Isolation And Characterization Of Suppressor Of Hypocotyl Non-phototropism Mutant Rpt2 In Arabidopsis

To adapt to the environment,plants have evolved a series of photoreceptors that performed special function and sensed lights of different wavelengths in natural world.For example,phytochromes(Phys)specifically sense red and far-red light,cryptochromes(CRYs)and photochromes(PHOTs)can effectively sense blue light,while UVR8 senses ultraviolet light.Among them,PHOTs(PHOT1 and PHOT2)are important blue-light receptors,which are involved in regulating plant movements,such as phototropism,chloroplast movement,stomatal aperture,and leaf positioning and flattening.Previous study had elucidated the mechanism of PHOT1 in mediating LBL(Low Blue Light)-induced phototropism.However,the mechanism that PHOT1 and PHOT2 functional redundantly mediate the HBL(High Blue Light)-induced phototropic reaction remains unclear.Given that Arabidopsis thaliana showed an enhanced phototropism phenotype with the mutation of PHOT1,and the over-expression of PHOT2 in phot1 mutant could also enhance the curvature of hypocotyl,however,the phot1 phot2 double mutant lacked phototropism.These results demonstrate that PHOT1 initiates a dual reaction in mediating and inhibiting phototropism to adapt to light environment.Furthermore,it has been reported that rpt2 mutant lacks phototropism,while phot1 rpt2 double mutant restores phototropism,which indicated that PHOT1-mediated bypass inhibition pathway is located upstream of RPT2.To analyze the process of plant optimal growth orientation to avoid damage from high blue light,the subject aims at screening double mutant plants that restore hypocotyl phototropism from EMS(Ethylmethane sulphonate)mutagenized rpt2 mutant,and then cloning the suppressor of RPT2 gene to explain the mechanism that PHOT1-mediated inhibition of phototropism.With unilateral HBL treatment to rpt2-EMS-M2,eighteen stable genetic and restoring phototropic mutants had been obtained.The homozygous identification demonstrated that 18 mutants all were homozygous mutation of rpt2.The identification of phototropic curvature showed that the curvature of line-40 and line-124 appeared smaller than WT under unilateral HBL,and the curvature of other mutants was similar to WT.Under unilateral LBL,line-40,line-101,line-128 and line-155 appeared phototropism,while the other mutants lacked phototropic reaction.Genetic analysis indicated that restoring phototropism was recessive mutation in line-12,40,43,52,56,101,145,155 mutants,moreover,the recessive mutation was controlled by one gene individually in line-52 and line-56 mutant,therefore the map-based cloning could be applied to these two mutants.By BSA analysis to line-52 mutant,seven candidate genes were obtained,namely AT1G027 XX,AT3G467XX,AT3G518 XX,AT3G149XX,AT3G191 XX,AT3G280XX and AT3G526 XX.We ordered the T-DNA inserted seeds of these seven candidate genes from ABRC.After homozygous identification,phenotype in HBL of single mutants detect found that at3g149 XX and at1g027 XX did not have phototropic response.In order to identify whether these genes regulate HBL suppression,we constructed complement and overexpression vectors of these candidate genes and transformed the line-52 double mutant.Currently,the transgenic T2 generation materials were obtained for six genes.Meanwhile,these single mutants was hybrided with rpt2 mutant,now all of these double mutants are the case that RPT2 gene is homozygous mutation and the other gene is heterozygous mutation.Whether these candidate genes are the target suppressor genes is necessary to verify after obtaining the homozygous double mutant material.By coarse and fine positioning,the mutation site of line-52 was first located at F9K21 which locates at the middle and lower part of the chromosome ?,from the aspect of inhibitor of RPT2 gene.It was found that line-52 lacked the phototropic reaction under LBL,while phenotype of rpt2 single mutant was consistent with the WT,indicating that the mutation of the unknown gene individually leads to dis-phototropic response to LBL,after excluding the possibility of mutation of the PHOT1 gene,so next the map clone of line-52 mutant was constructed from a LBL angle.By expanding the sample size to finely position,the mutation interval was narrowed to two adjacent BACs including “H6P9” and “H9P21”.Through the amplification and sequencing of the localization interval,it was found that the gene “H9-7” has a base inserted mutation.The gene is involved in the regulation of the transcription process.Combined with the literature,we speculate that this gene may be related to the regulation of the transcription process by alternative promoter,which affects the expression mode of associated protein,resulting in the occurrence of phototropic response.Next,we will transform the rpt2 with the CAS9 vector of the gene,meanwhile,construct double mutant to verify the function of the gene.In addition,the characteristics of various stages of growth and development were also recorded in line-52.The rosette leaves in line-52 are more broad and stretch than in rpt2,suggested that the suppressor gene may be also involved in the regulation of leaf location and flatten.With the experiments under different level blue light treatments showed that there was not chloroplast accumulation in the rpt2 single mutant,while there was accumulation movement to a certain extent in line-52,suggested that the inhibitor gene might also be involved in the regulation of chloroplast accumulation.In conclusion,we established a screening system of suppressor of hypocotyl non-phototropism mutant rpt2 in Arabidopsis and successfully screened out 18 stable mutant materials,of which 8 mutants' traits were caused by recessive mutation and 2 mutants' traits were caused by recessive single gene mutation.Through BSA analysis and map-based cloning,the possible mutation gene of line-52 double mutant was obtained.By phenotypic analysis of the above mutants and functional prediction of the candidate genes,It was preliminarily proved that the RPT2 inhibitor gene may be involved in the regulation of hypocotyl phototropism,chloroplast accumulation and leaf positioning and flatten process,which provided an important basis for understanding the mechanism of regulating phototropic movements involved in adapting to changing light environment.