The Cellular And Molecular Mechanism Of Peanut Embryo Abortion Under Low Calcium In Soil

Peanut(Arachis hypogaea L.)is an important oil and economic crop.Calcium plays key roles in peanut growth and development.Calcium deficiency in soil results in empty or unfilled pods which seriously affects yields and qualities.However,the underlying cellular and molecular mechanism is not clear.In this study,plant physiology and biochemistry,cytology and molecular biology methods were employed to reveal the celluar and molecular mechanism underlying peanut embryo abortion under calcium deficiency conditions from cellular level,transcriptional level(mRNA),post-transcriptional level(miRNA)and translational level(protein).The main results are as follows.1.Calcium deficiency resulted in decreased Ca2+ contents,impaired cellular structure and hampered accumulation of reserve substancesCa2+ contents in gynophores(5,10 and 15 days after pegging,DAP),pods(15 DAP)and seeds(25 DAP)of peanut variety MH6 under calcium sufficiency conditions were significantly higher than those under calcium deficiency.Especially,Ca2+ contents in seeds and pods decreased 46.19%and 55.66%under calcium deficiency,respectively.Conformed to the calcium contents,the seeds showed high rate of abortions before 30 DAP.Furthermore,we also used different peanut varieties with distinct tolerance to calcium deficiency growing in the soil with medium level calcium to measure the Ca2+ contents and.Among them,no significant difference in seeds was observed in seeds although gynophores and pods were different for peanut varieties ZH16 and GH21(high calcium deficiency-tolerance).On the other hand,Ca2+contents in gynophores,pods and seeds significantly decreased under calcium deficiency for peanut varieties 19-3 and 819(sensitive to calcium deficiency).As a result,the tolerant varieties produced much less aborted embryo,but the sensitive varieties produced more abortive embryos with ill-filled seeds.Apparently,different levels of tolerance to calcium deficiency in various cultivars were due to different ability to assimilate/accumulate Ca2+in seeds and the underlying mechanisms should be illuminated in the future study.No obvious differences were observed for the cell morphology at 15 DAP,except calcium deficiency cells containing less and smaller starch granules in cytoplasm.At 20 DAP,the cell wall was obviously thickening under calcium sufficiency condition while calcium deficiency embryo cells stayed thinner.Moreover,the central vacuoles were compartmented into smaller ones surrounded by cytoplasm in calcium sufficiency.There were more vacuole proteins with different sizes developing inside the vacuoles.Small lipid bodies began forming between the vacuoles and inside the cell walls.Nevertheless,there were no clear such developmental events in calcium deficiency embryo cells.Their endomembrane systems seemed somewhat disintegrated,and much less lipid bodies presented inside the cells.At 30 DAP,the embryo cell morphology with calcium deficiency stress was further distorted as the cell wall inclined to degrade.Both the protein and lipid bodies exhibited damaging without newly formation under calcium deficiency.Still more,as the endomembrane systems tended to decompose,the organelles appeared disorder arranged inside the embryo cells.By contrast,massive lipid bodies kept forming around many vacuole protein bodies in an orderly way in embryo cells under calcium sufficiency.Therefore,calcium deficiency condition caused the irregular embryo development,especial the membrane systems,at early stages,which impaired the accumulation of reserve substances including starch,proteins and lipids,which led to the abortion of seeds.2.Gene expression profile was changed in peanut embryos under calcium deficiencyRNA-seq for peanut embryos under calcium deficiency and sufficiency conditions showed the gene expression profile was significantly changed with abundant genes involed in various metabolic pathways differentially expressed.GO annotation showed differentially expressed genes(DEGs)maily participated in plant hormone biosynthesis and signaling transduction,carbohydrate metabolism and response,protein transport,ion transport,embryo development,programmed cell death(PCD),defense response,photosynthesis,etc.Several KEGG pathways including photosynthesis,flavonoid biosynthesis,fatty acid biosynthesis,starch and sucrose metabolism,glycolysis/gluconeogenesis,amino sugar and nucleotide sugar metabolism,plant and pathogen interaction were significantly enriched.The expression levels of a number of important DEGs including embryo development related genes,plant hormone biosynthesis and signaling transduction related genes and transcription factors were evidently changed.Futher analysis showed the changed expression level of genes controlling Ca2+ efflux and influx(CNGC1 and CNGC17showed down regulation while ACA4?ACA12?ACA13?ECA4 and MCU2 up regualtion)probably decreased the cytoplasmic Ca2+ level,which disrupted the cytoplasmic Ca2+ homeostasis and affected the important components in calcium signal system(EGFR?PLC2 and PLC4 showed down regulation while CPK28?CPK10?CRCK2?CRCK3?CML11 and CML39 up regulation.).Besides,some genes related to plant hormone biosynthesis and signaling transduction were also changed(ALDH3H1?GH3.1?GH3.6?AUX1/LAX2?PILS3?SAUR32 in auxin signal pathway were up regulated;CKX3 and CKX7 in cytokinin signal pathway were up regulated;CYP707A1?CYP707A3 and CYP707A4 in ABA signal pathway were up regulated while ABA2 and ABA1 down regulated;GA1?GA3 and KAO1 in GA signal pathway were down regulated;CYP90A1?CURL3?BSK3 and BAK1 in BR signal pathway were down regulated;ACS1?AC03?ETR?ABR1 and ERF098 in ethene signal pathway were up regulated.),which probably decreased ABA,GA,CTK and BRs and increased IAA and ETH.Therefore,the