The Mechanism Of Callose Synthetase PbrCalS1B.1 In Maintaining Pollen Tube Lifecycle Of Pear

Pear（Pyrus L.）belongs to Rosaceae family,which is one of the three major fruit trees in China.In the production process of pear,as the pollen tube cannot grow fast and healthy in the stylophore group and reach the ovule,resulting in fertilization failure,the fruit set rate is greatly reduced,which seriously affects the fruit yield.The callose is widely distributed in the pollen tube cell wall,providing sufficient mechanical strength for the lateral walls of the pollen tube to resist expansion pressure,and also forming callose plugs to maintain rapid elongation of the pollen tube and timely release of sperm cells to complete double fertilization.However,little is known about the molecular mechanism of callose regulation of pollen tube life cycle in this process.Therefore,it is important to analyze the fine regulation mechanism of callose synthesis in the whole life cycle of pear pollen tube in order to eventually overcome the low fruit set rate and select high-yielding pear varieties.This study uses’Dangshansuli’as pollen material for culture in vitro.Take’Cuiguanli’as the mother and’Dangshansuli’as the father to conduct in vivo pollination experiment to investigate the function of callose metabolism in regulating the life cycle of pear pollen tubes during pollination and fertilization.The main results are as follows:1.Continuous callose plug deposition maintains the pear pollen tube life cycle.In this study,we found that under in vitro culture conditions,pear pollen tubes started to grow rapidly after 3 hours post-culture（HPC）and then declined at 15 HPC.At the same time,pollen tubes in the late growth stage gradually accelerated senescence under the initiation of programmed cell death（PCD）,and the semi-growth duration(GD₅₀)of pollen tubes was 16.16 h.The overall growth-senescence curve was S-shaped.In addition,continuous deposition of callose plugs was accompanied by up to four or more throughout the pollen tube life cycle,and changes in relative callose content were highly correlated with pollen tube elongation rate（R=0.991）.In this study,we also found that treatment of pear pollen with the exogenously applied callose synthesis inhibitor 2DDG（2-deoxy-D-glucose）under in vitro culture conditions significantly reduced the number of callose plugs and relative callose content in pear pollen tubes;it also significantly inhibited the growth of pear pollen tubes and shortened GD_50.Furthermore,in vivo pollination experiment,by 0.5 m M2DDG treatment of pear pollen,the actual density of callose plug in the stylar tissue is significantly reduced,and the length of pollen tube only grows to 53.6%of the stigma length,finally unable to complete fertilization.These results indicate that continuous callose plug deposition is the essential basis for maintaining the pollen tube life cycle of pear,and the obstruction of callose synthesis may lead to premature senescence and loss of fertility of pear pollen tube.2.PbrCalS1B.1 is a key gene regulating callose synthesis in pollen tubes.In this study,we systematically identified 100 CalS genes in nine species of Rosaceae（M.domestica,P.communis,P.bretscheideri,P.persica,Pavium,P.mume,R.chinensis,R.occidentalis,F.vesca）.By constructing phylogenetic trees,CalS members of Rosaceae species were divided into 3 subgroups and 9 clusters,which were highly homologous to the Arabidopsis CalS gene and did not evolve additional clusters.The transcriptome data,GUS（β-glucuronidase）tissue mapping and RT-q PCR（Real-Time quantitative Polymerase Chain Reaction）analysis showed that PbrCalS1B.1 was specifically expressed in pollen tubes of pear.Finally,by subcellular localization analysis,PbrCalS1B.1 was found to be a protein localized to the plasma membrane.In this study,atcals5-1/PbrCalS1B.1 overexpressed transgenic lines were constructed to restore the fertility function of atcals5-1 mutants.It was found that the pod length,seed number,seed loss rate,pollen cell wall morphology and callose plug number almost recovered to the wild-type level.In addition,an Antisense Oligonucleotide（as-ODN）technique was used to inhibit the expression level of PbrCalS1B.1 during the growth of pear pollen tube,and it was found that the quantity of callose plug and relative callose content in pear pollen tube decreased sharply.At the same time,the PCD process of pear pollen tube was accelerated,resulting in a significant decrease of GD₅₀.The vitality of its pear flower powder is also greatly reduced.These results indicate that PbrCalS1B.1 is a key gene regulating callose synthesis in pollen tube and mediating pollen tube fertility function.3.PbrbZIP52 transcriptionally regulates PbrCalS1B.1 to promote callose plug deposition and maintain pollen tube life cycle in pear.Transcription analysis showed that there was a bZIP member PbrbZIP52 in the pollen tube growth process of pear,and its expression level was closely related to that of PbrCalS1B.1（R=0.975）,and the two had similar tissue expression patterns.By inhibiting the expression level of PbrbZIP52 during the pollen tube life cycle,the expression level of PbrCalS1B.1was significantly down-regulated.In addition,specific silencing of PbrbZIP52 results in a reduced number of callose plugs and a significant decrease in the relative callose content,leading to growth restriction and even premature senescence of the pollen tube.It similar to specific silencing PbrCalS1B.1 treatment.In addition,two A-box elements were found in the PbrCalS1B.1 promoter sequence.The Yeast one hybrid（Y1H）,Electrophoretic Mobility Shift Assay（EMSA）and transient transformation activity analysis all indicated that PbrbZIP52 interacted with the promoter of PbrCalS1B.1.In order to further verify this interaction,site-specific mutations were performed on the A-box element in the PbrCalS1B.1 promoter.The results showed that A single A-box mutation greatly weakened the binding strength of PbrbZIP52 and PbrCalS1B.1 promoters,and the simultaneous mutation of two A-boxes resulted in almost complete inhibition of their binding.These results confirmed the specificity of PbrbZIP52 binding to the promoter of PbrCalS1B.1.These results reveal a new regulatory mechanism by which callose regulates the pollen tube life cycle of pear and affects its fertility:the transcription factor PbrbZIP52 regulates the transcription by binding to A-box elements in the PbrCalS1B.1 promoter,in order to promote callose deposition to maintain normal pear pollen tube life cycle and finally achieve double fertilization.This finding provides theoretical guidance for breeding high-yielding pear varieties and stabilizing the seed setting rate of commercial orchards.