Research On The Regulation Of WUSCHEL-related Homeobox1 (CsWOX1)in Leaf Development Of Cucumis Sativus L.

Leaf,an important source organ,is the main place for photosynthesis in plants.Its morphology can directly affect the photosynthetic efficiency of plants,and is also an important part of plant morphogenesis,which plays an important role in plant growth and development.Leaf morphology,including shape and size,as part of plant structure,can directly affect plant formation and agricultural productivity.In previous studies,cucumber mango fruit(mf)mutant showed sterility defects in both male and female flowers,and we found that the mutant phenotype was caused by the lack of protein in cucumber WUSCHEL-related homeobox1(CsWOX1).In addition,mf mutant also showed obvious defects in leaf middle-lateral axis polarity development,which was manifested by significantly narrower leaf width.Therefore,in this study,we mainly revealed the mechanism by which CsWOX1 regulates vein development and size of cucumber leaves.Relevant research is mainly carried out from the following aspects:1.Leaf developmental defects in mf mutant were systematically identified.Leaves of mf mutant with continuous leaf positions showed obvious defect in the middle axis polarity of true leaves,especially in the distal region of leaves,and the leaf width became narrower with the increase of seedling age relative to the wild type.The width measurement of the distal region of the blade showed that mf leaves were significantly smaller than those of AM218 at each leaf position.At the same time,the ratio of leaf width to leaf length of mf mutant was also significantly smaller than that of wild type AM218,and the difference gradually widened from the sixth leaf position to the first leaf position.The longitudinal profile results of AM218 and mf plants showed that the palisade cells of wild-type AM218 plants were denser and thicker than those of mf plants.The shape of spongy cells in mf plant was more irregular and larger than that in AM218 plant.The leaf epidermal cells of mf were observed by scanning electron microscope,and the leaf cell size of mf was significantly larger than that of wild-type AM218.The number of cells of mf mutant was significantly less than that of AM218 plant under the same view.This showed the deficiency of cell development on the proximal and distal axis of mf mutant leaves.2.The role of CsWOX1 in leaf polarity development was investigated.By in situ hybridization analysis,CsWOX1 expression was confirmed in the middle area of the leaf margin.Atwox1 prs double mutant was constructed in Arabidopsis,and ectopic expression analysis confirmed that CsWOX1 could greatly restore the defect of leaf extension in the Atwox1 prs double mutant,which proved the role of CsWOX1 in leaf extension.3.It was found that CsWOX1 affected cucumber leaf shape development by regulating auxin polar transport gene.Transcriptome sequencing of mf mutant and AM218 plant leaves showed that there were some differential genes related to auxin polar transport.mf rl double mutant was constructed by mf mutant and round leaf(rl)mutant,which was caused by the mutation of Cs PID1,a maintenance gene of auxin polar transport in cucumber.Leaf shape of mf rl was similar to that of rl mutant.These results indicate that CsWOX1 is involved in normal leaf shape formation by relying on the function of Cs PID1.We further identified the auxin polar transport carrier genes in cucumber.It was found that the most of auxin polar transporter genes PINs and AUX/LAXs were down-regulated in mf mutant,while these genes were up-regulated in CsWOX1-overexpressed(OE)plants.CsWOX1 was found to directly activating the expression of Cs PIN2.All these evidence indicates that CsWOX1 affects leaf shape development depends on auxin polar transport process.4.The influence of CsWOX1 on the pattern formation of cucumber leaf vein was studied using mutant material.Through the mf rl double mutant,we found that CsWOX1 may influence the vein formation pattern by relying on auxin polar transport.The vein building patterns of different leaf shapes in this study,including wild-type,mf mutant,rl mutant,and OE plants,were statistically analyzed.It was found that the length,density and angle between primary vein and secondary veins were strongly correlated with the change of leaf shape.Therefore,it is speculated that CsWOX1 may be involved in vein pattern building by regulating auxin polar transport carrier gene,and affects leaf shape development.5.CsWOX1 affected cucumber leaf size by regulating TCP gene.Leaves of OE plants were significantly smaller than those of the wild type.Compared with wild-type plants,the cell volume of mf and OE plants was significantly larger and smaller,respectively.Through yeast two-hybrid library screening,it was found that CsWOX1 could interact with CIN-TCP4 a,which controls the process of cell development.At the same time,CIN-TCP genes in OE plants were also significantly up-regulated,while those in mf plants were significantly downregulated.suggesting that CSWOX1 has an effect on CIN-TCP genes at the transcription level and protein level,resulting in the opposite trend of cell volume changes in leaves of mf and OE plants.In addition,the study also found that CIN-TCP4 c could inhibit the promoter activity of CsWOX1,suggesting that there may be a feedback regulation between CIN-TCP4 c and CsWOX1.Both of them jointly coordinate the development state of leaf cells and thus affect the process of leaf expansion.In conclusion,CsWOX1 can influence the establishment of cucumber leaf shape and vein pattern by regulating auxin polar transport process.At the same time,CsWOX1 regulated the development state of leaf cells and the process of leaf expansion by establishing feedback regulation with CIN-TCP4 c.The discovery of these interaction factors promoted the regulatory mechanism study of WOX1 in leaf development.