Characterization Of MiRNAs Associated With Spike Development And H₂O₂ Response In Wheat

Common wheat(Triticum aestivum L.)is the important food crop in the world,and increase of wheat production has always been the main goal of breeders.Wheat grain yield consist of three components:spikes per plant,grains per spike and grain weight,grain number per spike can be dissected into two subcomponents:spikelets per spike and grain number per spikelet.An increase in any of these components will directly contribute to grain yield.Therefore,it is of great significance to improve wheat yield by identifying genes regulating spike development and studying their molecular regulation mechanism.It has been found that overexpressed wheat miR156 can regulate the tillering and spike morphology of wheat[1].In our study,we ectopically expressed miR156 in wheat spikes to specifically explore its function and molecular mechanism in spike development.Hydrogen peroxide(H2O2)is the most stable reactive oxygen species(ROS),which altered expression of genes that response to H2O2 encoding proteins involved in cell defence,signal transduction,redox homeostasis and energy homeostasis.In plants,many miRNAs related to H2O2 stress have been discovered,61 miRNAs have been found to be afected by H2O2 in Brachypodium distachyon,and 7 are afected in rice[2,3]However,miRNAs in response to H2O2 in wheat were still unclear.Thus,we identified miRNAs and their target genes in reponse to H2O2 in wheat by small RNA sequencing and degadome sequencing.The main results are as follows:1.MiR156 affects wheat spike development through negative regulating TaSPL genesWe generated the wheat transgenic lines with specifically higher expression of miR156 in spikes,and the spike length of transgenic lines was significantly decreased compared with wild type CB037.Moreover,the average length of rachis internode and spikelet number in transgenic lines was significantly decreased compared with CB037.Based on the published degradome sequencing data and modified RNA ligase-mediated 5'-rapid amplification of cDNA ends[4],8 TaSPL genes targeted by miR156 were identified.TaSPL3,TaSPL13,TaSPL14,TaSPL 16 and TaSPL 18 were significantly down-regulated in spikes of independent transgenic lines compared with CB037.It is indicated that miR156 regulated wheat spike development through mediating TaSPL genes cleavage.2.TaSPL14 with highly expression in spikes and stem affects spike length and plant height TaSPL14 were mostly abundant in wheat stem and young spikes.We generated TaSPL14 knock out mutants using the CRISPR/Cas9 system,transgenic plants with three homoeologous genes mutated simultaneously with frameshift mutations were obtained by analysised the sgRNA target sites of transgenic plants.taspl14 mutants showed decreased plant height and shortened spikes compared to wild-type Fielder.Moreover,the average length of rachis internode and spikelet number of taspll4 mutants was significantly decreased compared with Fielder.It is suggested that TaSPL14 affected wheat spike development through affecting rachis internode elongation and spikelet formation.3.TaSPL14 regulated the expression of genes involved in ethylene response signalingRNA sequencing was performed with wild-type Fielder and taspl14 mutants,and we identified 641 down regulated genes and 191 up regulated genes contained cis-element that TaSPL14 could combained in 2-kb promoter sequence of each gene by FIMO tool,it is indicated that these genes maybe the target genes of TaSPL14.Among them,TaSPL14 could directly and dramatically elevate the driving activities of promoters of TaEILl,TaERFl and TaRAP2.11,which involved in ethylene signal transduction pathway.3.Identification of the H2O2-responsive miRNAs and their target genesWe identified a total of 70 miRNAs had a minimum 1.5-fold difference between treated samples and untrated samples(P<0.05)by small RNA aequencing,and 86 unique target genes of 28 H2O2-responsive miRNAs were identified by degradome sequencing.The target gene Ta#S58897680 of miR3493b encoded a thioredoxin-like protein which is a component of the antioxidant system,indicated that miR3493b responsed to H2O2 stress by regulating esxpression levels of Ta#S58897680.