叶酸多聚谷氨酸合成酶AtDFC的生物学功能研究
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摘要
叶酸是指四氢叶酸及其衍生物这一类物质,是介导一碳单位转移的重要的辅助因子,参与了生物体内许多重要反应。叶酸由三部分构成:蝶啶、对氨基苯甲酸(pABA)和谷氨酸盐。植物和微生物能够自身合成叶酸,二者的合成途径基本相同。植物中叶酸的合成过程涉及两个分支:pABA分支和蝶啶分支,其中pABA分支在质体中进行,蝶啶分支在细胞质中进行,随后这两个分支在线粒体中汇合,形成叶酸分子的主体,在具有谷氨酸加尾功能的酶的作用下,形成四氢叶酸,随后通过其他酶的作用,形成各种不同形式的四氢叶酸衍生物。在拟南芥中,具有谷氨酸加尾功能的酶有4个,分别为AtDFA、AtDFB、AtDFC、AtDFD。AtDFA为二氢叶酸合成酶(dihydrofolate synthase,DHFS),定位于线粒体,在它的催化作用下,一个谷氨酸分子与二氢蝶酸(dihydropteroate , DHP)相结合,形成二氢叶酸;AtDFB、AtDFC、AtDFD为多聚谷氨酸合成酶(folylpolyglutamate synthase,FPGS),分别定位于质体、线粒体和细胞质,在多聚谷氨酸合成酶的作用下,谷氨酸分子可以逐一连接到四氢叶酸的谷氨酸γ位。
多聚谷氨酸形式的叶酸是叶酸依赖型酶所偏好的底物,并且多聚谷氨酸尾可以增加叶酸的稳定性,同时线粒体又是叶酸合成的重要场所,因此探讨线粒体中的多聚谷氨酸合成酶的功能是非常重要的。我们以拟南芥突变体Atdfc为材料,来探讨线粒体中的多聚谷氨酸合成酶AtDFC的生物学功能。
AtDFC在拟南芥生长的各个时期各个组织中普遍表达。Atdfc突变体在营养生长阶段与野生型并无明显差异,但其抽薹的时间要比野生型晚。对其抽薹前后叶数目进行统计,发现其叶数目与野生型相比并无明显变化,这与典型的晚花植物不同。微生物法测定总叶酸含量,Atdfc的总叶酸含量是野生型的64.4%;液相色谱/质谱法对野生型和Atdfc突变体中不同的叶酸衍生物含量进行分析,在突变体中,5-甲基四氢叶酸的含量降低了62.6%,5-甲酰四氢叶酸的含量降低了32.7%。基因芯片的统计结果显示,10.8%的差异基因与代谢途径相关,有4个基因涉及氮代谢,3个基因涉及谷胱甘肽代谢及P450介导的外源物质代谢,另外7个基因都涉及氨基酸的代谢。对野生型和Atdfc突变体中游离氨基酸的含量进行分析,大部分的氨基酸在Atdfc中的含量都有所增加,其中天冬酰胺、精氨酸、谷氨酰胺等参与铵和氨基酸的合成;丝氨酸参与一碳代谢的转移;以分支酸为底物可以合成酪氨酸和苯丙氨酸,这些氨基酸的含量在突变体中均有明显提高。在黑暗诱导叶片凋亡的过程中,在只用水处理的情况下,Atdfc的凋亡速率要比野生型快;用MTX(一种叶酸合成抑制剂)处理时,野生型和Atdfc的凋亡速率都明显加快,而且Atdfc突变体凋亡的速率要比野生型更快一些。对野生型、Atdfc和nia1/nia2(硝酸还原酶双突变体)进行MTX和氮饥饿处理,观察主根长度变化时,发现在正常条件下,野生型与Atdfc的主根长度无明显差异,nia1/nia2的主根长度略短;当用MTX处理时,野生型、Atdfc和nia1/nia2的主根生长都受到明显的抑制,Atdfc和nia1/nia2受到的抑制更明显;氮饥饿处理时,野生型、Atdfc和nia1/nia2的根长也都受到明显的抑制,但Atdfc受到的抑制程度更明显,野生型和nia1/nia2受抑制的程度无明显差别。
以上结果表明,AtDFC对叶酸的合成是非常重要的,线粒体中多聚谷氨酸合成酶(FPGS)功能的缺失导致拟南芥体内叶酸水平的下降,并且会影响氮代谢及氨基酸代谢,引起Atdfc突变体中游离氨基酸水平上升。叶酸合成途径受到阻断后会加速叶片衰老并抑制植物生长,并且在缺乏氮源的情况下,植物生长受抑制程度更加明显。
Tetrahydrofolate (THF) and its derivatives-commonly grouped under the name of folates-are vital cofactors for enzymes that mediate one-carbon transfer reactions. Folates are involved in a wide range of key metabolic functions. Bacteria and plants can synthesize folate de nove. Human and animals don’t have this ability and depends entirely on dietary supply. THF is a tripartite molecule, comprising pteridine, p-aminobenzoate (pABA) and glutamate moieties. The plant folate synthesis pathway is essentially the same as in bacteria. There are two branches in the plant folate biosynthesis pathway: pABA and pteridine branch. The pABA branch is in the plastid, and the pteridine branch is in the cytosol. Then they are coupled together in the mitochondria, glutamylated and reduced to produce THF. Other enzymes can catalyse THF to form THF derivatives. In plants, the four enzymes AtDFA, AtDFB, AtDFC and AtDFD have the ablity of adding glutamate. AtDFA is dihydrofolate synthase (DHFS) localized in mitochondria, it catalyzes one glutamate added to the dihydropteroate and produces dihydrofolat. AtDFB, AtDFC and AtDFD are folylpolyglutamate synthase (FPGS) localized in plastid, mitochondria and cytosol respectively. A short chain ofγ-linked glutamates can be added by FPGS.
