| The citrus gametic sterility is associated with male sterility and embryo sac sterility.Studies on citrus gametic sterility can clear out the gametophyte developmental process in citrus and supply theory basis for citrus seedlessness breeding.?Qianyang Seedless‘ Ponkan mandarin(QS)(Citrus reticulata Blanco)and a common seedy Ponkan mandarin ?Egan NO.1‘(EG)were used in the current study.The cytological work was performed on male and female gametophyte of both EG and QS,and the comparative proteomics was investigated by 2-DE to identify differentially expressed proteins(DEPs)in EG and QS early developmental flower buds.1.The cytological works were performed to unveil developmental progress of gametophytes in EG and QS.The detail process of microsporogenesis was compared in QS and EG anthers.Based on the transverse section result,abnormal phenotype of QS was firstly detected at the tetrad stage when uniformly four haploid microspores were enclosed in one tetrad of EG but more than four haploid microspores were observed in one tetrad of QS.The microspores further developed to form vacuole,enlarge with visible exine wall and gem pores at the middle microspore stage.In ES,a ball of dense cytoplasm was in the middle of the cell.In contrast,there were many variable sizes of hollow microspores in the mutant QS.Also,remains of degenerated abnormal microspores were visible in QS.Finally,compared to EG in which most of the mature pollen grains were full of cell solute,in QS defective microspores developed into variable sized pollen grains defaulted in cell solute accumulation at the mature pollen stage.The detail process of embryo sac development performed in QS and EG from stage megasporocyte formation to stage eight-nucleate embryo sac revealed that QS embryo sacs were abnormal in succession after the functional megaspore formation,displayed abnormal mitosis or pre-stopped development to generate degenerated nucellar remnants.Ultimately,QS embryo sac was smaller and empty with degenerate traces in it at eight-nucleate embryo sac stage.2.The chromosomal behavior during meiosis stage in pollen mother cells of EG and QS was compared.The male meiosis defects in QS initiate before or at zygotene stage,behaves in the thinner chromatin threads at the zygotene stage,no formation of the telomere bouquet structure,univalents at the diakinesis stage,chromosome bridge at telophase? and chromosomal fragments after telophase ? stage.To make clear the number of microspores in one microsporangium of ES and QS,the microspores at tetrad stage were observed after squashing and staining with modified carbol fusin.The tetrad in ES was consisted of four uniform microspores.While in QS,there were heterogeneous microspore populations from dyads to octoploids in one microsporangium.More than 400 cells were counted,the statistical data revealed that about 77.5% of the QS tetrads were abnormal.Moreover,24.5% were aberrant tetrads with four different sized microspores and 18.6% were pentrads.3.Histological analyses and TEM observation was conducted on microspores in EG and QS.The microspore section of EG and QS stained by PAS further demonstrated that pollen starch filling process which initiated at early two-nucleate microspore stage was impaired in QS.There was indistinguishable difference in polysaccharide distribution pattern between EG and QS anthers before the early two-nucleate microspore stage.While at the early 2-nucleate microspore stage,EG microspores began to accumulate starch grains yet QS microspores had no starch deposited.At the mature pollen stage,EG pollen had abundant starch while almost all the QS pollens were hollow.The pollen wall development was compared between EG and QS.The pollen wall development is defective in QS,especially the exine.At the tetrad stage,primexine and probacula are formed around the microspore,while a thin primexine is formed in QS.At early uniucleate microspore stage,the complete patterned exine including bacula,nexine and tectum is produced in EG and QS.Every part of exine in EG is obviously thicker than that in QS.The exine is supposed to be PAS negative reaction.Compared to EG,exine layer of QS mature pollen was thicker.The blue autofluorescene of microspores observed under UV light showed that blue auto fluorescence was scattered in EG microspore cavity while it was concentrated in the wall area of QS microspores.These results indicated that the development of pollen wall was affected in QS.4.A comparative proteomic study was performed to investigate the differentially expressed proteins(2-DE)in QS.2DE combined with MALDI-TOF-TOF Mass Spectrum was conducted to investigate the DEPs in EG and QS flower buds at three stages,when the anthers were undergoing tetrad to uninucleate microspore stages.97 spots corresponded to 87 unique proteins with high confidence were identified.1)The DEPs were mainly including proteins related to stress defense,carbohydrate/energy metabolism,protein synthesis,modification and degradation,and flavonoid and lignin related secondary metabolism.LAP6,ACOS5 and DRL1 which are potential sporopollenin precursor biosynthesis regulating proteins were identified.Phenylpropanoid metabolism,response to abiotic stress especially response to oxidative stress and energy metabolism were enriched biological processes of DEPs at early developmental stages.2)Of all the DEPs,the protein expression of DEPs related to response to oxidative stress and pollen wall development showed relatively stable up or down-expression mode in QS.This suggests that most of biological processes in QS are disordered.3)5 proteins involved in protein synthesis,modification and degradation,and 5 proteins related to in ROS regulating signal pathway,flavonoid metabolism,cell and chloroplast division were only detected in MF1 and MF2 flower buds,but not in QS MF2 flower buds.Protein ORP3 A was only detected in EG MF1 flower buds and ORP3 A is involved in lipid transport.The 11 mentioned proteins might play important roles in vacuole formation and tapetum PCD(programmed cell death)which specially initiated in microspore at MF1 stage. |
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