12-Oxophytodienoate Reductase Overexpression Compromises Tolerance to Botrytis cinerea in Hexaploid and Tetraploid Wheat

Abstract

12-Oxophytodienoate reductase is the enzyme involved in the biosynthesis of phytohormone jasmonates, which are considered to be the major regulators of plant tolerance to biotic challenges, especially necrotrophic pathogens. However, we observe compromised tolerance to the necrotrophic fungal pathogen Botrytis cinerea in transgenic hexaploid bread wheat and tetraploid emmer wheat plants overexpressing 12-OXOPHYTODIENOATE REDUCTASE-3 gene from Arabidopsis thaliana, while in Arabidopsis plants themselves, endogenously produced and exogenously applied jasmonates exert a strong protective effect against B. cinerea. Exogenous application of methyl jasmonate on hexaploid and tetraploid wheat leaves suppresses tolerance to B. cinerea and induces the formation of chlorotic damages. Exogenous treatment with methyl jasmonate in concentrations of 100 µM and higher causes leaf yellowing even in the absence of the pathogen, in agreement with findings on the role of jasmonates in the regulation of leaf senescence. Thereby, the present study demonstrates the negative role of the jasmonate system in hexaploid and tetraploid wheat tolerance to B. cinerea and reveals previously unknown jasmonate-mediated responses.

Endogenously Produced Jasmonates Affect Leaf Growth and Improve Osmotic Stress Tolerance in Emmer Wheat

“…The phytohormone content was analyzed 30 min after the wounding (Figure 3). As expected, the overexpression of AtOPR3 (that encodes a peroxisome-localized enzyme) led to an increase in wounding-induced jasmonic acid levels in leaf tissues, as previously shown [37]. However, against expectations, in cases of AtAOS overexpression encoding a chloroplast-localized enzyme, no increase in levels of the chloroplast-formed OPDA was observed, while the level of JA in mechanically damaged plants was increased significantly, in comparison to non-transgenic plants.…”

“…For quantitative real-time RT-PCR, total RNA was extracted from the 4th young leaf of non-transgenic and transgenic emmer wheat with the Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol and used to synthesize cDNA as previously described [37]. According to the sequence of AtAOS, the primers were designed: 5′-AAATCCAACGGCGGAGAACT-3′ as forward primer and 5′-TCGTCGCCAACGGTTGATAA-3′ as reverse primer.…”

“…The analysis of phytohormones in the third leaves of transgenic and non-transgenic emmer wheat at the four-leaf stage was carried out exactly as previously described [37]. All wounding experiments were accomplished in six independent repetitions, i.e., the plants were randomized and wounded at different times (with fixed time interval between wounding and harvest).…”

“…The genetic transformation of emmer wheat Runo was performed using the biolistic method [35]. Transformation vector pBAR-GFP.UbiAOS, carrying AtAOS and selective genes, used in this study (Figure 1) was essentially similar to the vector we used previously to generate transgenic hexaploid and tetraploid emmer wheat overexpressing the OPR gene [37].…”

“…To better understand the functionality of individual AOS and OPR genes of the JAs biosynthesis pathway when overexpressed in emmer wheat, we performed a comparative analysis of newly established transgenic lines with AtAOS overexpression (RA3, RA4, and RA9) and previously generated transgenic emmer wheat plants with AtOPR3 overexpression (RC12, RC26, and R29) [37]. First, we analyzed the content of the main jasmonates in leaves of transgenic and non-transgenic plants, including OPDA, JA, and JA-Ile conjugatethe most active form of jasmonate, which performs most of the signaling functions.…”