Binding of the transcription factor MYC2-like to the ABRE of the OsCYP2 promoter enhances salt tolerance in Oryza sativa

Abstract

Cyclophilins, a type of peptidyl-prolyl cis-trans isomerase, function as important molecular chaperones in a series of biological processes. However, the expression pattern and signal transduction pathway of cyclophilins are still unclear. Here, we showed that the promoter of OsCYP2 could function as a tissue-specific promoter by GUS staining. Moreover, we found that the promoter sequence contained not only core elements but also inducible elements. Then, the ABA-responsive element was used for cDNA library screening, and the transcription factor MYC2-like was identified by a yeast one-hybrid assay and confirmed through an electrophoretic mobility shift assay. Furthermore, the relative expression showed that MYC2-like was induced by abscisic acid. In addition, MYC2-like overexpression enhanced salt tolerance in transformants and partially restored the cyp2-RNAi line. In summary, we explored a novel transcriptional signal mediated by MYC2-like, a potential regulator of salt stress-related physiological processes in rice.

Fig 1. Staining showed expression patterns of OsCYP2 indicated by the GUS reporter gene which was driven by the promoter of OsCYP2.

A-D: seed, leaf, stem and root of wild type; E-H: seed, leaf, stem and root of the pOsCYP2:GUS transgenic plants.

Fig 2. Cotransformation and serial dilution (1:5) with a initial optical density OD₆₀₀) of 0.5 on selective media to verify the interaction of candidate transcription factor (No. 11).

SD/-Leu: synthetically defined medium with dropout leucine, SD/-Leu+AbA: synthetically defined medium with dropout leucine and plus 750 μg/L Aureobasidin A; Y1H (p62-AbAi): bait strain; Y1H (p63-AbAi): mutant bait strain.

Fig 3. MYC2-like protein bind to the ABRE element by EMSA assays.

1: no MYC2-like protein for biotin labeled probe (p62) to bind, establishes the position of an unshifted probe band; 2: the MYC2-like protein was incubated with the biotin labeled probe (p62); 3: 200-fold excesses of unlabeled probes were used for competition; 4: biotin labeled mutant probe (p63) were used as a negative control.

Fig 4. Relative transcript level of the MYC2-like induced by salt and ABA.

Error bars represent the standard deviation.

Fig 5. Phenotype of wild type, MYC2-like overexpression (L1), MYC2-like overexpression in cyp2-RNAi (L2) and cyp2-RNAi (L3) under salt stress.

Fig 6. Comparison of antioxidative enzymes (SOD, POD, CAT), membrane lipid peroxidation index (MDA), osmolyte (proline), and Na⁺/K⁺ ratio among wild type and transformants under salt stress.

Values are mean ± standard deviation followed by the same letter did not significantly differences at P≤0.05 according to least significant difference test.