Regulation Of BBM And WOX On Adventitious Root Development In Larix

Transcription factors play important regulatory roles during plant growth and development.APETALA2(AP2)subfamily genes are involved in the growth and development of plants.WUSCHEL-related homeobox(WOX)transcription family,being unique to plant,plays an important role in the regulation of stem cell maintenance,lateral organogenesis and adventitious root regeneration.Larix is an important fast-growing and ecological afforestation species,but is difficult to root,and the rooting mechanism remains uncovered.Larix kaempferi × Larix olgensis hybrid clones-day wing 8 × long mix 18-10 with both good growth traits and rooting capacity,which was breded by Forestry Research Institution,Chinese Academy of Forestry,was taken as marerials in this study.By integrating the genomics and bioinformatics,genetics and molecular biology methods,the larch AP2 subfamily and WOX gene family were identified,and six genes,including Lk BBM1,Lk BBM2,Lk WUS,Lk WOX66,Lk WOX65 and Lk WOX52 were selected and investigated for their roles in the regulation in the development of adventitious roots of larch.The main results were as follows:(1)By using bioinformatics methods,a total of 38 AP2 genes were identified in larch genome,which were unevenly distributed on the 27 scaffolds,which classified into three major branches,eu AP2,base NT and eu ANT.Tandem repeats may be the main pattern of the AP2 subfamily gene expansion.The structure of those genes was quite different,with the exons number ranging from 1 to 10.Some introns were obviously longer,which may contain inserted transposon.71 WOX family genes were identified,which were distributed unevenly on 39 scaffolds and divided into four branches(modern branch/WUS branch,middle branch,ancient branch and peculiar branch).21 WOXs genes belonged to the modern/WUS branch and contained a conserved WUS-box domain.Members with similar phylogenetic relationships had similar intron/exon structures,gene lengths,and conserved motifs,which may have similar functions.(2)Lk BBM1 and Lk BBM2 were highly expressed in adventitious root tips,and Lk BBM2 was expressed in the formation layer of stems during the initiation of adventitious root primordia.The fused protein Lk BBM1-GFP was only localized in the nucleus whereas Lk BBM2-GFP was localized in both the nucleus and cytoplasm.Over-expression of Lk BBM2 and Lk BBM1 in Arabidopsis significantly elongated the roots.Furthermore,over-expression those two genes in the hybrid poplar(Populus alba × P.glandulosa)significantly increased ARs number,plant height,aboveground and root biomass.(3)Lk WUS,Lk WOX66,Lk WOX65 and Lk WOX52 are nuclear localization proteins that play roles in nucleus.Lk WOX65 was mainly expressed in the layer formation area of the stem and around the epidermis of the ARs,and the level of expression in the middle and end of the ARs were higher than that in the ARs tip.Over-expression of Lk WOX65 and Lk WOX52 in poplar significantly increased ARs number,the root length became shorter,plant height and roots biomass of ten-week-old seedlings were significantly higher than controls.Over-expressed of Lk WUS and Lk WOX66 significantly increased ARs number,roots biomasses were significantly higher than controls,while the difference between seedling height and ground diameter is not significant.(4)The endogenous hormones contents of JA and ABA in Lk WOX65-OE stems were significantly decreased,while the contents of IAA,JA and ABA in roots were significantly increased,the contents of IAA and ABA in Lk WOX52-OE stems were significantly increased,and JA and ABA levels in roots were significantly increased,indicating that those two genes affect AR development by regulating these hormone levels.Differentially expressed genes of Lk WOX66,Lk WOX65 and Lk WOX52 overexpression plants were analyzed by RNA sequencing.We found that the expression of plant hormone signaling genes(ARF2,ARF3,ARF7 and ARF18 et al.),rooting-related transcription factors(NAC 89-like,WOX5,LBD29 and SCR et al.),ARs or root development related genes(CYCD3,GRF1 and TAA1 et al.)were affected,which preliminary reveal the mechanism of those three WOX genes during adventitious root development.Yeast two-hybrid c DNA libraries of larch ARs were constructed and library capacity was greater than 1.15*10^7CFU.Three proteins interacting with LKWOX65 were screened,namely palmitoyltransferase(PAT18),acyl-Co A binding protein6(ACBP6)and COP1-interacting protein7(CIP7).It indicating that LKWOX65 may play a regulatory role in adventitious root development by interacting with these three proteins.Here,the role of BBM and WOXs genes in ARs development were studied in larch.Those results will provide basis for comprehensive research on the mechanism of BBM and WOXs genes in different species and also provide theoretical significance and potential application value for larch genetic improvement and cutting propagation.