Regulation Of Trichome Formation In Arabidopsis By DUF538 Proteins SVBs

Trichome formation in Arabidopsis is regulated by several key regulators including the WD40 protien TRANSPARENT TESTA GLABRA1(TTG1),the R2R3 MYB transcription factor GLABRA1(GL1),the b HLH transcription factor GLABRA3(GL3)or ENHANCER OF GLABRA3(EGL3),the homeodomain protein GL2,and the seven R3 MYB transcription factors.Plants hormones such as gibberellin,salicylic acid,jasmonic acid,and cytokinins have been shown to regulate trichome formation by affecting the transcription or activities of the key regulators.DUF538 protein SMALLER TRICHOMES WITH VARIABLE BRANCHES(SVB)has been shown to regulate trichome morphology in Arabidopsis.We report here the identification of SVB and its closely related genes SVB2 to SVB6 as abscisic acid(ABA)responsive genes.We found that the expression levels of SVB,SVB2,SVB4,SVB5,and SVB6 were increased in response to ABA treatment and their expression levels were reduced in the ABA biosynthesis mutant aba1-5.However,the expression level of SVB3 was decreased in response to ABA treatment.Characterization of mutants and overexpression transgenic plants of SVBs gene,suggest that SVBs function redundantly to regulate trichome formation in Arabidopsis.In addition to the trichome defects reported previously,we found that even though the trichome numbers were largely unaffected in all the svbs single mutants and the svb3 svb4 and svb5 svb6 double mutants generated by using CRISPR/Cas9 gene editing,the trichome numbers were greatly reduced in the svb svb2 double mutants and svb svb2 svb3 triple mutants.On the other hand,plants overexpressing SVB,SVB2 and SVB3 produced more trichomes.To provide further evidence on the roles of SVB,SVB2,and SVB3 in regulating trichome formation in Arabidopsis,we generated transgenic plants expressing SVB and SVB3 in the svb svb2 mutant plants.We found the number of trichomes in 35S:SVB/svb svb2 transgenic plants was fully restored,while the 35S:SVB3/svb svb2 transgenic plants showed a partial recovery of trichomes.The result indicates that SVB,SVB2 and SVB3 function redundantly to regulate trichome formation in Arabidopsis.RT-PCR results show that the expression level of the trichome formation key regulator gene EGL3 was increased in the svb svb2 double mutants;whereas the expression levels of all other key regulators genes remained largely unchanged.These results suggest that SVBs may not function by affecting the key regulator genes to regulate trichome formation in Arabidopsis.Considering that SVBs and R3 MYB transcription factors have opposite roles in regulating trichome formation,we examined if SVBs may function downstream the R3 MYB transcription factors to regulate trichome formation.We therefore edited SVB and SVB2 in the try cpc double mutant plants and observed that the loss of function of SVB in the try cpc double mutant plants resulted in decreased trichome formation,and trichome numbers were further decreased,when SVB2 was also mutated.These findings indicate that SVB and SVB2 may function downstream of TRY and CPC in regulating trichome formation.Since SVBs are ABA responsive genes,we examined if ABA signaling is required for SVBs regulated trichome formation by generating 35S:SVB transgenic plants in the ABA signaling mutants abi5-7 and abi5-1,and we found that the transgenic plants are still able to produce more trichomes.These results suggest that ABA signaling is not required for SVBs regulated trichome formation.On the other hand,we found that the svb svb2 double and svb svb2 svb3 triple mutants are more sensitive to ABA in the cotyledon greening assay,suggest that SVBs are involved in the regulation of ABA responses in Arabidopsis.In addition to regulate trichome formation,we also found that SVBs function redundantly to regulate plant growth and development in Arabidopsis,as the svb svb2 double mutant showed a stunted plant growth,whereas overexpression of SVB,SVB2,and SVB3 resulted in taller plants compared to the Col wild type plants.In summary,the results of this study indicate that SVBs are ABA responsive genes,that SVBs function redundantly to regulate plant growth and trichome formation in Arabidopsis,and that SVBs may function downstream of R3 MYB transcription factors to regulate trichome formation in Arabidopsis.