Map-based Cloning And Functional Analysis Of Hairy Leaf 6 In Rice

Trichomes are specialized plant epidermal cells,which develop as hair-like projections that extend from the epidermal surface.Trichomes serve a diverse range of protective functions such as lessening heat load of leaves,reducing water transpiration rates,enhancing tolerance to UV radiation,and increasing herbivore resistance in plant species.Trichome formation has been extensively studied as a mechanistic model for epidermal cell differentiation and cell morphogenesis in Arabidopsis.As the research model in monocot plants,most studies in rice focused on the plant type,yield production,grain quality and resistance,etc.Little research about trichome in rice was reported.To analyze the genetic and molecular mechanisms underlying trichome development in rice,genome-wide association study and linkage analysis were conducted to map key genes response for trichome development.The function of HL6,the key factor controlling hairy leaf through map-based cloning was analyzed and a regulatory network was proposed.The main results are as follows:1.There are three kinds of leaf trichomes in rice germplasm:glabrous,hairless and hairy leaf.Genome-wide association study was performed in the 253 accessions using the genotype of 50 K SNP array.Two significant SNPs were detected in chromosome 6 responsible for the hairy leaf phenotype.Transgenic lines over-expressing two candidate genes,Loc_Os06g44300 and Loc_Os06g44320,respectively,showed no significant change in the trichome phenotype compared with the wild type indicating there maybe other genes responsible for the hairy leaf in rice germplasm.2.The hairy leaf phenotype was controlled by a single dominant gene in the F₂population constructed from the hairy parent W39 and the hairless parent ZS97.The gene responsible for the hairy leaf was named as HL6 which was mapped in chromosome 6 using BSA strategy.HL6 was fine mapped to the region of 55 kb between the markers S5 and S9.Complementary test showed Loc_Os06g44750was HL6.The variations in the promoter and coding region of HL6 caused difference in transcription level and amino acid,respectively.The complementary transgenic lines with the promoter of HL6^W3939 replaced by HL6^ZS97S97 remain to be hairless and the lines over-expressing different HL6 alleles all showed hairy leaf suggesting that HL6 transcriptionally regulates hairy leaf.3.HL6 encodes an AP2/ERF protein containing a single AP2 domain,localizing in the nucleus and the C terminal region plays the important role in transcription activation.Phylogenetic analysis revealed that HL6 is closely related to EMBRYOMAKER which controlling ectopic trichome formation on cotyledons in Arabidopsis,supporting that a conserved function of AP2 proteins in the regulation of trichome development in monocot and dicot plants.4.HL6 had different response to auxin treatment in NILs,with a slight repression to HL6^ZS97S97 but an approximate 2-fold induction to HL6^W39.Two AuxREs showed sequence variations between HL6^W3939 and HL6^ZS97,where one SNP at-2375 site and a 19 bp at-2361 site leads two AuxREs mutated in HL6^ZS97.EMSA showed OsARF25 could bind to the two Aux REs in HL6^W3939 but did not bind to the two mutated AuxREs in HL6^ZS97.These two putative AuxREs with variations in HL6 may be responsible for different responses to auxin in the NILs.5.Genetic analysis in the population from hairy parent W39 and glabrous parent LEMONT showed OsWOX3 B which controls glabrous leaf genetically interacted HL6 and the HL6 controlled the hairy phenotype is dependent on functional OsWOX3 B.OsWOX3B regulates the expression of HL6 through binding to its promoter directly.HL6 also interacts with OsWOX3 B to form complex through AP2 and HD.HL6 and OsWOX3 B regulated the expression of OsYUCCA5,OsPIN1 a,OsARF4 and OsARF7 simultaneously.Both of HL6 and OsWOX3 B could bind to OsYUCCA5 directly and the protein interaction between HL6 and OsWOX3 B could enhance the binding of HL6 to OsYUCCA5.6.HL6 controlled hairy leaf could increase the resistance to the hot stress and repress the water transpiration rate in leaves,however HL6 also had negative effect on the yield production.7.There are at least 10 haplotypes for HL6 in rice germplasm.The W39 allele HL6^W3939 is belong to the haplotype H10 and occurs with a rare frequency of 0.4%.The nucleotide diversity of HL6 in the assayed cultivated rice was much less（one-tenth）than that in the wild rice.The parameters of Tajima’s D test and other tests indicated that HL6 in indica rice deviated significantly from the neutral selection expectation.These results suggest that the HL6 for hairy leaf in the cultivated indica rice was from wild rice,which was under strong selection during rice domestication.Our findings provide new insights in the molecular network of HL6 and OsWOX3 B involved in the auxin-mediated pathway that regulates trichome formation in rice and may be beneficial to the genetic improvement of leaf trichome and yield traits through HL6.