Differential Proteomic Analysis Of Ovule During The Period Of Free Nuclei Mitosis Of Megagametophyte In Pinus Tabulaeformis Carr.

The megagametophyte(female gametophyte)is the core structure of ovule.The period of free nuclei mitosis of megagametophyte is the critical stage in the development process of ovule.The failure of nuclei mitosis will hinder the maturity of megagametophyte and the production of egg cell,potentially leading to female sterility of plants.So it is of important theoretical and practical significance to study the molecular regulatory mechanism of ovule development in this period.At present,the molecular mechanisms that regulate ovule development have been reported,but mainly focused on angiosperms.There are few studies on gymnosperms.The ovule development of gymnosperms is different from that of angiosperms.The difference is especially apparent for woody plants.In previous studies,our research group found that the Pinus tabulaeformis Curr.ovules of No.28 Female-Sterile Line collected from P.tabulaeformis Seed Garden in Xingcheng,Liaoning Province,China aborted mainly because of the megagametophyte free nuclei stopped-mitosis in the midway,and considered that the phenomenon of female sterility was a result from common regulation and expression of multiple genes.Proteins,as the products of the comprehensive expression of functional genes,directly reflect the regulating characteristics of plants growth and development.Therefore,with the use of two-dimensional electrophoresis(2-DE),isobaric tags for relative and absolute quantification(iTRAQ)and other proteomics technologies,and by means of morphology,anatomy and molecular biology,this paper gave a study on differentially expressed proteomes of P.tabulaeformis FL(fertile line)and SL(sterile line)ovules during the period of free nuclei mitosis of megagametophyte,so as to investigate the molecular regulatory mechanism of ovule abortion in No.28 SL.The following results were obtained:1.In the generation and development process of P.tabulaeformis ovulate strobilus,the morphological and anatomical features were systematically observed.The development stages of ovule were accurately grasped.The main results were as follows.In the first autumn,when the longitudinal cliameter(LD)of bud was about 7.0 mm,the axillary bud primordium developed.With the arrival of winter,the bud lay dormant.In the spring of the second year,when the new ovulate strobilus LD(LDOs)was about 5.0 mm,ovule cells differentiated.When LDOS was about 6.0 mm,megaspore mother cell differentiated:about 11.0 mm,dozens of free nuclei were in ovule.After that,the ovule lav dormant throughout the winter.In the spring of the third year,when LDOs about 21.0 mm,there had been several thousands of free nuclei.When the megagametophyte began to form cell walls,LDOs was about 31.0 mm.When LDOs was about 37.0 mm,archegonial initial cells differentiated;about 43.0 mm,archegonia and egg cells got mature,and the cellularization of megagametophyte completed;about 45.0 mm,fertilization occurred.In autumn,when LDOs was about 53.0 mm,mature embryoes can be observed.The generation and development process of P.tabulaeformis ovulate strobilus lasted 26 months over a span of three years.2.The separation and analysis based on 2-DE was conducted for ovule proteomes of the two P.tabulaeformis lines in the vigorous period of free nuclei mitosis of megagametophyte.Finally,1,424 and 1,224 proteins were separated in FL and SL ovule,respectively,among which,42 proteins were differentially expressed in two lines(ratio>1.5).Compared to FL ovule,the expression levels of 9 proteins in SL were decreased and 33 proteins increased.By mass spectrometry analysis,9 proteins were successfully analyzed,including serine/threonine protein kinase,calmodulin,pantothenate kinase,UDP-glucose pyrophosphorylase,DNA-directed RNA polymerase subunit beta,heat shock cognate protein 70-1,ATP synthase subunit beta and two uncharacteristic proteins.They were involved in substance and energy metabolism,cell cycle,transcription regulation and stress resistance,potentially indicating that the substance and energy metabolism was abnormal and SL ovule might be under stress.3.The separation and analysis based on iTRAQ was conducted for ovule proteomes of the two P.tabulaeformis lines in the early and late stages of free nuclei mitosis of megagametophyte.Finally,4,192 proteins were separated,and more than 400 differentially expressed proteins were obtained in four comparisons(ratio>1.2).Mass spectrum and bioinformatics analysis results indicated that at least 37 differentially expressed proteins were involved in the regulation of ovule development of P.tabulaeformis,including cinnamoyl-CoA reductase,peroxidase precursor protein,glycerol-3-phosphate acyltransferase 6,electron transfer flavoprotein:ubiqionone oxidoreductase,glutathione S-transferase,1-aminocyclopropane-1-carboxylate oxidase,DNA-dependent protein kinase catalytic subunit,calcium B-like protein,etc.Their expression levels indicated that in SL ovules,(1)substances and energy might be deficient,perhaps leading to abnormal DNA replication.(2)Because of the incomplete antioxidant system and the abnormal expression levels of enzymes involved in cell signal transduction,DNA DSBs(Double-Strand Breaks)probably occured.(3)Facing the abnormities of DNA replication and damage,the cell cycle was arrested and the DNA damage failed to be repaired,potentially resulting in the occurrence of programmed cell death and ovule abortion in the female-sterile line of P.tabulaeformis.4.The gene expression patterns of 10 proteins,for instance,DNA-dependent protein kinase catalytic subunit,glutathione S-transferase,replication protein A et al.,were explored by reverse transcription qPCR,which further verified the results of aforementioned proteomics study.