Folate molecules exist in vivo mainly as polyglutamates and these are preferred by folate-dependent enzymes. For this reason, FPGS are very important especially the one localized in mitochondria which is a major site for folate synthesis. In this study, Atdfc mutant was used to investigate the biological function of AtDFC.
AtDFC was expressed ubiquitously both in different development stage and in various tissues. There was nearly no difference in visible phenotypes between wild type plants and Atdfc mutant plants during vegetable growth. However, Atdfc mutant plants bolted about one week later than wild type. Typical late-flowering mutants had more leaves than wild type, but the Atdfc mutants plants contain the same leaf number as the wild type . Total folate content of the Atdfc was 35.6% less than that of the wild type measured by microbiological method. The content of different folate derivatives was analyzed by HPLC/MS, and the results showed that 5-methyl-THF in Atdfc mutant was62.6% less than that of the wild type; 5-formyl-THF in Atdfc mutant was 32.7% less than that of the wild type. Microarray results showed that there were 10.8% differentially expressed genes involved in metabolism pathway. Four genes were involved in nitrogen metabolism; three genes were involved in metabolism of xenobiotics by cytochrome P450; seven were involved in amino acid metabolism. Then we measured the free amino acid content. Most of these amino acids have a higher content in Atdfc than in wild type. Among these amino acids, Asn, Arg and Gln participate in the synthesis of ammonium and amino acid; Ser participates in one-carbon metabolism; chorismic acid can be used to synthesize Tyr and Phe. In the process of dark-induced senescence, Atdfc mutant leaves senescence rapidly than do wild type leaves when treated with ddH2O. Treatment of the Atdfc mutant and wild type leaves with methotrexate (MTX,inhibitor of the folat synthesis pathway) accelerated dark-induced senescence. The Atdfc mutant showed severe yellowing and cell death. We recorded the length of primary root when treated the Atdfc mutant, nia1/nia2 (nitrate reductase double mutant) and wild type seedling with (MTX) or nitrogen starvation. The length of primary roots almost had no difference between the Atdfc mutant and the wild type in the normal condition. The primary root of nia1/nia2 mutant is a little shorter than the wild type primary root. Treatment of the Atdfc mutant, nia1/nia2 and wild type with MTX or nitrogen starvation inhibited the primary root growth. In MTX treatment, the percentage of inhibition of wild type was 65%; the ones of Atdfc and nia1/nia2 mutant were 85% and 86.7%. In nitrogen starvation treatment, the percentage of inhibition of Atdfc was 85%; the ones of wild type plants and nia1/nia2 mutant were 72% and 67.7%.
