Mechanism Of Peptides-receptor Kinases Regulates Pollen-stigma Interaction

In plant sexual reproduction,pollen-stigma communication is the first stage of male-female interaction,ensuring the germination of compatible pollen,which is one of the barriers in distant hybridization.So far,little is known about the molecular mechanism of pollen-stigma recognition.In Arabidopsis thaliana,PCP-B(Pollen Coat Protein B-class),a cysteine-rich peptide family in pollen coat has been found playing a role in the regulation of pollen hydration and germination.Nevertheless,how the stigma recognizes PCP-Bs peptides and regulates downstream signaling pathways remain elusive.This study focused on how stigma perceiving and responding to pollen PCP-Bs signals.The main results are as follows:1.Pollen PCP-B? peptide induces the reduction of the reactive oxygen species levels in stigma papilla cells for the regulation of pollen hydration.The results showed that the levels of ROS in the papilla cells of stigma reduced by pollination in Arabidopsis.The pollen hydration rate and germination were accelerated on the stigma fed by ROS inhibitors and scavengers.The ROS levels of stigma treated with PCP-B?peptide were reduced.Furthermore,the pollen hydration was also accelerated on the stigma treated with PCP-B? peptide.Therefore,PCP-B? induced the decrease of stigma ROS levels which promoted pollen hydration.2.PCP-Bs-FERONIA/ANJEA regulated the ROS level of stigma and affected the pollen hydration.In this study,we found that FERONIA/ANJEA,members of the Cr RLK1 L family,were expressed in the stigma.The mutation of FERONIA or ANJEA led to a decrease of ROS levels in stigma papilla cells.The pollen grains adhered to the related mutants of FERONIA and ANJEA can complete hydration in a short time.It was found that the effect of PCP-B? on pollen hydration was mediated by FERONIA/ANJEA in genetic experiment.Also,PCP-B? could interact with the extracellular domain of FERONIA or ANJEA in biochemical experiments,respectively.Therefore,FERONIA/ANJEA,acting as PCP-B? receptors,jointly regulated the ROS level of stigma and affected pollen hydration.3.Stigma maintained ROS level via RALF23/33-FERONIA/ANJEA/LLG1-ROPs-RBOHD signaling pathway before pollination.The results showed that RALF23/33,LLG1,ROP2 and RBOHD genes were highly expressed in stigma.It was found that the mutants of these genes all showed low ROS levels in stigma,and the pollen hydration rate on stigmas of these mutants were accelerated.Biochemical experiments showed that RALF23/33 could interact with FERONIA/ANJEA extracellular domains as well.The level of ROS in stigma was increased by RALF33 peptide treatment,but not in the stigma of anj fer,llg1 and rbohd mutants.Therefore,this signaling pathway maintained the stigma ROS level before pollination.4.PCP-B? and RALF33 were competitively bound to FERONIA/ANJEA.PCPB? inhibited the function of RALF33 which could revert the ROS level of ralf33 in a manner of concentration-depended.There was no significant difference in pollen hydration rate between the ralf33 stigma treated by PCP-B?/RALF33 and the control ralf33.Biochemical experiments further showed that PCP-B? could compete with RALF33 in FERONIA-RALF33 complex with higher affinity.Our findings uncovered an intricate mechanism that PCP-B peptides could competitively combine with stigma autocrine RALF33 to FERONIA,thereby repressing stigma ROS levels to facilitate pollen hydration.As Cr RLK1 L,RALFs,PCP-Bs,RAC/ROPs and NADPH oxidases are all conserved in the plant kingdom,the antagonistic control of ROS levels by PCP-Bs and RALFs through Cr RLK1 L might represent a fundamental mechanism by which compatible pollen disengages stigma gating in flowering plants.This study demonstrates that receptor kinases are multifunctional and can sense and switch interactions with different types of small peptides,which is another important example of antagonistic small peptide regulating plant development.