【作者】 孟红岩；

【导师】 张春义；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2014， 博士

【摘要】 叶酸在植物体内主要以多谷氨酸尾形式存在,参与很多重要的生命活动。单谷氨酸尾形式的叶酸可以在叶酰聚谷氨酸合成酶(FPGS)的作用下生成多谷氨酸尾形式的叶酸。本研究利用质体定位的FPGS (又称FPGS1或AtDFB)功能缺失的突变体解析叶酸在拟南芥发育和代谢中的生物学功能。主要结果如下：1.当培养基中氮源充足时,atdfb突变体主根明显短于野生型,培养11天时突变体的主根长度只有野生型的23%。突变体对低氮胁迫反应比野生型更敏感：在低氮条件下,突变体发育几乎停滞,培养11天时突变体的主根长度只有野生型的4%。不管在氮源充足还是低氮条件,互补植物的表型均恢复至野生型水平。另外,低氮条件下突变体发育几乎停滞的表型可以被5-甲酰四氢叶酸(5-F-THF)恢复。同时,在添加5-F-THF的条件下突变体的静止中心细胞排列正常。2.土壤培养条件下,播种后5天的突变体主根比野生型短,15天时其主根长度与野生型类似。另外,突变体的抽薹时间比野生型要晚一周。3.成熟的突变体种子体积比野生型小,千粒重比野生型降低5%。突变体种子总碳(C)含量比野生型低,而总氮(N)含量高于野生型,C/N比值比野生型低23%,说明突变体种子的碳氮分配能力不足。进一步分析发现突变体种子中储存的营养物质含量发生变化,碳含量丰富的化合物如糖类、有机酸、脂肪酸等含量偏高,而氮含量丰富的化合物如氨基酸、可溶蛋白和N03含量降低。4.黑暗培养条件下,突变体只能利用培养环境中的硝态氮(N03),不能利用铵态氮(NH4+)或有机氮(例如Asn或Gln)。在硝酸根(N03)充足条件下,突变体暗形态建成异常,下胚轴长度只有野生型的80%,进一步研究发现突变体下胚轴伸长的障碍可能与种子胚中储存的物质不足有关。在N03不足时,突变体的下胚轴随着培养基中N03的减少而逐渐缩短。在上述两种NO3条件下,5-F-THF均能使突变体短下胚轴长度恢复至野生型水平,但是在铵态氮条件下,5-F-THF不能使突变体下胚轴伸长恢复。5.暗形态建成过程中突变体的叶酸代谢发生紊乱,主要表现为：总叶酸和5-甲基四氢叶酸(5-M-THF)含量低于野生型,5-M-THF占总叶酸的比例较野生型低,七谷氨酸尾形式的5-M-THF和5-F-THF含量较野生型高。氮源充足的条件下突变体中多种代谢物含量异常,如氨基酸、菜油甾醇、S-腺苷甲硫氨酸等；低氮条件下突变体中积累了更多的代谢物,如氨基酸、糖类、豆甾醇、有机酸和脂肪酸等。转录组数据分析表明,这些代谢物含量的变化与对应代谢途径关键酶基因的表达改变有关。5-F-THF在一定程度上可以恢复突变体的叶酸代谢和其他代谢。总之,我们的研究结果表明,(1) AtDFB的功能缺失影响了拟南芥根的发育及种子储存物的积累；(2)种子储存物积累的异常影响了突变体的暗形态建成；(3)突变体中叶酸代谢的紊乱导致多条代谢途径基因表达变化及代谢网络中多种代谢物含量的异常,最终导致了拟南芥暗形态建成的异常。该研究阐述了叶酸代谢对植物种子储存物积累的影响,建立了叶酸代谢与其他代谢网络及暗形态建成之间的联系。更多还原

