Identification And Functional Analysis Of Wheat OPR And LOX Gene Families In Response To Stresses

The JA(jasmonic acid)is an important phytohormone in plants.It can act as not only an endogenous hormone to regulate the growth and development of plants,but also signaling molecules to modulate responses to biotic and abiotic stresses.The biosynthesis of JA is via the AOS branch of Oxylipin signaling pathway,and the OPR and LOX are two key enzymes in the JA biosynthesis pathway.The OPR can reduce the OPDA to produce precursors of JA,thereby controling the final step of JA synthesis.Besides,the LOX regulates the initial step of JA synthesis through oxidizing unsaturated fatty acids to hydroperoxides.The identification and functional analysis have been conducted in Arabidopsis,tomato and a few other species,but the roles of OPR and LOX gene families in wheat were still not entirely clear.Hence,the whole genome identifications of OPR and LOX gene families were performed in this study.We analyzed the phylogenetic relationships,gene structure and protein conserved motifs of OPR and LOX gene families,and explored the cis-acting elements of promoters,the tissue-specific expression patterns as well as the responses to biotic and abiotic stresses.Finally,the biological functions of TaOPRIII-7 and TaLOX11-6A genes were systematically studied using transgenic wheat materials.The main findings are presented as follows:1.A total of 48 OPR genes were identified from the whole genome of wheat,and the Oxidored_FMN protein conserved domains were found in all of these genes.Based on the phylogenetic analysis,these 48 OPR genes could be divided into five subfamilies(sub.I-V),and there were 6,4,33,3 and 2 OPR genes in sub.I-V,respectively.The subfamily III contained the largest number of OPR genes.In addition,similar gene structures and protein conserved motifs were discovered in the OPR genes from the same subfamily.According to the analysis of promoter cis-acting elements,the wheat OPRs were highly related with growth and development,phytohormone-mediated processes as well as the biotic and abiotic stresses.The 48 wheat OPR genes were located on 15 of the 21 chromosomes in wheat,and no OPR gene were found on chromosomes 3A,3B,3D,4A,5D and 6A.Among these OPR genes,14 were tandem duplication genes,and 37 were segmental duplicated genes,indicating that the gene duplication played a vital role in the expansion of OPR gene family in wheat.The expression patterns of OPR genes are tissue-specific,and they expressed in at least one of the five tissues(leaf,root,stem,spike and grain).Besides,the expression pattern of OPR genes were stress-specific,and their expression could be regulated or induced by phytohormones(MeJA,ABA and SA)and various stresses(drought,heat,salt,wounding and aphids).To sum up,the wheat OPR is a multifunctional gene family.2.The TaOPRIII-7 gene is located on chromosome 2 in wheat.The TaOPRIII-7 is expressed in roots,stems,leaves,spikes and grains,and is especially highly expressed in leaves and roots(root > leaf).According to the qRT-PCR results,TaOPRIII-7 could respond to biotic(aphid)and abiotic stresses(drought,heat,salt and wounding).After silencing the TaOPRIII-7 using a hairpin silencing vector,the anthers of transgenic plants could not be normally cracked,and the pollen fertility and seed setting rate were significantly reduced,manifesting that the TaOPRIII-7 was involved in the development of male organs.Silencing the TaOPRIII-7 improved the resistance to greenbug in wheat,such as: 1)compared with transgenic plants,aphids were more preferred to WT(wild-type)plants,and the preference decrease rate in transgenic plants were significantly higher than that of WT;2)the non-puncturing time and xylem feeding time of aphids were increased,while the phloem feeding time was decreased significantly;3)the callose deposition in the phloem of transgenic plants was increased,and the expression of TaGSL,a defense-related gene,were increased as well.Thus it could be inferred that the TaOPRIII-7 gene activated the phloem-induced aphid defense;4)Silencing of TaOPRIII-7 gene resulted in increased OPDA content and increased tolerance to aphids,indicating that the OPDA might play an important role in the resistance to aphids.In addition,enhanced resistance to drought stress were observed in the TaOPRIII-7-silenced wheat plants.3.A total of 50 members in wheat LOX gene family were identified and could be divided into two subfamilies,9-LOX and 13-LOX.The gene structure and protein motifs are conserved,but the differences in several structures and the deletion of some protein motifs might be an important cause for the functional diversity.The distribution of wheat LOX on chromosomes was non-random,and they were located on 19 of the 21 wheat chromosomes.42 TaLOX genes were located on chromosomes 2,4,5 and 6.Among the 50 LOX genes,40 genes of them were replicated genes,suggesting that the gene duplication was of significance in the expansion of the LOX gene family.The TaLOX promoter contained multifarious cis-acting elements associated with transcription,cell cycle,development,phytohormones and stresses,and many of them were phytohormone-and stress-related elements(MeJA,ABA,GA,SA,IAA,ET,anoxic induction,wounding,pathogens,drought,light,low temperature and light-responsive elements).The TaLOXs were expressed in at least one of the five tissues(leaves,roots,stems,ears and grains),and they presented a tissue-specific expression pattern.The results of RNA-seq and qRT-PCR indicated that the wheat LOX genes could respond to various phytohormones and stresses with different expression patterns,and the expression of LOX could be induced by drought,heat,salt,aphids,ABA and SA.4.The TaLOX11-6A was located on chromosome 6A,and it belonged to the subfamily 9-LOX.The expression of the TaLOX11-6A gene were found in five tissues(leaves,roots,stems,ears and grains)of wheat.Based on the qRT-PCR tests,the TaLOX11-6A gene were responsive to some treatments,such as drought,salt,aphids,ABA and SA.Overexpression of TaLOX11-6A gene in wheat improved the resistance to drought stress significantly,and the roots grew faster than WT plants under drought stress.Besides,the overexpressed TaLOX11-6A increased the expression of TaLOX,TaOPR and TaPLD genes.Moreover,the overexpression of TaLOX11-6A could reduce the rate of chlorophyll decline,increase accumulation of proline,decrease the production of malondialdehyde as well as increase the activity of SOD,thus enhancing the scavenging ability of ROS.Therefore,the increased tolerance to drought after the overexpression of TaLOX11-6A was attributed to the enhanced stability of cell membrane,the reduced membrane damage as well as the alleviated oxidative damage by ROS,and the JA and other related signaling pathways were involved in these biological processes.