Function Analysis Of Phospholipase D/phosphatidic Acid Involves In Self Incompatibility Of Pear

Self-incompatibility(SI)is an important mechanism to prevent inbreeding and promote cross fertilization in plants.S-RNase-based gametophytic SI mechanism is an important SI type and exists in account for more than half of known SI plant species.S-RNase has been shown to be acting as the pistil determinant in SI.After entering the SI pollen,S-RNase become cytotoxic and inhibits the growth of pollen tube.A wide range of potentially cytotoxins are found in the environment of plants.Cytotoxin is recognized by the target cell then exerts its bio-destructive activity.Simultaneously,the cells exert a spectrum of predetermined strategies constitutes resistance mechanism to counter the cytotoxic effect.Programmed cell death(PCD),often as the ultimate endpoint of extreme defense,terminates the spread of cytotoxin.However,the presence of a defense mechanism exerted by SI pollen tube for the cytotoxic effect of S-RNase is largely unknown.We propose a novel mechanism in pollen during the response to the S-RNase cytotoxical attack,which advances the understanding of signal transduction in SI.To explore this scientific problem,this study makes many progresses as follow:1.The study provides a valuable reference gene resource for gene expression analysis in pear pollen.Ten candidate reference genes were chosen and examined in 36 pear samples.Among these,PbrEF1?,PbrGAPDH and PbrAPT were developed to detect the purity of cDNA.Three qRT-PCR analysis programs,geNorm,NormFinder and BestKeeper,were applied for systematic determinations.The results showed that all the candidate reference genes could be used for qRT-PCR normalization in pear pollen.PbrEF1? was the most stable reference gene across all abiotic stresses of pollen samples,especially for high temperature,1-butanol and NaCl treatments,and also during pollen development.PbrTUB was the optimal reference gene for pear tissue expression.PbrGAPDH was suitable for low temperature treatment and PbrPP2A was suitable for abscisic acid and also for NaCl treatment.For CuCl2 treatment,either of reference genes PbrCYP and PbrUBI was suitable.To illustrate the suitability of these reference genes,the expression profile of PbrVHA-?was used to analyze the expression patterns in different pear tissues,pollen developing stages and NaCl treatment.The results provide a valuable resource for gene expression characterization in pear pollen.2.Here we report that PLD and PA have a role in the process of polarised plant cell expansion as represented by pollen tube growth.Totally 18 PLD genes were identified in pear.These PLD genes can be divided into six groups ?,?,?,?,? and ? through domain and phylogenetic analysis.Pear PLDs originates from 6 ancestral genes,include PbrPLD?1,PbrPLD?1,PbrPLD?1,PbrPLD?,PbrPLD?1,and PbrPLD?,then,expanded by fragment replication through 4 replication events strings.Gene expression analysis showed that PbrPLDs were expressed differentially in various organs.Seven PLD genes were predominantly expressed in pollen,including PLD?1,PLD?3,PLD?4,PLD?1,PLD?1,PLD?2 and PLD?1.Among them,only PbrPLD?1 showed a marked enhancement in the expression level in response to self-S-RNase challenge.PbrPLD?1 located on the plasma membrane.These studies not only resolve the evolutionary path of the PLD gene family in the pear genome,but also provide us with a perspective on the potential role of PbrPLD?1 in the SI process.3.The incompatible S-RNase activates PbrPLD?1 and increasing the concentration of PA and reduced SI-induced PCD.The specific inhibition of PbrPLD?1 enzyme activity by antisense oligonucleotide silencing impaired SI-induced PA production and accelerated PCD in SI pollen tubes.Moreover,complementation assays by adding PA into the culture solution alleviated the accelerated death of antisense oligonucleotide PbrPLD?1 pollen tubes in SI response.This evidence is important for determining the mechanism of action of PLD is an important step to increase the defense ability of incompatibility pollen tube of pear.4.S-RNase directly interacts with the actin protein,PbrAct1,eliciting the depolymerization of the actin cytoskeleton and,consequently,inducing PCD in SI pollen tubes.The RC4 as the S-RNase converse domain serves for interaction between S-RNase and PbrAct1.P156 as the key amino acid of RC4 regulates the interaction with actin in pear.The PbrPLD?1 produced PA,which postponed actin cytoskeleton depolymerization,thus delaying the death of the SI pollen tube.The specific inhibition of PbrPLD?l enzyme activity abolished this protective function.Furthermore,pharmacological experiments show that PA is located upstream of the actin filament signaling pathway.In summary,we propose a protective strategy for pollen tube against the S-RNase signalling in the early steps of SI response.An increased titer of PA,elicited by membrane PbrPLD?1,prevents depolymerization of the actin cytoskeleton,delaying PCD for SI pollen tube.This is probably the evolutionary mechanism for pollen tube to try its utmost to transport the male gametes for fertilization.These results provide an extra dimension in signalling control upon S-RNase challenge in pollen tubes during SI.