Apple Glycosyltransferase MdUGT73AR4 Glycosylates ABA to Regulate Stomatal Movement Involved in Drought Stress

Abstract

Abscisic acid (ABA) is a drought-stress-responsive hormone that plays an important role in the stomatal activity of plant leaves. Currently, ABA glycosides have been identified in apples, but their glycosyltransferases for glycosylation modification of ABA are still unidentified. In this study, the mRNA expression of glycosyltransferase gene MdUGT73AR4 was significantly up-regulated in mature apple leaves which were treated in drought stress by Real-Time PCR. It was hypothesised that MdUGT73AR4 might play an important role in drought stress. In order to further characterise the glycosylation modification substrate of glycosyltransferase MdUGT73AR4, we demonstrated through in vitro and in vivo functional validation that MdUGT73AR4 can glycosylate ABA. Moreover, the overexpression lines of MdUGT73AR4 significantly enhance its drought stress resistance function. We also found that the adversity stress transcription factor AREB1B might be an upstream transcription factor of MdUGT73AR4 by bioinformatics, EMSA, and ChIP experiments. In conclusion, this study found that the adversity stress transcription factor AREB1B was significantly up-regulated at the onset of drought stress, which in turn positively regulated the downstream glycosyltransferase MdUGT73AR4, causing it to modify ABA by mass glycosylation and promoting the ABA synthesis pathway, resulting in the accumulation of ABA content, and displaying a stress-resistant phenotype.

Keywords: ABA; AREB1B; MdUGT73AR4; drought tolerance; glycosyltransferase; stomatal.

Figure 1

Real-Time PCR detection of glycosyltransferase genes MdUGT73AR3 (Md00G1046200), MdUGT73AR4 (Md07G1007600), MdUGT73AR5 (Md07G1007400), MdUGT73AR6 (XP_008375157.2), MdUGT73AB13 (Md00G1055100), and MdUGT73AC7 (Md05G1085600) induced expression of mRNA levels in “Gala” apple sample seedlings after drought stress treatment for 0 h, 3 h, 6 h, 12 h, and 24 h. Note: * p < 0.05, ** p < 0.01, n = 3.

Figure 2

Expression pattern of different parts of apple glycosyltransferase gene MdUGT73AR4 detected by Real-Time PCR. Note: * p < 0.05, ** p < 0.01, n = 3.

Figure 3

HPLC detection of MdUGT73AR4 glycosylation modification substrate ABA. (A): HPLC detection of MdUGT73AR4 glycosylation modification product ABA glucoside; (B): mass spectrometry detection of MdUGT73AR4 glycosylation modification product ABA glucoside molecular weight.

Figure 4

Obtained MdUGT73AR4 overexpression lines and phenotypic observation. (A) Detection of gene MdUGT73AR4 mRNA expression level in overexpression lines; (B) determination of relative water content in mature leaves of MdUGT73AR4 overexpression lines OE10 and OE15; (C) determination of relative conductivity in mature leaves of MdUGT73AR4 overexpression lines OE10 and OE15; (D) detection of ABA and ABA glycoside contents in MdUGT73AR4 overexpression lines OE10 and OE15; (E) determination of stomatal opening in MdUGT73AR4 overexpression lines OE10 and OE15 after drought stress; (F) determination of stomatal expression levels of stomatal-related genes in MdUGT73AR4 overexpression lines OE10 and OE15 after drought stress. Note: * p < 0.05, ** p < 0.01, n = 3.

Figure 5

MdUGT73AR4 upstream transcription factor identification. (A) EMSA identification of MdUGT73AR4 upstream transcription factor AREB1B; (B) ChIP assay identification of MdUGT73AR4 upstream transcription factor AREB1B. 1, MYC; 2, AREB1A; 3, AREB1B; 4, AREB2A; 5, AREB2B. Note: * p < 0.05, ** p < 0.01, n = 3.