【Abstract】 Folates take part in many important physiological processes during plant development, andpolyglutamylated species are dominant form of folates in plants. Folylpolyglutamate synthetase (FPGS)catalyzes the addition of glutamate residues to the folate molecule to form folylpolyglutamates. In thisstudy, the T-DNA insertion mutant of the plastidial FPGS (named FPGS1or AtDFB) was use to explorethe role of folates during seedling development and metabolism in Arabidopsis. The results are asfollows.1. The atdfb mutant displayed shorter primary root than the wild type even under nitrogen(N)-sufficient conditions, and the length of primary root of11-day-old atdfb seedlings was23%of thewild type. Under N-limited conditions, the development of atdfb almost stagnated, with the primary rootlength of11-day-old atdfb seedlings was only4%of the wild type. Phenotypes of the complementedplants were similar to the wild type under both N-sufficient and limited conditions. Under N-limitedconditions, exogenous5-formyl-tetrahydrofolate (5-F-THF) restored the phenotype of atdfb to the wildtype. At the same time, the quiescent center in atdfb was well organized as the wild type.2. When grown in the soil, the mutant seedling of5day after sowing (DAS) displayed shorterprimary root than the wild type, while the length of primary root of atdfb was similar to the wild type inseedlings of15DAS. Besides, the bolting time of atdfb was one week later than the wild type.3. The dimensions of mature atdfb seeds were smaller than the wild type, and the weight per1000seeds in atdfb was5%lighter than the wild type. The mutant seeds had lower carbon (C) content andhigher nitrogen (N) content. The C/N ratio in atdfb was23%less than the wild type, indicative of analtered C and N partitioning capacity. Further analysis revealed that the seed storage in atdfb was altered,with higher content of C-rich metabolites, such as sugars, organic acids and fatty acids, and less N-richmetabolites, such as amino acids, soluble protein and nitrate (NO-3). These results indicated significantchanges in seed storage in the mutant.4. During the skotomorphogenesis, the mutant could use NO-3but not ammonium (NH+4) ororganic N (such as Asn or Gln) as the sole N source. Even under N-sufficient conditions, the mutantdisplayed defects in skotomorphogenesis, with80%of the hypocotyl length of the wild type. Furtheranalysis indicated possible relation between hypocotyl elongation defects and shortage of storage inembryo in atdfb. Under N-insufficient conditions, the hypocotyl length of atdfb reduced when theconcentration of NO-3decreased. Exogenous5-F-THF restored the hypocotyl length in atdfb seedlingsto that of the wild type under both N-sufficient and limited conditions with NO-3not NH+4as the sole Nsource.5. Folate profiling was altered in atdfb, with lower total folates content,5-methyl-tetrahydrofolate(5-M-THF) content, and proportion of5-M-THF in total folates, and higher level of heptaglutamylatedforms of5-M-THF and5-F-THF. The metabolites profiling in atdfb was also perturbed, with alteredcontents of amino acids, campesterol and S-adenosylmethionine even under N-sufficient conditions.Under N-limited conditions, many metabolites accumulated in atdfb, such as amino acids, sugars, stigmasterol, organic acids and fatty acids. Transcriptome analysis indicated altered expression of genesinvolved in multiple metabolic pathways. Exogenous application of5-F-THF restored the amounts ofmany metabolites to wild-type levels in atdfb seedlings or significantly reduced differences in the levelsof many metabolites between atdfb and the wild type, including the5-M-THF content and theproportion of5-M-THF in total folates.In brief, our results indicated that (1) AtDFB was required for primary root development and seedreserves accumulation in Arabidopsis;(2) The altered seed accumulation in atdfb led to defectivehypocotyl elongation in darkness;(3) The perturbed folates profiling led to altered gene expressionabundance and metabolites content involved in multiple metabolic pathways, resulting in defectiveskotomorphogenesis with shortened hypocotyls in atdfb. Taken together, this study demonstrated thatfolate metabolism played an important role in seed reserve accumulation, seed germination andpost-germinative hypocotyl elongation, providing novel insights into potential associations amongfolate metabolism, metabolic networks, and skotomorphogenesis.更多还原