Effects of Glutamine on Vitamin B6 Biosynthesis in Arabidopsis thaliana

穀氨醯胺(Glutamine)是植物體內含量最豐富的胺基酸之一，做為初級氮同化作用最初始的有機氮產物，也是合成其他胺基酸、核酸、葉綠素等含氮化合物時氮的提供來源。我們發現以glutamine作為唯一氮源時，阿拉伯芥可以有效的吸收利用以供植物生長發育，但過量的glutamine則會抑制植物生長，因此我們使用能使野生種正常生長的glutamine濃度，篩選對glutamine過度敏感而出現生長抑制的突變株。突變株23844/pdx3-3為其中之一，在以glutamine作為唯一氮源時出現主根生長被顯著抑制、細胞大小排列異常、細胞分裂數量較少且在根部表面出現不正常的細胞凋亡等性狀，最終造成根系發育受到影響。根據map-based cloning及next generation sequencing定位pdx3-3之突變基因，發現一個點突變在At5g49970基因上，使Gln226轉換成終止密碼子，影響了維生素B6生合成補救途徑(vitamin B6 salvage pathway)中pyridoxine/ pyridoxamine phosphate oxidase (PDX3/PPOX)的表現。從遺傳互補及基因靜默植株的性狀觀察中，顯示pdx3-3對glutamine過度敏感的性狀確實來自於PDX3失去功能所造成。在另一個維生素B6補救途徑突變株sos4 (salt overly sensitive 4)也觀察到和pdx3-3相似的性狀，而在pdx3-3/sos4雙重突變株有性狀加乘的效果，此外在pdx3-3及sos4中則有維生素B6衍生物含量不正常累積的現象，顯示維生素B6補救途徑的缺失會導致對glutamine過度敏感的性狀。由於PDX3及維生素B6補救途徑的功能尚未被完全了解透徹，因此我們會再進一步研究glutamine和維生素B6補救途徑的關係，以及對維生素B6恆定的調控。Glutamine is one of the most abundant free amino acids in plants. As the first organic nitrogen from primary nitrogen assimilation, glutamine is a major amino donor for the synthesis of amino acids, nucleotides, and other nitrogen-containing compounds. We showed that glutamine could efficiently support Arabidopsis growth when used as a sole nitrogen source in the growth medium, but the addition of excess glutamine significantly inhibited Arabidopsis growth. We thus used appropriate glutamine concentration as sole nitrogen source to screen for “glutamine hypersensitive” mutants. One of the mutants, line 23844/pdx3-3, had dramatically reduced root growth, irregular cell arrangement, less cell proliferation and abnormal cell death in glutamine-containing medium. Line 23844 is defective in At5g49970 that encodes pyridoxine/ pyridoxamine phosphate oxidase (PDX3) of the vitamin B6 salvage pathway. The mutant has a point mutation that changes Gln226 to a stop codon. In addition to the mutant, we have obtained the genetic complementation line and PDX3 gene silencing lines that show similar phenotypes. These results suggested that the growth defects in 23844 are caused by loss-of-function in the PDX3 gene. sos4 (salt overly sensitive 4), another mutant involved in vitamin B6 salvage pathway, had similar phenotypes with pdx3-3, and pdx3-3/sos4 double mutant had more severe phenotypes. Furthermore, by analysis vitamin B6 contents in pdx3-3 and sos4, we found abnormal vitamin B6 levels in mutants. These data indicated that defects in vitamin B6 salvage pathway might result in glutamine hypersensitive phenotypes. Nevertheless, the functions of PDX3 and the vitamin B6 salvage pathway are not well understood in plants. We will further study the relationship between glutamine and vitamin B6 salvage pathway, and the regulation of vitamin B6 homeostasis