Molecular Mechanisms Regulating Pz Ipt1 Expression In Male Gamete And Studies Of Cytokinin Controlling Female Gametophyte Development In Arabidopsis Thaliana

Male and female gametophyte development,double fertilization and embryo development in angiosperms are keys to the alternation of generations.Mature pollen is male gametophyte in angiosperms,and Arabidopsis mature pollen contains a vegetative cell and two sperm cells.In the past decade,many researches indicate that sperm cells have rich gene expression that is critical for the development and differentiation of sperm cells and embryo development after double fertilization.However,the regulatory mechanisms controlling gene expression in sperm cells have not been elucidated completed.In Arabidopsis,the megaspore mother cell undergoes meiosis to produce four megaspores in the ovule.Only one functional megaspore out of the four megaspores survives,and undergoes three rounds of mitotic divisions and subsequent cellularizations to produce a seven-celled mature embryo sac,including two synergids,an egg,two polar nuclei and three antipodal cells.Cytokinin,an important phytohormone,plays critical roles in regulating plant growth and development.However,the molecular mechanisms that cytokinin regulates the female gametophyte development are still largely unknown.In previous study we identified an isopentenyltransferase gene known as PzIPT1 that is highly selectively transcribed in one sperm cell morphotype(S_vn)of Plumbago zeylanica.In transgenic Arabidopsis,PzIPT1 promoter displays activity in both sperm cells and upon progressive promoter truncation from the 5'-end results in a progressive decrease in reporter production,consistent with occurrence of multiple enhancer sites,which contribute to the gene's high level of expression.Cytokinin-dependent protein binding?CPB?motifs are identified in the promoter sequence,which respond with stimulation by cytokinin in an apparent feed-forward mechanism.Expression of reporter GFP could be enhanced when transgenic pollen grains of?130::GFP and?154::GFP were treated with 100 nM cytokinin for 15 min,30 min,45 min,and60 min.The expression of GFP decreased after the transgenic pollen grains were treated for 120min.When the cytokinin-treated pollen grains were washed with pollen medium,retreatment with cytokinin for 15 min could enhance the expression of GFP again.However,GFP expression did not change when transgenic pollen grains of?89-95::GFP with CPB sites deleted were treated with cytokinin.Expression of PzIPT1 promoter in sperm cells confers specificity independent of previously reported Germline Restrictive Silencer Factor?GRSF?binding sequence.Instead,a cis-acting regulatory region consisting of two duplicated 6-bp Male Gamete Selective Activation?MGSA?motifs occurs near the site of transcription initiation.Disruption of this sequence-specific site inactivates GFP expression in sperm cells,but not other sporophytic tissues or ogans.Multiple copies of the MGSA motif fused with the minimal CaMV35S promoter elements can confer reporter gene expression in sperm cells.Similar duplicated MGSA motifs are also identified from37 promoter sequences of sperm cell-expressed genes in Arabidopsis,and expression of 6 genes in sperm cells was already confirmed,suggesting selective activation is possibly a common mechanism for regulation of gene expression in sperm cells of flowering plants.Moreover,a pollen cDNA library was constructed for yeast one-hybrid screening of MGSA binding proteins,and several genes possiblely regulating sperm gene expression have been identified,providing a startpoint for further studies of regulatory mechanisms of gene expression in angiosperms sperm cells.To investigate the effects of cytokinin on embryo sac development,low-level cytokinin mutant ES::CKX1 and high-level cytokinin mutant ES::IPT1 were constructed.Both of the two mutants are sterile.Microscopic observation found that embryo sac development in ES::CKX1 and ES::IPT1 was retarded,resulting in arrested female gametophytes at the one-,two-,or four-nucleate stage when normal female gametophytes develop to the eigth-nucleate stage.Female gametophyte development requires three times of perfect mitosis that is finely regulated by a series of cyclins and cyclin-dependent protein kinases.We crossed all cell cycle regulating genes into transgenic plants of ES::CKX1 and ES::IPT1,and found many of them were up-or down-regulated when compared with wildtype plants.For example,G₂ checkpoint marker genes CDKA;1,CDKB2;1 and CDKB2;2 were down-regulated in embryo sacs of ES::CKX1 and ES::IPT1;CYCB1;1,a cyclin should be degraded when cell cycle moves from G₂ to M phase,was up-regulated in embryo sacs of ES::CKX1 and ES::IPT1;the cytokinin response regulator genes ARR10,ARR12 and ARR18 were down-regulated in ES::CKX1 and ES::IPT1.The above results show that cytokinin is required for female gametophyte development through regulating the expression of important cyclins and cyclin-dependent protein kinases in the cell cycle.