海藻糖增加阿拉伯芥葉片中APL3與GPT2的表現與澱粉的累積

Abstract

植物葉片白天合成暫存性澱粉，再於夜晚分解成葡萄糖提供植物的生長。糖類除了做為澱粉的前驅物之外，也參與了澱粉代謝基因表現的調控。前人研究指出，海藻糖可以使得澱粉合成相關基因APL3的表現量大幅增加，使得澱粉合成缺失株adg2恢復合成澱粉的能力，同時也調降了澱粉降解蛋白基因SEX1、BAM3以及GLT1的表現，因此使阿拉伯芥幼苗的子葉部分累積大量的澱粉。本研究以碘液染色觀察到pgi澱粉合成突變株可因應海藻糖的處理使得葉片澱粉含量上升；由microarray初步分析海藻糖處理所造成的基因變化後，以RT-PCR檢測GPT表現量，發現阿拉伯芥中具功能性的磷酸葡萄糖運輸蛋白基因之一，AtGPT2 (At1g61800)，會因海藻糖處理而提高基因的表現量；且海藻糖無法使pgi1-2/gpt2-1雙重突變株累積大量澱粉，顯示海藻糖除了增強APL3表現並提高AGPase活性以促進合成澱粉外，同時也藉由GPT2蛋白開啟葉綠體以及細胞質之間的G6P通道，而擴大了澱粉前驅物G6P的來源，先前研究觀察到海藻糖使得野生型阿拉伯芥葉片澱粉含量增加的效應，應是由於APL3以及GPT2兩蛋白質的共同作用。本研究檢測海藻糖誘導GPT2表現所需要的時間，結果顯示在3小時海藻糖處理之後，即可由RT-PCR檢測到被誘導表現的GPT2。本研究也發現蔗糖與海藻糖均能誘導GPT2以及APL3的表現，但蔗糖使pgi突變株累積澱粉效應較海藻糖弱，顯示兩種雙糖對於澱粉累積可能有不同的效應。

Transitory starch is synthesized during the day and degraded during the night to sustain the growth of plants. Sugars are not only the precursors of starch but also regulate expression of genes involving in starch metabolism. Trehalose can both induce the expression of starch synthesis gene APL3 and reduce the expression of starch degradation genes, SEX1, BAM3 and GLT1. The effects therefore enhance the starch accumulation in cotyledons of wild type Arabidopsis and rescue the starch deficient phenotype of adg2 mutant but not pgm and adg1 mutants. In this study, pgi mutant is found to accumulate more starch in leaves after trehalose treatment compared with pgi mutant without sugar treatment. In microarray analysis, one of the functional GPT genes, AtGPT2 (At1g61800), is shown to be up-regulated by trehalose feeding. RT-PCR is performed to comfirm this result. pgi1-2/gpt2-1 double mutant cannot accumulate as much starch as pgi1-2 after trehalose treatment, which suggests the accumulation by trehalose feeding in pgi is due to both induction of APL3 and GPT2. By time course experiments, trehalose can rapidly induce the expression of GPT2 after 3-hour treatment. Both sucrose and trehalose can induce GPT2 and APL3 expression, but the sucrose effect on starch accumulation is weaker than trehalose in pgi mutants, which suggests the effects on starch accumulation of the two sugars are not exactly the same.