Functional Characterization Of Jasmonate Signaling Regulatory Genes NINJA And DFR From Gossypium Hirsutum L.

Plant growth and defense always keep a balance,resulting from the re-allocation of energy and nutrients.Some evidences documented that jasmonic acid(JA)signaling pathway is associated with this balance.Despite the importance of growth-defense tradeoffs in shaping plant productivity in natural and agricultural ecosystems,the molecular mechanisms underlying growth and defense are poorly understood.In this study,the two genes regarding plant growth and defense were addressed in JA signaling pathway.Firstly,a DFR cDNA homolog encodes a 355 amino acid peptide was cloned from early developing fibers of upland cotton,designated as GhDFR1.Silencing GhDFR1 in cotton by virus-induced gene silencing(VIGS)led to significant decrease in accumulation of anthocyanins and proanthocyanins(PAs).Based on LC-MS analysis,two PA monomers,(–)-epicatachin and(–)-epigallocatachin,remarkably decreased in content in fibers of GhDFR1-silenced plants,but two new monomers,(–)-catachin and(–)-gallocatachin were present compared to the control plants agroinoculated with empty vector.The ectopic expression of GhDFR1 in an Arabidopsis TT3 mutant allowed for reconstruction of PAs biosynthesis pathway,leading to accumulation of PAs in seed coat.A cotton NINJA(novel interactor of JA ZIM-domain)gene was isolated by PCR methods,named as GhNINJA.GhNINJA contains a 1305 bp open read frame,and encodes a 434 amino acid peptide.According to quantitative real-time PCR(qRT-PCR)analysis,GhNINJA is preferentially expressed in roots.And its expression level is greatly induced by Verticillium dahliae infection.Through VIGS technique,we developed GhNINJA-silenced cotton plants.Based on qRT-PCR analysis,the expression level of the target gene in GhNINJA-silenced plants only reaching an 6% average expression level of the control.Phenotypic observation showed that the regenerating lateral root growth of silenced plants was largely inhibited compared to the control.Analysis by microscopy demonstrated that the cell length of the root differentiation zone in GhNINJA-silenced plants is significantly shorter than those of the control.Moreover,the silenced plants exhibited higher tolerance to V.dahliae infection compared to the control,which was linked to the increased expression of the defence marker genes PDF1.2 and PR4.Taken together,these data demonstrate that GhDFR1 contributes to the biosynthesis of anthocyanins and PAs in cotton.GhNINJA represses the root growth and enhances the tolerance to V.dahliae.The dissecting function of the two genes should facilitate in well understanding the potential mechanism of plant growth and defense,and provided some information to selective defense genes and breeding.