【作者】 张莹；

【导师】 董家强；

【作者基本信息】 山东大学 ， 遗传学， 2025， 博士

【摘要】 在植物生长发育过程中,氨基酸不仅是蛋白质合成的基本单元,还参与多种信号通路的调控。丝氨酸(L-serine)作为重要的代谢中枢,兼具代谢底物和信号分子的双重功能,在植物中发挥关键作用。在拟南芥(Arabidopsis thaliana)等C3植物中,丝氨酸主要通过光呼吸相关的乙醇酸途径合成;而在光呼吸速率较低的C4植物,如玉米(Zea mays L.)中,其丝氨酸合成途径可能有所不同,但具体机制及其生物学功能尚不明确。玉米作为全球产量最高、经济价值最重要的粮食作物,其产量和品质高度依赖籽粒内部代谢网络的精细调控。因此,系统阐明玉米中丝氨酸的合成机制及其在籽粒发育中的作用,不仅有助于揭示C4植物中丝氨酸的代谢路径,也为提升玉米的养分利用效率和籽粒产量提供理论依据与遗传基础。本研究以玉米经典突变体dek20(defective kernel 20)为研究对象,系统揭示了 C4植物中丝氨酸生物合成,以及丝氨酸缺乏条件下籽粒发育调控的分子机制,主要发现如下:1.与WT籽粒相比,dek20籽粒体积变小、表皮皱缩,籽粒无法正常萌发。胚和胚乳的发育严重受损,细胞周期紊乱。突变体籽粒的百粒重、淀粉含量和储藏蛋白含量均显著下降。2.Dek20基因编码磷酸甘油酸脱氢酶 1(phosphoglycerate dehydrogenase 1,PGDH1),为丝氨酸生物合成的磷酸化途径(phosphorylated pathway of serine biosynthesis,PPSB)中的限速酶,催化该代谢路径的起始反应。初级代谢组检测显示dek20籽粒中丝氨酸含量降低了 80%,外源补充丝氨酸可部分恢复其表型。3.蛋白结构解析与体外酶活实验表明,dek20突变破坏了 Ser282与His284之间的相互作用,增强了 His284与底物NAD+的结合能力,影响了 NAD+的释放,从而抑制DEK20蛋白的催化活性,进而导致丝氨酸合成受到阻碍,最终导致DEK20酶活性下降。4.dek20中丝氨酸缺乏诱导tRNASer-TGA与tRNASer-TCG发生降解,致使核糖体在丝氨酸密码子处发生停滞,翻译延伸受阻。核糖体的停滞激活了 GCN2(general control nonderepressible 2)激酶,进而促进 eIF2α(eukaryotic initiation factor2α)磷酸化,抑制翻译起始过程。同时,丝氨酸的缺乏造成TOR活性的降低,抑制了其下游靶蛋白S6K1(ribosomal protein S6 kinase 1)的磷酸化水平,进一步阻碍翻译的起始。5.Ribo-seq和RNA-seq联合分析显示,DEK20通过调控与氨基酸代谢、储藏物质合成及细胞周期相关的关键基因的翻译,从而影响籽粒正常的发育过程。综上所述,本研究综合运用遗传学、结构生物学、生物化学和分子生物学等多种手段,系统阐明了 DEK20/PGDH1在玉米丝氨酸合成及籽粒发育中的关键作用,填补了 C4植物丝氨酸代谢机制研究的空白。研究成果不仅为深入理解C4植物的氨基酸代谢通路提供了新视角,也为籽粒产量提升和品质改良提供了坚实的理论基础和宝贵的遗传资源。更多还原

【Abstract】 Amino acids play essential roles in plant growth and development,serving not only as fundamental substrates for protein synthesis but also as key participants in diverse signaling pathways.Among them,serine functions as central metabolic hub,acting both as substrate and signaling molecule with significant influence on plant development.In C3 plants such as Arabidopsis thaliana,serine is primarily synthesized via the photorespiration-related glycolate pathway.However,Zea mays,a representative C4 plant with a low rate of photorespiration,likely employs distinct mechanisms for serine biosynthesis.As the highest-yielding and most economically important C4 crop globally,maize growth and yield are tightly linked to nutrient metabolism.Therefore,elucidating the mechanisms of serine biosynthesis and its role in maize kernel development is not only critical for understanding serine metabolism in C4 plants but also provides a theoretical foundation and genetic resources for improving nutrient use efficiency and grain yield in maize.This study investigates the classical maize mutant defective kernel 20(dek20)to uncover the mechanism of serine biosynthesis in C4 plants and the molecular pathways underlying kernel development under serine-deficient conditions.The main findings are as follows:1.Mature dek20 kernels exhibit reduced size and a wrinkled pericarp.Embryo and endosperm development is severely impaired,accompanied by disrupted cell cycle progression,ultimately leading to germination failure.The hundred-kernel weight,starch content,and storage protein levels are all significantly reduced in dek20.2.The Dek20 gene encodes phosphoglycerate dehydrogenase 1(PGDH1),the rate-limiting enzyme in the phosphorylated pathway of serine biosynthesis(PPSB),which catalyzes the initial step of this metabolic pathway.Metabolomic analysis reveals that serine level in dek20 kernels is reduced by 80%,and exogenous serine supplementation can partially rescue the phenotype.3.Structural analysis of DEK20WT and DEK20S282L proteins indicates that the S282L mutation disrupts the interaction between Ser282 and His284,leading to the release of His284.This residue then forms a stable interaction with NAD+,thereby inhibiting enzymatic activity and dramatically decreasing serine content in dek20 kernels.4.Serine deficiency in dek20 induces the degradation of tRNASer-TGA and tRNASerTCG,causing ribosome stalling at serine codons and impairing translation elongation.This ribosome stalling activates the kinase GCN2(General Control Nonderepressible 2),which promotes phosphorylation of eIF2α(eukaryotic initiation factor 2α),thereby inhibiting translation initiation.Simultaneously,serine depletion reduces TOR activity and decreases the phosphorylation level of its downstream target S6K1(ribosomal protein S6 kinase 1),further suppressing translation initiation.5.Integrated Ribo-seq and RNA-seq analyses demonstrate that Dek20 regulates kernel development by modulating the translation of key genes involved in amino acid metabolism,storage compound biosynthesis,and cell cycle control.In summary,this study integrates genetics,structural biology,biochemistry,and molecular biology to comprehensively elucidate the critical role of DEK20/PGDH1 in serine biosynthesis and maize kernel development.It fills a key gap in our understanding of serine metabolism in C4 plants and provides novel insights into amino acid metabolic regulation.These findings offer valuable theoretical and genetic resources for enhancing nutrient use efficiency and grain yield in maize.更多还原