banlance among the various endogenous hormones was striked.Notably,some key genes related to embryo development such as APIFA?TCP4?ANT?WRI1?FUS3?ABI3 were differentiallt expressed,which should play important roles in peanut embyo abortion under calcium deficiency.Overexpression of AhAPIFA can induce seed abortion in Arabidosis.Hence,a large number of genes responded to calcium deficiency,intracellular iron transport was abnormal,then affected various hormones levels after complex signal transduction,as well as important genes related to development and PCD changed,led to abnormal metabolism and transport of intracellular reserve substances and at last resulted in embryo abortion.Besides,552 common DEGs were analyzed using gene chip of peanut early embryo development(5,10 and 15DAP)and 218 DEGs were differentially expressed.Among them,defense responsive genes,cell wall degradation related genes were up regulated.However,nutrient metabolism,cell wall biogenesis and organization,embryo development and responsive to hormone related genes were down regulated.This was consistent with cellular morphology and structure loosed and distorted under calcium deficiency and hindered accumulation of reserve substances.This study expicited peanut embryo abortion under calcium deficiency was regulated on mRNA level,which laid foundation for investigating the mechanism underlying this important and interesting scientific issue.3.miRNA expression profile was changed in peanut embryos under calcium deficiencymRNA level regulation of peanut embryo abortion under calcium deficiency was clarified by RNA-seq and gene chip analysis.miRNA expression profile was performed to reveal the post transcriptional regulation mechanism.A total of 87 miRNAs with 117 targets were differentially expressed under calcium deficiency and sufficiency.These target genes participated in signal transduction,celluar communication,transcription,RNA processing and modification,translation,ribosomal structure and biogenesis,post translation modification,protein turnover,chaperones,inorganic ion transport and metabolism,cell cycle control,cell division and carbohydrate transport.KEGG pathway analysis showed plant hormone signal transduction,starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism,RNA transport and ubiquitin mediated proteolysis,etc.were enriched.Integrated analysis of miRNAs and RNA-seq identified 52 differentially expressed target genes of 20 miRNAs.The gene chip expression profile for some differentially expressed targets was consistent with the RNA-seq results.Our results demonstrate that seed/embryo development related genes,such as TCP4,AP2,EMB2750 encoding PPR protein,GRFs,cell division and proliferation related genes HsfB4 and DIVARICATA,and plant hormone signaling pathway related genes CYP707A1 and CYP707A3 for ABA,and BR1 for brassionsteroids,ubiquitin-conjugating enzyme E2(UBC19)and E3 ubiquitin-protein ligase(UBC20)were actively modulated by miRNAs during early embryo development,and resulted in embryo abortion.This study expicited peanut embryo abortion under calcium deficiency was also regulated on post transcriptional level,which was significant for further investigating the mechanism underlying peanut embryo abortion under calcium deficiency.4.Protein expression profile was changed in peanut embryos under calcium deficiencyA comparative proteome analysis between developing embryos under calcium deficiency and sufficiency at 15 and 30DAP was performed using 2-DE approach and obtained high density protein atlas with 3000 protein spots.A total of 113 differentially expressed proteins were identified by MALDI-TO MS,suggesting that many identified proteins were modified in post-translation.Functional annotation revealed these proteins were involved in posttranslational modification,protein turnover,chaperones,energy production and conversion,amino acid transport and metabolism,carbohydrate transport and metabolism,translation,ribosomal structure and biogenesis,cell wall/membrane/envelope biogenesis,cytoskeleton,signal transduction mechanisms and lipid transport and metabolism.RNA-seq analysis showed the expression levels of 48 differentally expressed protein genes were changed with 27 up regulation and 21 down regulation.Among them,genes related to reserve substances accumulation(CRA1),cell redox homeostasis(PRXIIB?2CysPrx?IFR),protein biosynthesis,process and modification TIF3H1?PDI?SKP1B),regulation of gene expression,epigenetic(SUVH1),nucleosome assembly(NAP1;4),photosynthesis(PSBQ2)were down regulated.Genes related to glyoxylate cycle(aconitate hydratase)?carbon fixation(PPC16)?male-female gamete recognition during double fertilization forming a zygote and endosperm(At3g02360)and sucrose metabolism(SS)were up regualted.mRNA expression level of some differentially expressed protein genes related to protein dephosphorylation(TOPP4),proteolysis(SCPL48),protein folding(HSP70)was opposite to protein protein level.It was speculated these genes were regulated possibly by post transcription,translation or posttranslation modification.Calcium deficiency hindered reserve substances accumulation,affected protein biosynthesis,metabolism and modification,disturbed cell redox homeostasis,disordered ATP metabolism and degraded cell wall,which resulted in embryo abortion finally.Peanut embryo abortion under calcium deficiency was also regulated at translational level.Overall,the cellular and molecular mechanism of peanut embryo abortion caused by calcium deficiency was elucidated from multiple perspectives,including cell level,mRNA level,post-transcriptional regulation and translation level,which laid a foundation for further identifying the key genes regulating peanut embryo development and also laid a theoretical foundation for peanut genetic improvement of low calcium tolerance.