A single nucleotide substitution of GSAM gene causes massive accumulation of glutamate 1-semialdehyde and yellow leaf phenotype in rice

Qian Wang#, Baiyang Zhu#, Congping Chen, Zhaodi Yuan, Jia Guo, Xiaorong Yang, San Wang, Yan Lv, Qingsong Liu, Bin Yang, Changhui Sun, Pingrong Wang*, Xiaojian Deng*

Rice (IF=4.783, TOP), 2021

https://pubmed.ncbi.nlm.nih.gov/34089406/

Abstract

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.

Keywords: Rice, Tetrapyrrol biosynthesis, GSAM gene, Protein interaction, Chloroplast development, Yellow leaf mutant, Gene cloning