Molecular Mechanism Analysis Of ZmCSLD1 And Map Based Cloning Of QLA2-1 For Plant Architecture Development

Maize is a staple crop in the world.It is necessary to enhance maize yield to meet the market demand due to the increased population in future.Plant architecture,which mainly includes plant height,leaf length,leaf width,leaf angle and tassel architecture traits,is critical to increase maize production.Therefore,dissecting the genetic basis of plant architecture traits,cloning the genes with major effects,and subsequently elucidating the molecular mechanism of the formation of plant architecture,will contribute to the genetic improvement of maize breeding.Previously,our group identified a cellulose synthase like protein,ZnCSLD1,controlling maize organ size and QTL for leaf angle in a tesosinte-maize population.In this study,the molecular mechanism of ZmCSLD1 regulating maize organ size was elucidated,and the major QTL--qLA2-1 for leaf angle was fine-mapped.The major results are in the following:1.In our previous study,ZmCSLD1,which encoded a cellulose synthase like protein,was identified as a candidate gene for a major QTL-qLW10 controlling leaf width.To confirm this,three different ZmCSLD1 alleles,qlw10MTL,qlw10BYK and Zmcsld1Mo17 was used to carry out allelism test with a known Mu mutant,Zmcsld1W22.As expect,the decreased leaf width of Zmcsld1W22 couldn't be complemented by three different alleles,which confirmed ZmCSLD1 is the real causal gene for qLW10.2.ZmCSLD1 was conserved in the 32 species by phylogenetic analysis.Transient expression in maize protoplasm showed ZmCSLD1 was subcellular localized in the trans-Golgi.Cell wall composition analysis in Zmcsld1W22 and ZmCSLD1W22 show that ZmCSLD1 played an important role in the cell wall biosynthesis.3.ZmCSLD1 had a pleiotropic effect that affected the size of multiple maize organs.Knocking out ZmCSLD1 repressed plant growth.Interestingly,allelism test show that mutant phenotype was complemented in F1 of Zmcsld1Mo17 and qlw10BYK,which might be caused by intragenic complementation because the causal sites occurred in different protein domains.This hypothesis was further validated by yeast-two hybrid system,indicating that ZmCSLD1 participated in cell wall biosynthesis by homodimer or homomultimeric complex.4.Cell size and number analysis indicated that the narrowing of the qlw10MTL leaf was mainly caused by a decrease in cell number because of a reduction in cell division.According to expression pattern,ZmCSLD1 was highly expressed in the rapid cell division region of different organs,consistent with cell circle related gene CycB1;4.Therefore,ZmCSLD1 might participate in cell division by affecting cell wall plate formation.5.A total of 827 differnntially expressed genes(DEGs)between ZmCSLD1W22 and Zmcsld1W22 were identified from three leaf tissues 9B,8B and 8M,and 379,298 and 396 DEGs for 9B,8B and 8M,respectively.Among these DEGs,8 DEGs were annonated to participate in cell wall biosynthesis.Cell wall composition analysis show that all monosaccharide and crystalline cellulose were decreased in 9B and 8B of leaf tissues in ZmcsldlW22.Thus,loss function of ZmCSLD1 might affect the formation of cell wall.6.The microscopy observation by light,transmission electron and scanning electron microscope found wart clusters occured on the leaf undersurface of Zmcsld1W22 and Zmcsld1Mo17,which were caused by abnormal cell extension.The immunofluorescence analysis showed the decreased xylan but increased mannan for the cell wall of wart cells.In addition,there were 9 DEGs related to cell expansion,which were highly expressed in 8M of Zmcsld1W22.Thus,the formation of wart cluster might be due to the changed cell wall composition in Zmcsld1W22.7.Previously,our group identified six QTLs for leaf angle in a teosinte-maize BC2F5 population.One of the major QTL for leaf angle on chromosome 2,qLA2-1,explained 9.16%of phenotype variation,and the teosinte allele had an increase effect on leaf angle.The effect of qLA2-1 was confirmed by using two heterogeneous inbred families,XM034 and XM024.Compared with the maize allele,the leaf angle in teosinte allele increased 21.36° and 12.16° in the populations derived from XM034 and XM024,respectively.qLA2-1 was subsequently narrowed down to 39-kb region in the upstream of ZmLG1 by using 3000 BC1F2,4500 BC2F2 and 2000 BC3F2 of XM034 and 1200 BC1F2 of XMO24.8.Sequence analysis identified 424 polymorphic sites in 39-kb region in 508 maize diverse line.Two SNPs and three In-Dels were significantly associated with leaf angle at P ? 1.0 × 10-3,among which four polymorphic sites were in complete LD.9.The measurement of leaf auricle areas,cell size and cell number in NIL34A and NIL34B showed that the leaf auricle areas of NIL34B was significantly higher than NIL34A,which was mainly caused by the increase of cell number.In addition,the expression level of ZmLG1 in NIL34B was higher than that of NIL34A at the cell division phase of leaf auricle.In summary,ZmCSLD1 was subcellular localized in the trans-Golgi,highly expressed in the early stage of organ development.Loss-function of ZmCSLD1 affected the cell wall polysaccharides biosynthesis,which led to decreased cell division and increased cell expansion,and finally repressed plant growth.In addition,a major QTL for leaf angle,qLA2-1,was narrowed down 39-kb region in the upstream of ZmLG1.The teosinte allele increased the expression level of ZmLG1,and promoted cell division of leaf auricle,which consequently results in flat leaf angle.