing stage (120 kg/hm2), and then evaluated the effects of this continued stress through RNA-seq and GC-MS metabolomics profiling of grain at the mid-filling stage. The results showed that except for an increase in grain size and weight, and in the content of total sugar, starch, and fiber in bran fraction and white flour, the other metrics were all decreased under N-deficiency conditions. A total of 761 differentially expressed genes (DEGs) and 77 differentially accumulated metabolites (DAMs) were identified. Under N-deficiency, 51 down-regulated DEGs were involved in the process of impeding chlorophyll synthesis, chloroplast development, light harvesting, and electron transfer functions of photosystem, which resulted in the SPAD and Pn value decreased by 32 and 15.2% compared with N-sufficiency, inhibited photosynthesis. Twenty-four DEGs implicated the inhibition of amino acids synthesis and protein transport, in agreement with a 17–42% reduction in ornithine, cysteine, aspartate, and tyrosine from metabolome, and an 18.6% reduction in grain protein content. However, 14 DEGs were implicated in promoting sugar accumulation in the cell wall and another six DEGs also enhanced cell wall synthesis, which significantly increased fiber content in the endosperm and likely contributed to increasing the thousands-grain weight (TGW). Moreover, RNA-seq profiling suggested that wheat grain can improve the capacity of DNA repair, iron uptake, disease and abiotic stress resistance, and oxidative stress scavenging through increasing the content levels of anthocyanin, flavonoid, GABA, galactose, and glucose under N-deficiency condition. This study identified candidate genes and metabolites related to low N adaption and tolerance that may provide new insights into a comprehensive understanding of the genotype-specific differences in performance under N-deficiency conditions.

氮素（N）的补充对面包小麦（Triticum aestivum L.）的产量与品质至关重要。关于氮素缺乏对小麦幼苗期影响的研究已有报道，然而，在幼苗期施加不同氮素管理方案并持续施用对小麦籽粒充实与成熟的影响则鲜为人知，尽管充实阶段对最终籽粒产量和面粉品质至关重要。本研究对比了在播种前（120 kg/hm²）和分蘖期（120 kg/hm²）施加氮素缺乏和充足条件下小麦的表型特征，如籽粒产量、籽粒蛋白质与糖质、植物生长、叶片光合作用，随后通过RNA测序和GC-MS代谢组学分析评估了持续施加该压力的影响。结果显示，除了籽粒尺寸和重量，以及麸皮部分和白色面粉中总糖、淀粉和纤维含量的增加外，其他指标均有所下降。共鉴定出761个差异表达基因（DEGs）和77个差异积累代谢物（DAMs）。在氮素缺乏条件下，51个下调的DEGs参与了阻碍叶绿素合成、叶绿体发育、光捕获和光合系统电子传递功能的进程，导致与氮素充足相比，SPAD和Pn值分别下降了32%和15.2%，抑制了光合作用。24个DEGs与氨基酸合成和蛋白质运输的抑制有关，与代谢组中精氨酸、半胱氨酸、天冬氨酸和酪氨酸含量降低17-42%，以及籽粒蛋白质含量降低18.6%相一致。然而，14个DEGs与细胞壁中糖分的积累促进有关，另外六个DEGs也增强了细胞壁的合成，显著增加了胚乳中的纤维含量，可能有助于提高千粒重（TGW）。此外，RNA测序分析表明，小麦籽粒可以通过在氮素缺乏条件下增加花青素、黄酮、GABA、半乳糖和葡萄糖的含量，提高DNA修复、铁吸收、抗病性和抗非生物胁迫的能力，以及清除氧化应激。本研究确定了与低氮适应性及耐受性相关的候选基因和代谢物，这些发现可能为深入理解氮素缺乏条件下基因型特异性的性能差异提供新的见解。