Taken together, AtDFC is very important for folat synthesis. The loss of mitochondiral FPGS decreases the level of folate and has an effect on the metabolism of nitrogen and amino acid. Blocking of the folate synthesis pathway accelerated leaf senescence and resulted in an increased sensitivity to nitrogen starvation .
多聚谷氨酸形式的叶酸是叶酸依赖型酶所偏好的底物,并且多聚谷氨酸尾可以增加叶酸的稳定性,同时线粒体又是叶酸合成的重要场所,因此探讨线粒体中的多聚谷氨酸合成酶的功能是非常重要的。我们以拟南芥突变体Atdfc为材料,来探讨线粒体中的多聚谷氨酸合成酶AtDFC的生物学功能。
AtDFC在拟南芥生长的各个时期各个组织中普遍表达。Atdfc突变体在营养生长阶段与野生型并无明显差异,但其抽薹的时间要比野生型晚。对其抽薹前后叶数目进行统计,发现其叶数目与野生型相比并无明显变化,这与典型的晚花植物不同。微生物法测定总叶酸含量,Atdfc的总叶酸含量是野生型的64.4%;液相色谱/质谱法对野生型和Atdfc突变体中不同的叶酸衍生物含量进行分析,在突变体中,5-甲基四氢叶酸的含量降低了62.6%,5-甲酰四氢叶酸的含量降低了32.7%。基因芯片的统计结果显示,10.8%的差异基因与代谢途径相关,有4个基因涉及氮代谢,3个基因涉及谷胱甘肽代谢及P450介导的外源物质代谢,另外7个基因都涉及氨基酸的代谢。对野生型和Atdfc突变体中游离氨基酸的含量进行分析,大部分的氨基酸在Atdfc中的含量都有所增加,其中天冬酰胺、精氨酸、谷氨酰胺等参与铵和氨基酸的合成;丝氨酸参与一碳代谢的转移;以分支酸为底物可以合成酪氨酸和苯丙氨酸,这些氨基酸的含量在突变体中均有明显提高。在黑暗诱导叶片凋亡的过程中,在只用水处理的情况下,Atdfc的凋亡速率要比野生型快;用MTX(一种叶酸合成抑制剂)处理时,野生型和Atdfc的凋亡速率都明显加快,而且Atdfc突变体凋亡的速率要比野生型更快一些。对野生型、Atdfc和nia1/nia2(硝酸还原酶双突变体)进行MTX和氮饥饿处理,观察主根长度变化时,发现在正常条件下,野生型与Atdfc的主根长度无明显差异,nia1/nia2的主根长度略短;当用MTX处理时,野生型、Atdfc和nia1/nia2的主根生长都受到明显的抑制,Atdfc和nia1/nia2受到的抑制更明显;氮饥饿处理时,野生型、Atdfc和nia1/nia2的根长也都受到明显的抑制,但Atdfc受到的抑制程度更明显,野生型和nia1/nia2受抑制的程度无明显差别。
以上结果表明,AtDFC对叶酸的合成是非常重要的,线粒体中多聚谷氨酸合成酶(FPGS)功能的缺失导致拟南芥体内叶酸水平的下降,并且会影响氮代谢及氨基酸代谢,引起Atdfc突变体中游离氨基酸水平上升。叶酸合成途径受到阻断后会加速叶片衰老并抑制植物生长,并且在缺乏氮源的情况下,植物生长受抑制程度更加明显。
Tetrahydrofolate (THF) and its derivatives-commonly grouped under the name of folates-are vital cofactors for enzymes that mediate one-carbon transfer reactions. Folates are involved in a wide range of key metabolic functions. Bacteria and plants can synthesize folate de nove. Human and animals don’t have this ability and depends entirely on dietary supply. THF is a tripartite molecule, comprising pteridine, p-aminobenzoate (pABA) and glutamate moieties. The plant folate synthesis pathway is essentially the same as in bacteria. There are two branches in the plant folate biosynthesis pathway: pABA and pteridine branch. The pABA branch is in the plastid, and the pteridine branch is in the cytosol. Then they are coupled together in the mitochondria, glutamylated and reduced to produce THF. Other enzymes can catalyse THF to form THF derivatives. In plants, the four enzymes AtDFA, AtDFB, AtDFC and AtDFD have the ablity of adding glutamate. AtDFA is dihydrofolate synthase (DHFS) localized in mitochondria, it catalyzes one glutamate added to the dihydropteroate and produces dihydrofolat. AtDFB, AtDFC and AtDFD are folylpolyglutamate synthase (FPGS) localized in plastid, mitochondria and cytosol respectively. A short chain ofγ-linked glutamates can be added by FPGS.
