拟南芥叶酰聚谷氨酸合成酶AtDFB调控下胚轴伸长的机理研究

Folates, termed of tetrahydrofolate and its derivatives, are vital substance in life development. Folylpolyglutamate synthetase (FPGS) catalyzes the addition of glutamate residues to the folate molecule to form folylpolyglutamates. The T-DNA insertion mutant of the plastidial FPGS displayed altered folate profiling that resulted in disturbed nitrogen metabolism and shortened hypocotyl. The shortened hypocotyl phenotype of the mutant was specifically sensitive to low nitrogen condition indicating that hypocotyl elongation was probably regulated by nitrogen metabolism. It is unclear that how the altered folate profiling disturbs nitrogen metabolism that inhibits hypocotyl elongation in the mutant. In this project, the measurement of the rate of exogenous nitrate transporting into root cell, enzyme activity assay of key enzymes in nitrogen metabolism, and the content of nitrogen-rich metabolites will all be taken to uncover the mainly altered nitrogen metabolism process which probably inhibits hypocotyl elongation in the mutant. Furtherly, the binding model and binding site of folate with the enzyme (protein) in the nitrogen metabolism process uncovered above, and the effect of folate on the expression and stability of the key enzyme (protein) will be implemented to explain the mechanism of folate in regulating nitrogen metabolism. Moreover, the hypocotyl phenotype and the reaction to exogenous applied folate of the mutant of key enzyme (protein) will be used to explore the mechanism of folate in regulating nitrogen metabolism inhibiting hypocotyl elongation. The successful implementation of this project will have great research value for straightening out the relationship of folate, nitrogen metabolism and hypocotyl elongation.

叶酸，是四氢叶酸及其衍生物的统称，是生命活动正常进行必不可少的重要物质。我们前期的研究发现，拟南芥质体定位的叶酰聚谷氨酸合成酶（AtDFB）功能缺失造成的叶酸代谢紊乱会导致黄化苗氮代谢的异常和下胚轴缩短。突变体下胚轴缩短的表型特异对低氮条件敏感，说明下胚轴伸长可能受氮代谢的调控。然而，叶酸如何调控氮代谢进而影响下胚轴伸长的机理尚未阐明。本研究将通过分析突变体吸收外源硝酸盐的能力、氮代谢关键酶活性和氮代谢物含量等指标的变化与缩短的下胚轴之间的关系，解析调控下胚轴伸长的主要氮代谢过程；进一步通过分析叶酸与该氮代谢过程关键酶（蛋白）的结合模式及结合位点，叶酸对该酶（蛋白）的表达量及稳定性的影响，该酶（蛋白）功能缺失突变体的下胚轴表型及对叶酸的反应等，剖析叶酸对该氮代谢过程的调控机理。本项目的顺利实施，将理顺叶酸、氮代谢和下胚轴伸长之间的关系，具有重要的研究价值。

拟南芥质体定位的叶酰聚谷氨酸合成酶AtDFB功能缺失突变体暗形态建成异常，下胚轴缩短，下胚轴表皮细胞伸长障碍。在氮限制条件突变体下胚轴急剧缩短，细胞壁成分之果胶在突变体中显著积累。细胞壁物质合成相关基因XTH26和细胞伸长相关基因EXP18等在突变体中高表达，表达量分别达到野生型的36和25倍。AtDFB功能缺失造成拟南芥黄化苗叶酸代谢紊乱及氮代谢异常。突变体对硝酸盐转运异于野生型，外源5-F-THF恢复突变体对硝酸盐的转运能力，说明叶酸参与植物根部硝酸盐转运。AtDFB功能缺失会造成大多数氨基酸合成增强（如Gln、Ser、Asn、Arg、Pro和Thr等），进而造成突变体总氨基酸的积累，5-F-THF能够恢复氨基酸含量。叶酸可能通过调控Gln积累影响下胚轴伸长。突变体中亚硝酸还原酶活性只有野生型的一半，5-F-THF能够恢复该酶活性。研究证实叶酸直接参与硝酸盐转运、还原及氨基酸合成代谢等氮代谢环节。.对氮源充足条件培养的黄化苗进行组学分析。共筛选出显著差异基因1275个，其中815个基因在突变体中表达上调，460个基因下调；筛选出差异代谢物80种，其中41种代谢物在突变体中含量显著提高，39种代谢物含量降低。富集分析发现差异基因集中被注释在参与光合作用、含硫化合物合成、三羧酸循环等生物过程，以及质体、叶绿体、类囊体等细胞组分。对80种显著差异代谢物进行富集分析，主要被注释到参与次级代谢物合成、氨基酸合成、抗生素合成及氨酰-tRNA合成等生物过程。这些研究结果与AtDFB定位在质体，叶酸参与一碳代谢完全吻合。组学结果进一步证实叶酸对植物代谢至关重要，叶酸代谢紊乱对植物代谢网络造成多方面多层次的扰动。.本项目初步开展了牛樟芝叶酸合成和代谢的研究。发现牛樟芝能够自身合成叶酸，具有亚甲基四氢叶酸还原酶和二氢叶酸还原酶等叶酸合成关键酶编码基因，合成和代谢途径有待进一步研究。