The Arabidopsis CrRLK1L Protein Kinases(CLPs) Are Required For Normal Growth Of Pollen Tubes In The Pistil

In flowering plants,the male gametophytes(pollen)and female gametophytes(embryo sac)contain male gametes(sperm cells)and female gamete(egg cell),respectively.They are produced in the anther of the stamen and the ovule of the pistil,respectively.During anthesis,the mature pollen grain is delivered to the stigma of the pistil,germinate after recognized by the stigmatic tissue and produce a pollen tube.The pollen tube invades into stigmatic cells,extends in the transmitting tract,finally enters the embryo sac and releases two sperm cells which were carried into the embryo sac for double fertilization.The polar growth of pollen tubes in the pistil is essential for the transportation of the sperms into embry sac and fertilization.It is one of the important steps in plant reproduction.Ensuring the normal polar growth in the pistil is regulated by interaction between pollen tube and the tissues in the pistil.However,little is known about the mechanisms that underlie the interaction between pollen tube and the pistil tissues.CrRLK1L subfamily is one of the supper RLK families in plants.They are involved in cell signaling.Especially,they have attracted more and more concerns by their involvement in female-male interaction.However,their roles in growth of pollen tubes in the pistil tissues remain poorly understood.This study characterized the roles and mechanisms of the pollen and pollen tube-expressed CrRLKIL RLKs in Arabidopsis.Expression patterns of the 17 members in the CrRLKIL subfamily were analysed using the microarray data provided by the TAIR website(www.arahidopsis.org).Five candidates were identified for the pollen tube-expressed CrRLKIL genes,namely:ANX1 and ANX2 which have been reported to be involved in pollen tube growth,CAPI involved in root development and the two functionally unknown CrRLKIL PROTEIN(CLP)genes CLP1 and CLP2.Real-time PCR analyses showed that CLP1 and CLP2 were expressed in pollen at a higher lever,while CAP1 was expressed at a lower level in pollen.The promoter activity and GFP-labeling assays also showed that CLP1 and CLP2 genes were highly expressed in pollen.Moreover,the CLP1 and CLP2 proteins were expressed in pollen and pollen tubes,and were localized on the plasma mcmbrance in the tip region of the pollen tube.These results indicated that the CLPs arc the RLKs expressed in the pollen tube and localized on the plasma membranes in the tip region of pollen tubes.The clp mutants in CLP1 and CLP2 genes were generated applying the CRISPR/Cas9 gene editing system.Four single mutant alleles:clp1-1,clp1-2,clp2-1,clp2-2 and two double mutants:clp1-1 clp2-1 and clp1-2 clp2-2 were obtained.The mutated sites were all targeted to the sequences within the extracellular domains of the CLP1 and CLP2,which caused a single base pair deletion or addition and resulted in premature generation of the stop codons corresponding to the regions within the extracellular domains,leading to premature interruption of the protein translation.The clpl mutants had a normal seed set,while the clp2 significantly reduced seed set,but the clpl clp2 double mutants did not enhance the phenotype in reduced fertility.Genetic analyses showed that the genetic transmitting efficiency of the male and female clpl gametes was normal,similar to that of wild type.The genetic transmitting efficiency of the female clp2 gametes was also normal,but transmition efficiency of male clp2 gametes was reduced significantly,and the clp1 mutants could slightly enhance the defect of clp2 in the genetic transmitting efficiency.The morphology,viability,nuclear development and in vitro germination of all clp mutant pollen tubes were similar to those of wild-type pollen grains.When germinated and grew in vivo,the clp1 pollen tubes could enter the transmitting tract and some of them exhibited slightly abormal surfaces,but the fertilization was not affected.However,the clp2 mutant pollen tubes exhibited a drastic depolarization in the pistil although they could enter the style tissues.They became swollen and unable to enter the transmitting tract,resulting in fertilization failure.The phenotypic complementation indicated that the defect of the clp pollen tubes in the pistil was caused by the mutations in the CLP genes.Furthermore,the dominant negative effect of CLP1 also exhibited a strong phenotype similar to that of the clp2 mutants.These results indicated that the CLP genes are important for ensuring the normal growth of pollen tubes in the pistil,and the CLP2 has a larger contribution than the CLP1.Y2H and BiFC assays showed that the CLP1 and CLP2 could interact with RopGEF1 or RopGEF12.In the plant cell assay system,the CLP-GEF interaction position was colocalized with the signals of plasma membrane-labeled marker.The assays also showed that RopGEFl and RopGEF12 could interact with ROP1,ROP3,ROP5 or ROP9.Furthermore,the CLP1 and CLP2 interacted with the autophosphated region of RopGEFl or RopGEF12,while ROP1,ROP3,ROP5 or ROP9 interacted with the PRONE domain of RopGEFl or RopGEF12,implying that the CLP,the GEF and the ROP may form a complex.The Y3H assays demonstrated that CLP1 or CLP2 and RopGEFl or RopGEF 12 could form a complex with ROP1,ROP3,ROP5 or ROP9.In addition,Y2H and BiFC assays showed that CLP1 could interact with CLP2 to form a heteropolymer,while CLP2 not only interated with CLP1 to form a heterogenous polymer,but also could form a homopolymer on the plasma membrane.In summary,the CLP genes play important roles in ensuring the normal polar growth of pollen tubes in the pistil.The CLP proteins may be involved in the interaction between pollen tubes and pistil tissues and regulate the polar growth of pollen tubes through ROP signaling pathway.These results could provide new cues for further study of the mechanisms that underlie pollen tube-pistil tissues interaction.