Folate molecules exist in vivo mainly as polyglutamates and these are preferred by folate-dependent enzymes. For this reason, FPGS are very important especially the one localized in mitochondria which is a major site for folate synthesis. In this study, Atdfc mutant was used to investigate the biological function of AtDFC.
AtDFC was expressed ubiquitously both in different development stage and in various tissues. There was nearly no difference in visible phenotypes between wild type plants and Atdfc mutant plants during vegetable growth. However, Atdfc mutant plants bolted about one week later than wild type. Typical late-flowering mutants had more leaves than wild type, but the Atdfc mutants plants contain the same leaf number as the wild type . Total folate content of the Atdfc was 35.6% less than that of the wild type measured by microbiological method. The content of different folate derivatives was analyzed by HPLC/MS, and the results showed that 5-methyl-THF in Atdfc mutant was62.6% less than that of the wild type; 5-formyl-THF in Atdfc mutant was 32.7% less than that of the wild type. Microarray results showed that there were 10.8% differentially expressed genes involved in metabolism pathway. Four genes were involved in nitrogen metabolism; three genes were involved in metabolism of xenobiotics by cytochrome P450; seven were involved in amino acid metabolism. Then we measured the free amino acid content. Most of these amino acids have a higher content in Atdfc than in wild type. Among these amino acids, Asn, Arg and Gln participate in the synthesis of ammonium and amino acid; Ser participates in one-carbon metabolism; chorismic acid can be used to synthesize Tyr and Phe. In the process of dark-induced senescence, Atdfc mutant leaves senescence rapidly than do wild type leaves when treated with ddH2O. Treatment of the Atdfc mutant and wild type leaves with methotrexate (MTX,inhibitor of the folat synthesis pathway) accelerated dark-induced senescence. The Atdfc mutant showed severe yellowing and cell death. We recorded the length of primary root when treated the Atdfc mutant, nia1/nia2 (nitrate reductase double mutant) and wild type seedling with (MTX) or nitrogen starvation. The length of primary roots almost had no difference between the Atdfc mutant and the wild type in the normal condition. The primary root of nia1/nia2 mutant is a little shorter than the wild type primary root. Treatment of the Atdfc mutant, nia1/nia2 and wild type with MTX or nitrogen starvation inhibited the primary root growth. In MTX treatment, the percentage of inhibition of wild type was 65%; the ones of Atdfc and nia1/nia2 mutant were 85% and 86.7%. In nitrogen starvation treatment, the percentage of inhibition of Atdfc was 85%; the ones of wild type plants and nia1/nia2 mutant were 72% and 67.7%.
Taken together, AtDFC is very important for folat synthesis. The loss of mitochondiral FPGS decreases the level of folate and has an effect on the metabolism of nitrogen and amino acid. Blocking of the folate synthesis pathway accelerated leaf senescence and resulted in an increased sensitivity to nitrogen starvation .
引文
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