Analysis of 5-Oxoproline level and upregulation of 5-Oxoprolinase (OXP1) gene
GGCT2;1 is involved in GSH degradation and the recycling of its constituent amino acids (Paulose et al ., 2013). The immediate product of GGCTs activity is 5-oxoproline (5-OP) resulting from the degradation of GSH or its precursor γ-glutamyl cysteine (γ-EC). We measured the 5-OP levels in WT and two CsGGCT2;1 OE lines #102 and #103 under normal and AsIII treatments using triple-quadrupole mass spectroscopy (UPLC/Xevo TQD QQQ-MS, Waters Corporation, Milford, MA). No significant differences were observed between WT and OE lines under normal or AsIII treatment. However, the OE lines had approximately 21-24% more 5-oxoproline levels under MS control conditions than the WT (Figure 6a). Additionally, the 5-OP level increased in both WT and OE lines by 19-33% under AsIII treatment relative to non-stress control conditions. Still, the OE lines maintained 10-20% higher 5-OP levels than WT under AsIII treatment.
Since the immediate product of GGCTs activity is 5-oxoproline (5-OP), significantly higher levels of 5-OP were expected in the lines overexpressing CsGGCT2;1 . 5-OP is further converted to glutamate (Glu) by the endogenous activity of Oxoprolinase1 (OXP1). Our findings prompted us to study the upregulation of the endogenous OXP1 gene in CsGGCT2;1 OE lines using qRT-PCR under normal and AsIII stress conditions. Our results showed that under control conditions the expression of CsOXP1 was upregulated 2 to 3.5-fold in the OE lines compared to WT plants and 1.5 to 3.5-fold under AsIII stress conditions (Figure 6b).