Background

Tetrapyrroles play indispensable roles in various biological processes. In higher plants, glutamate 1-semialdehyde 2,1-aminomutase (GSAM) converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA), which is the rate-limiting step of tetrapyrrole biosynthesis. Up to now, GSAM genes have been successively identified from many species. Besides, it was found that GSAM could form a dimeric protein with itself by x-ray crystallography. However, no mutant of GSAM has been identified in monocotyledonous plants, and no experiment on interaction of GSAM protein with itself has been reported so far.

Result

We isolated a yellow leaf mutant, ys53, in rice (Oryza sativa). The mutant showed decreased photosynthetic pigment contents, suppressed chloroplast development, and reduced photosynthetic capacity. In consequence, its major agronomic traits were significantly affected. Map-based cloning revealed that the candidate gene was LOC_Os08g41990 encoding GSAM protein. In ys53 mutant, a single nucleotide substitution in this gene caused an amino acid change in the encoded protein, so its ALA-synthesis ability was significantly reduced and GSA was massively accumulated. Complementation assays suggested the mutant phenotype of ys53 could be rescued by introducing wild-type OsGSAM gene, confirming that the point mutation in OsGSAM is the cause of the mutant phenotype. OsGSAM is mainly expressed in green tissues, and its encoded protein is localized to chloroplast. qRT-PCR analysis indicated that the mutation of OsGSAM not only affected the expressions of tetrapyrrole biosynthetic genes, but also influenced those of photosynthetic genes in rice. In addition, the yeast two-hybrid experiment showed that OsGSAM protein could interact with itself, which could largely depend on the two specific regions containing the 81th–160th and the 321th–400th amino acid residues at its N- and C-terminals, respectively.

Conclusions

We successfully characterized rice GSAM gene by a yellow leaf mutant and map-based cloning approach. Meanwhile, we verified that OsGSAM protein could interact with itself mainly by means of the two specific regions of amino acid residues at its N- and C-terminals, respectively.

中文翻译：

---

GSAM基因的单核苷酸取代导致水稻谷氨酸1-半醛大量积累和黄叶表型

背景

四吡咯在各种生物过程中发挥着不可或缺的作用。在高等植物中，谷氨酸1-半醛2,1-氨基变位酶（GSAM）将谷氨酸1-半醛（GSA）转化为5-氨基乙酰丙酸（ALA），这是四吡咯生物合成的限速步骤。截至目前，GSAM基因已从多个物种中陆续被鉴定出来。此外，X射线晶体学发现GSAM可以与自身形成二聚体蛋白。然而，在单子叶植物中尚未发现GSAM突变体，也未见GSAM蛋白与其自身相互作用的实验报道。

结果

我们在水稻（Oryza sativa ）中分离出黄叶突变体ys53。该突变体表现出光合色素含量降低、叶绿体发育受到抑制、光合能力降低。结果，其主要农艺性状受到显着影响。图位克隆显示候选基因是编码GSAM蛋白的LOC_Os08g41990。在ys53突变体中，该基因的单核苷酸替换导致编码蛋白的氨基酸发生变化，因此其ALA合成能力显着降低，GSA大量积累。互补分析表明ys53的突变表型可以通过引入野生型Os GSAM基因来挽救，证实OsGSAM中的点突变是突变表型的原因。OsGSAM主要在绿色组织中表达，其编码蛋白定位于叶绿体。qRT-PCR分析表明， OsGSAM突变不仅影响水稻四吡咯生物合成基因的表达，还影响水稻光合基因的表达。此外，酵母双杂交实验表明，OsGSAM蛋白能够与自身相互作用，这在很大程度上取决于其N端和C端分别包含第81-160和第321-400位氨基酸残基的两个特定区域。

结论

我们通过黄叶突变体和基于图位的克隆方法成功地表征了水稻GSAM基因。同时，我们验证了OsGSAM蛋白主要通过其N端和C端氨基酸残基的两个特定区域分别与自身相互作用。