Functional Analyses Of Plant ICKs During Megasporogenesis And Megagametogenesis

ICKs/KRPs(Inhibitors of cyclin-dependent kinase/Kip-related proteins,shorted as ICKs below)are a family of plant cyclin-dependent kinase(CDK)inhibitors,and considered as core cell cycle regulators,since ICK/KRP proteins can inhibit cell cycle progression and cell division.The Arabidopsis genome has seven ICK/KRP genes.Currently,we know little about the functions of the plant ICK/KRP genes and the specific role of each member of the ICK/KRP gene family,partly due to functional redundancy of multiple family members.Previously,five ICKs(1/2/5/6/7)were down-regulated and studied in our laboratory.The objective of this study is to create ICK T-DNA insertion hextuple and septuple mutants,and systematically analyze the effects on plant growth and development when all seven ICK genes are knocked out.Phenotypic analysis of the hextuple mutant ick123567 and the septuple mutant in which all seven ICK genes were inactivated,showed that the seedling and seed weights of the septuple mutant increased compared to those in the quintuple mutant and WT.Various abiotic stress treatments were tested including auxin(NAA),cytokinin(kinetin),abscisic acid(PBI425-an ABA analog),NaCl,sucrose,paraquat and MMS(ethyl methane sulfonate)to analyze the effects on the root length and seedling growth of the WT and septuple mutant,but no significant difference was observed between the WT and septuple mutant.Interestingly,the septuple mutant had much shorter siliques,and a reduced number of seeds per silique.Cytological observation showed that 47%of septuple ovules were aborted,while the ovules were normal in the WT,quintuple mutant ickl2567,and hextuple mutant ick123567.Results from reciprocal crosses showed that the short silique phenotype was observed only when the septuple mutant was used as the female parent,suggesting a defect in the female parent.Thus,embryo sac development was examined.At FG7(female gametophyte,FG)stage,the WT Arabidopsis embryo sacs normally had an egg cell,one central cell and two synergids after three antipodals disintegrated just before fertilization.Howerver,many embryo sacs of the septuple mutant contained extra nuclei,which could be recognized as two,three or four sets of egg,central and synergid nuclei,often with clear boundaries between different sets,suggesting the existence of separate embryo sacs.Using a pEC1.1::GUS marker for egg cells and pFIS2::GUS marker for central cells,we confirmed the presence of multiple egg and central cells in the mutant ovules.At functional megaspore(FM)stage,only one FM was observed in WT ovules,while about 30%of the septuple mutant ovules did not have an observable FM,but the remaining ones had one to three,in rare cases four,FMs.Further,at FG2 stage,the WT embryo sacs had one typical pair of nuclei while the septuple mutant ovules could have two to four pairs often with clear boundaries between the pairs,consistent with multiple FMs observed.However,in stage 2-I ovules,only one megaspore mother cell(MMC)was observed in the WT and the septuple mutant.The results lead us to conclude that knocking out all seven ICKs results in the the formation of multiple FMs which further develop into multiple female gametophytes in the septuple mutant.Three days after flowering,the WT ovules had one embryo and endosperm nuclei of uniform morphology distributed throughout the embryo sac,while the mutant ovules could have different types of nuclei in the endosperm,or two or three separate endosperm compartments and surprisingly two embryos.Furthermore,three days after flowering,the developing embryo expressed the pEC1.1::GUS marker,and in addition 1-2 other locations also showed GUS expression in some developing seeds,suggesting the presence of extra unfertilized egg cells.Consistent with the development of double embryos,twin seedlings were observed about 2%seeds of the septuple mutant.Genotyping of twin seedlings received the mixed pollen of WT and septuple mutant carrying the marker for central cell(pFIS2::GUS)showed that some twin seedlings had the same genotype,while other twin seedlings had different genotypes,indicating that the two seedlings were produced from fertilization by two different pollen tubes.The phenotypes of the septuple mutant could be complemented by reintroducing either ICK4 or ICK7 genomic fragment.Taken together,it is suggested that ICK/KRP genes not only function in the cell cycle regulation during plant growth,but also participate in the degeneration of the non-functional megaspores.In the normal plants,multiple ICK/KRP genes function redundantly to ensure that only one FM,one female gametophyte and consequently one embryo is developed normally.