Abstract
Alfalfa is widely grown worldwide as a perennial high-quality legume forage and as a good ecological landcover. The cysteine synthase (CSase) gene family is actively involved in plant growth and development and abiotic stress resistance but has not been systematically investigated in alfalfa. We identified 39 MsCSase genes on 4 chromosomes of the alfalfa genome. Phylogenetic analysis demonstrated that these genes were clustered into six subfamilies, and members of the same subfamily had similar physicochemical properties and sequence structures. Overexpression of the CSase gene in alfalfa increased alkali tolerance. Compared with control plants, the overexpression lines presented higher proline, soluble sugars, and cysteine and reduced glutathione contents and superoxide dismutase and peroxidase activities as well as lower hydrogen peroxide and superoxide anion contents after alkali stress. The relative expression of γ-glutamyl cysteine synthetase gene (a downstream gene of CSase) in the overexpression lines was much higher than that in the control line. The CSase gene enhanced alkalinity tolerance by regulating osmoregulatory substances and improving antioxidant capacity. These results provide a reference for studying the CSase gene family in alfalfa and expanding the alkali tolerance gene resources of forage plants.
Highlights
Cysteine is the first organic substance in plants found to contain both sulfur and nitrogen and is a precursor of sulfur-containing metabolites such as methionine (Takahashi et al, 2011), glutathione (GSH) and Fe-S clusters, which play an important role in plant development and metabolic processes (Droux, 2004; Van Hoewyk et al, 2008)
A total of 39 MsCSase gene sequences were retrieved from alfalfa using BLAST, PF00291 domain and PLP-binding site (PXXSVKDR) searches and named MsCSase01 to MsCSase39 according to their chromosome locations (Supplementary Table 2)
The phylogenetic tree results demonstrated that the MsCSase proteins could be divided into 6 subfamilies according to the clades and classification from Arabidopsis, including 14, 9, 5, 4, and 4 members in the CysA subfamily, CysB subfamily, sulfocysteine synthase (SCS) subfamily, CysD subfamily and CysC subfamily, respectively (Figure 1)
Summary
Cysteine is the first organic substance in plants found to contain both sulfur and nitrogen and is a precursor of sulfur-containing metabolites such as methionine (Takahashi et al, 2011), glutathione (GSH) and Fe-S clusters, which play an important role in plant development and metabolic processes (Droux, 2004; Van Hoewyk et al, 2008). Cysteine synthase (CSase) is involved in the final step of cysteine synthesis; this enzyme catalyzes the synthesis of cysteine from H2S and O-acetylserine (OAS) (Jez and Dey, 2013; Romero et al, 2014). The CSase gene family was previously identified in Arabidopsis (Arabidopsis thaliana) and was found to comprise nine genes divided into five subfamilies, all of whose members contain PLP-binding sites (PXXSVKDR) that are highly conserved across species (Yamaguchi et al, 2000). When plants need to enhance GSH biosynthesis under heavy metal stress, CSase can increase cysteine production and subsequently affect the synthesis of downstream substances and achieve improved plant tolerance
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