Functional Studies On The Regulation Of Cell Elongation By AtWOX11,12 In Arabidopsis Thaliana

WOX gene family is a family of transcription factors unique to plants.It participates in the development of flowers,leaves,roots,callus formation,embryo formation and other different growth and development processes.However,there is a lack of systematic studies on the function of WOX family genes in A.thaliana.Therefore,based on subcellular localization analysis and functional studies of previously reported WOX family genes,we found that WOX11 mainly participates in the development of roots,stems,and buds,while WOX12 research is related to root development.However,the fact that WOX11 and WOX12 belong to the same intermediate clade in evolution,with high degree of homology.We speculate that WOX12,similar to WOX11,may participate in other parts of plant growth and development,such as stem and bud development,in addition to participating in root development.And in the study,it was found that AtWOX11 and AtWOX12 were expressed to varying degrees in other parts.Therefore,we conducted further functional exploration in A.thaliana WOX11 and WOX12.The main results of this study are:1.Subcellular localization of WOX family proteinsSubcellular localization analysis was performed for the WOX family proteins,and it was found that AtWOX1,2,3,4,5,6,7,8,9,10,11,12 were expressed in the nucleus and cell membrane in N.benthamiana and A.thaliana protoplasts,and less in the cytoplasm,indicating that the WOX family proteins are mainly expressed locally in the nucleus and cell membrane,thus exerting stable biological functions.2.Bioinformatics analysis of AtWOX11,12AtWOX11（AT3G03660.1）has a full length of 2946 bp and a CDS region of 807 bp.It has 4 exons and 3 intron regions,encoding a protein sequence of 268 amino acids.AtWOX12（AT5G17810.2）has a full length of 4328 bp and a CDS region of 807 bp.It has 3 exons and 2 intron regions and encodes a protein sequence of 268 amino acids.According to the physical and chemical properties and signal peptide analysis of AtWOX11 and AtWOX12 proteins,they are both relatively stable hydrophilic proteins without signal peptide.After multiple sequence alignment of AtWOX11,12,it was found that they have a high degree of similarity,especially in the highly conserved homologous structural regions of HD.Using protein secondary and tertiary structure analysis,AtWOX11,12 protein structure contains significantαhelical regions,which are highly similar,indicate that they are prone to form helix-turn helix（HTH）motifs that bind to DNA,and they have similarities in binding sites,which may play a similar role in gene expression regulation.3.Expression pattern analysis of AtWOX11,12q RT-PCR quantitative analysis of AtWOX11 and AtWOX12 was performed on the roots,stems,rosette leaves,flowers,and pods of 35 days wild type A.thaliana.It was found that AtWOX11 was highly expressed in the roots and pods of A.thaliana,followed by the stem and flowers,and the rosette leaves were the least expressed;AtWOX12 is expressed in various parts of A.thaliana above ground,with the highest expression in roots and flowers,followed by rosette leaves and pods,and lower expression in stems.It can be seen that AtWOX11 and AtWOX12 are constitutively expressed in A.thaliana.4.Identification and phenotypic analysis of AtWOX11,12 overexpressing plantsOE-AtWOX11 and OE-AtWOX12 overexpression vectors were constructed and transformed into wild-type A.thaliana.The stable expression lines of transgenic A.thaliana were selected from T₃generation.RT-PCR identification results showed that the expression levels of different individual strains of OE-AtWOX11 and OE-AtWOX12were up-regulated,with AtWOX11-3 being the highest upregulated by 400 times and OE-AtWOX12-3 being the highest upregulated by 300 times.We conducted phenotype statistics and analysis on three A.thaliana overexpression transgenic lines of OE-AtWOX11 and OE-AtWOX12.The results showed that compared with wild-type A.thaliana（Col-0）,OE-AtWOX11 and OE-AtWOX12 overexpression lines were inhibited to varying degrees in plant height,stem thickness,pod length,petiole length,rosette leaf length,rosette leaf width,and flower development.This indicates that overexpression of AtWOX11 and AtWOX12 may be inhibit the growth and development of aboveground organs in A.thaliana.5.AtWOX11,12 affects plant cell elongationThrough scanning electron microscopy observation of the main stem and rosette leaves of OE-AtWOX11 and OE-AtWOX12,it was found that compared with the wild-type control（Col-0）,the main stem cells and rosette leaf epidermal cells were significantly smaller,and the higher the overexpression,the more significant the inhibition of cells,indicating that cell elongation was inhibited to varying degrees in the OE-AtWOX11 and OE-AtWOX12 lines.Subsequently,we conducted q RT-PCR analysis on the cell elongation genes（EXPA1,EXPA8）,cell elongation related hormones IAA,and BR synthesis pathway genes（IAA7,IAA17,BR6ox1,BR6ox2）in OE-AtWOX11 and OE-AtWOX12.The results showed that the expression levels of these related genes were significantly down-regulated in OE-AtWOX11 and OE-AtWOX12.It is speculated that overexpression of AtWOX11,12 will lead to significant inhibition of IAA and BR synthesis pathways.Thus,it may be inhibit the expression of genes related to cell elongation and inhibits cell elongation.