SNPs within the glutathione S-transferase genes as markers for the identification of more or less stress responsive sweet orange varieties

The glutathione S-transferases (GSTs) are ubiquitous dimeric enzymes catalyzing the nucleophilic addition of glutathione (GSH) to the electrophilic groups of a large variety of toxic chemicals. The resulting GS-conjugates are sequestered into the vacuoles where further transformation processes can promote their partial salvage, substitution or degradation. Subunits of all known GSTs exhibit a two-domain fold, the N-terminal domain, including the highly conserved GSH-binding site (G-site), and the C-terminal domain which represents the more divergent cosubstrate-binding site (H-site). Previously, two GST genes sharing 98.6% homology (U1 and U2) have been isolated from sweet orange. The encoded enzymes differ in three amino acids, all of them localized in the H-site of the proteins. Despite the fact that the enzymes share very high sequence similarity, they have been found to show different substrate specificity as well as different catalytic efficiency towards 1-chloro-2,4-dinitrobenzene (CDNB). In the present work, the analysis of single nucleotide polymorphisms (SNPs) among members of nine Citrus sinensis varieties was performed by genotype sequencing. The data showed that all SNPs, which lead to one or to the other GST isoform, are as different alleles in a heterozygosis status at the GST locus thus suggesting that all the varieties under investigation could potentially express both isoforms in their organs. The analysis of gene expression highlighted that the GST isoforms show different distribution between leaf and flesh as well as that they are differently involved in abiotic stress response. Therefore, the regulation of GST gene expression certainly fulfils a crucial role in citrus plant defense mechanisms. Moreover, gene sequencing revealed the presence of other still unidentified SNPs, most of them leading to conservative amino acid substitutions. In a particular case (LSQUOTarocco dal musoRSQUO), the newly individuated SNP inserts an in frame stop codon within the protein coding region which prematurely interrupts the mRNA translation process producing an incomplete, most likely inactive, form of enzyme. Therefore, SNP genotyping might represent a powerful tool to identify among varieties those potentially more responsive to environmental stresses.