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.

In light of recent climate change, with its rising temperatures and precipitation changes, we are facing the need to increase the valuable crop’s tolerance against unfavorable environmental conditions. Emmer wheat is a cereal crop with high nutritional value. We investigated the possibility of improving the stress tolerance of emmer wheat by activating the synthesis of the stress hormone jasmonate by overexpressing two genes of the jasmonate biosynthetic pathway from Arabidopsis thaliana, ALLENE OXIDE SYNTHASE (AtAOS) and OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3). Analyses of jasmonates in intact and mechanically wounded leaves of non-transgenic and transgenic plants showed that the overexpression of each of the two genes resulted in increased wounding-induced levels of jasmonic acid and jasmonate-isoleucine. Against all expectations, the overexpression of AtAOS, encoding a chloroplast-localized enzyme, does not lead to an increased level of the chloroplast-formed 12-oxo-phytodienoic acid (OPDA), suggesting an effective conversion of OPDA to downstream products in wounded emmer wheat leaves. Transgenic plants overexpressing AtAOS or AtOPR3 with increased jasmonate levels show a similar phenotype, manifested by shortening of the first and second leaves and elongation of the fourth leaf, as well as increased tolerance to osmotic stress induced by the presence of the polyethylene glycol (PEG) 6000.

‘Isabel’ grape (Vitis labrusca x V. vinifera L. hybrid) is one of the main grape cultivars in Russia and some other countries for processing, due to its vigor, tolerance to the main fungal diseases, high yield and potential for sugar accumulation. The stilbene synthase gene VlvSTS was isolated from the hybrid grape cv. Isabel and cloned into a pSS plant transformation vector under the control of a constitutive 35S RNA double promoter of the cauliflower mosaic virus, CaMV 35SS. VlvSTS-gene containing transgenic tobacco lines were obtained and analyzed. For the first time plants expressing the VlvSTS gene were shown to have an enhanced resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora B15. Transgenic plants were tested for resistance to a number of fungal pathogens. The plants were resistant to the grey mould fungus Botrytis cinerea, but not to the fungi Fusarium oxysporum, F. sporotrichioides, or F. culmorum. According to the results of a high performance liquid chromatography-mass spectrometry analysis, the amount of trans-resveratrol in leaves of transgenic plants with the highest expression of the VlvSTS gene was in a range from 150 to 170 μg/g of raw biomass. Change in the color and a decreased anthocyanin content in the flower corollas of transgenic plants were observed in transgenic lines with the highest expression of VlvSTS. A decrease in total flavonoid content was found in the flower petals but not the leaves of these tobacco lines. High expression of the VlvSTS gene influenced pollen development and seed productivity in transgenic plants. The size of pollen grains increased, while their total number per anther decreased. A decrease in the number of fertile pollen grains resulted in a decreased average weight of a seed boll in transgenic plants.

The transformation protocol based on the dual selection approach (fluorescent protein and herbicide resistance) has been applied here to produce transgenic plants of two cereal species, emmer wheat and bread wheat, with the goal of activating the synthesis of the stress hormone jasmonates by overexpressing ALLENE OXIDE SYNTHASE from Arabidopsis thaliana (AtAOS) and bread wheat (TaAOS) and OXOPHYTODIENOATE REDUCTASE 3 from A. thaliana (AtOPR3) under the strong constitutive promoter (ZmUbi1), either individually or both genes simultaneously. The delivery of the expression cassette encoding AOS was found to affect morphogenesis in both wheat species negatively. The effect of transgene expression on the accumulation of individual jasmonates in hexaploid and tetraploid wheat was observed. Among the introduced genes, overexpression of TaAOS was the most successful in increasing stress-inducible phytohormone levels in transgenic plants, resulting in higher accumulations of JA and JA-Ile in emmer wheat and 12-OPDA in bread wheat. In general, overexpression of AOS, alone or together with AtOPR3, negatively affected leaf lamina length and grain numbers per spike in both wheat species. Double (AtAOS + AtOPR3) transgenic wheat plants were characterized by significantly reduced plant height and seed numbers, especially in emmer wheat, where several primary plants failed to produce seeds.

The presence of several sets of chromosomes in polyploid crops is a serious problem for the application of gene and genome editing systems. Efficient CRISPR/Cas-based mutagenesis of series of genes involved in the grain starch biosynthesis of hexaploid triticale has been developed. Triticale (×Triticosecale),is a hybrid of rye (Secale) and wheat (Triticum) and consists of three subgenomes. Four genes were targeted and to ensure efficient editing of all subgenomes, a trio of guide RNAs for each target genes were designed. To enable simultaneous editing of 36 genetic loci at once (three sgRNAs × four genes × three subgenomes), an expression cassette was constructed, assembled as an array of twelve sgRNAs. The polysitron vector was delivered to morphogenic calli using a gene gun [1] together with a vector encoding Cas9 nuclease [2] to induce mutations. A number of transgenic plants of spring and winter triticale carrying both Cas9 and sgRNAs inserts have been generated. The efficiency of native gene editing varied depending on the target gene and sgRNA activity. Using a trio of sgRNAs for each target gene, we successfully mutated all three subgenome copies, thereby modifying seed starch synthesis. It can be expected that the described approach will make an important contribution to the future breeding of polyploid crops to produce various combinations of new genetic alleles encoding desired traits. The study is supported by Kurchatov Genomics Center of All-Russia Research Institute of Agricultural Biotechnology, agreement No. 075-15-2019-1667.