ROXY1 Interacts With BHLH74 To Mediate Root Growth In Arabidopsis Thaliana

ROXY1 encodes a CC-type land plant specific glutaredoxin(GRX)that is mainly involved in cellular redox and other life processes and plays an important role in plant growth and development.bHLH74,a member of the bHLH family of transcription factors,is widespread in plants and is involved in important biological processes such as plant growth and development,stress resistance,and disease resistance.In a yeast two-hybrid screening,bHLH74 was picked up as a new interactor of ROXY1.To further validate relationship between ROXY1 and bHLH74 in planta and to explore the function of ROXY1 and bHLH74 in Arabidopsis development,we performed Bimolecular Fluorescence Complementation(BiFC),pull-down in vitro assays,analysis of the expression patterns and mutant phenotypes.The main findings of this paper are as follows:1.The interaction of ROXY1 with bHLH74 was verified.Two different experimental approaches,in vivo BiFC and in vitro GST pull-down,were used to further validate the interaction between ROXY1 and bHLH74.2.The promoter activity and subcellular localization of bHLH74 were analyzed.Expression constructs containing fused Gus reporter and fused GFP reporter into Arabidopsis showed that the bHLH74 promoter was expressed in stigma,petals,leaves,stems and lateral roots,and was highly expressed in primary roots;Subcellular localization bHLH74 was found to be predominantly distributed in the nucleus.bHLH74 partially overlaps with ROXY1 at the sites of expression in roots,thereby inferring that bHLH74 interacts with ROXY1 in roots,collectively participating in root growth and development.3.The biological function of ROXY1 with bHLH74 involved in Arabidopsis root growth and development was investigated.By comparing the root growth of the roxy1-2,bhlh74-1 and bhlh74-2 mutants to that of wild-type plants under the same conditions,it was found that the roots of the roxy1-2,bhlh74-1 and bhlh74-2 mutants were all shorter than those of the wild type,whereas the roots of the bhlh74-1 roxy1-2 double mutant were much shorter.These results indicate that both ROXY1 and bHLH74 are involved in root growth in Arabidopsis and positively regulate root growth.4.Identification and analysis of important cysteines in bHLH74.We used point mutation to mutate the three cysteines(C75,C101 and C242)in bHLH74 to serine,respectively,and showed that only when the C242 was mutated,bHLH74(C242S)no longer interacted with ROXY1 and could not rescue the phenotype of the bhlh74-1 mutant,and the root did notgrow longer when it was overexpressed.These results indicate that C242 is essential for proper bHLH74 function.5.The conservation of the interaction of ROXY1 with bHLH74 was analyzed.BiFC was used to verify that ROXY1 interacts with bHLH74 in tomato,indicating that this interaction of ROXY1 with bHLH74 is conserved across species.Meanwhile we also verified with BiFC that bHLH74 could form homodimer by itself,heterodimer with its cognate bHLH49,and ROXY1 interacted with bHLH49 as well.6.To explore whether bHLH49 is also involved in root development,bhlh49 mutants were obtained by CRISPR-cas9 technology.The finding that the length and morphology of the roots of bhlh49 mutants were indistinguishable from those of wild type,but that the roots of bhlh74-1 bhlh49 double mutant plants were shorter compared with those of bhlh74-1 mutants,suggests that bHLH49 is also involved in root growth.7.Downstream target genes of ROXY1 and bHLH74 were investigated.Using RNAseq,we analyzed the downstream target genes of ROXY1 and bHLH74,and found that 173 genes were up-regulated in both roxy1-2 and bhlh74-1,223 genes were down-regulated in both roxy1-2 and bhlh74-1,85% downstream target genes of bHLH74 were regulated by ROXY1,compared with only 24.3% downstream target genes of ROXY1 were regulated by bHLH74,indicating that both ROXY1 and bHLH74 are involved in the regulation of some same physiological processes or pathways,such as phenylpropanoid biosynthesis,phytohormone signaling processes,starch and sucrose metabolism and the MAPK signaling pathway,which in turn affect root growth and development.Previous studies reported that ROXY1 interacts with TGA-b ZIP TF family members to participate in floral organ development and disease resistance,whereas in this study,ROXY1 was found to interact with bHLH74 in roots,and some downstream target genes and biological pathways regulated by ROXY1 in combination with bHLH74 were initially analyzed.These findings provide a foundation for a comprehensive dissection of the molecular mechanisms by which ROXY1 and bHLH74 together participate in the regulation of root growth and development,and provide a scientific basis for the study of their same genes in important crops.