Function Research Of Annexin Genes ZmAnn33 And ZmAnn35 During Maize Seed Embryo Germination

A rapid and proper seedling establishment after sowing plays a crucial role in maize productions.However,as one kind of thermophilic crops,maize is susceptible to chilling injury when sowed in early spring,leading to severely inhibit seed germination and seedling growth.Thus,it is of great significance to research on the physiological events during seed germination,especially under chilling conditions.Germination is a complex process referring to all kinds of metabolic reactions and signal transduction pathways.It has been well known that macromolecules and organelles often undergo a repair process due to absorption of water upon early imbibition in dry seeds,while how the damaged membrane is repaired still need farther investigation.Studies on animal plasma membrane had shown that the membrane binding protein-annexins-directly participate in the repair process of membrane lesions,indicating the similar function in its plant's counterparts.Therefore,this research carried out the function analysis of two annexin genes,ZmAnn33 and ZmAnn35,during maize seed germination.Chilling-sensitive inbred lines Mo 17 of maize was used to analyze the expression profiles of ZmAnn33,35 during seed germination and responses to chilling conditions.Then,two maize annexins were transformed to Arabidopsisthaliana to further investigate the possible membrane repair function in maize.Main results of present study were as follows:1.Five stages during the period of maize germination were divided,including dry seed,imbibition,protrusion,germination and the end of germination.The change of antioxidant enzymes activities(SOD,POD,CAT)and the expression profiles of annexin genes were investigated at 250C and 15? germination.The results indicated that the activities of antioxidant enzymes varied during the germination process,but showed the increased pattern overall.Compared with 25?,the activity of superoxide dismutase(SOD)was higher under chilling stress.while the activities of both peroxidase(POD)and catalase(CAT)were significantly inhibited.The highest value of electrolyte leakage(EL)was detected at 9 hour after imbibitions(HAI)and then decreased under both normal and chilling conditions,demonstrating that impaired membrane system during early germination could undergo a repair process.Besides,the EL of chilling germinating seeds was significantly higher contrast to normal condition.The results indicated that seed cell membrane systems were under impaired state during early germination and the damage will be more severe at low temperature.Expressions of both annexin genes showed similar tendency under 25? and 15?conditions,which were gradually up regulated along with germination time course.While the increase of maize annexins levels was dramatically suppressed by low temperature.2.The changes of physiological characteristics and expression analysis of two annexin genes upon chilling imbibition were examined.The content of malondialdehyde(MDA)in the embryo under each treatment showed a transient increase at the first imbibition hours,followed by gradually decrease.It suggested that the membrane lipid peroxidation increased in early imbibition due to the rapid absorption of water,which caused damage to the cell membrane.MDA levels from chilling imbibitional embryo were significantly higher than those under optimal temperature,implying a more severe damage to cell membrane.The expression pattern of ZmAnn33,35 was in accordance with the degree of membrane damage,and low temperature significantly reduced both genes levels.Annexin is a kind of Ca2+-binding protein,the presence of Ca2+ inhibitor EGTA significantly inhibited their expression.Meanwhile,presence of EGTA caused higher MDA contents compared to other treatments at whole imbibitions stages,suggesting the aggravated membrane damage under chilling condition.These results suggested the aggravated damage of membrane had a close relation to the suppressed ZmAnns expressions.To obtain a chilling injury,imbibitional seeds were subjected to a lower temperature at 50C for two hours,followed by transferred to 25? for recovery.The annexins expression profiles after one hour's recovery would undergo a prompt increase compared to those in non-recover embryos.3.To further investigate the function of maize annexins,full length cDNA of ZmAnn33 and ZmAnn35 genes were overexpressed in Arabidopsis.After 3 days chilling stress,no obvious morphological differences were noticed between the transgenic lines and wild type(WT)seedlings from the 1/2 MS medium.The result was consistent with the observations from root length and electrolyte leakage assay,which data showed no significant differences among transgenic lines and WT lines.However,the growth of both annexin overexpression seedlings were promoted during recovery period and showed less membrane damage indicating by lower EL.Besides,the CAT and POD activities were notably enhanced in both transgenic seedlings at recovery period after the chilling treatment and transgenic seedlings contained significantly lower MDA compared to WT.Present results implied that maize annexins might function in alleviation of cellular oxidative stress and thus played a protective role from chilling stress.7-day-old seedlings of transgenic lines and WT were placed at 1? for 3h to obtain a transient chilling stress,immediately followed by FM4-64(50 ?m)staining.Most cells in WT root were completely stained with FM4-64,while in two ZmAnn transgenic plants,cells from the elongation region showed intact plasma membrane,only cells of root tips were stained.The results suggested that cells from annexin overexpression plants possess more intact plasma membranes.The enhanced membrane integrity may be conferred by the accelerated intracellular exocytosis process in ZmAnns overexpression Arabidopsis.Besides,both annexin genes predominantly localized to the plasma membrane,but they also appeared in the vicinity of intracellular PM surface.In brief,these findings demonstrated that ZmAnn33 and ZmAnn35 play important roles in membrane recovery during maize seed germination.4.RNA sequencing technology was used to comprehensively investigate the gene expressions in chilling-imbibed maize embryo and to reveal the underlying mechanism of chilling injury at molecular level.Imbibed seeds for 2 hours at 5?(LT2)were selected and transcriptomic comparative analysis was performed.Among 327 DEGs indentified between dry seed(CKO)and LT2,15 specific genes with plasma membrane(PM)relevant functions belonging to lipid metabolism,stress,signaling and transport were characterized,and most of them showed down-regulation pattern under chilling stress.When transferred to 25? for recovery(LT3),remarkable changes occurred in maize embryo.There were 873 DEGs including many PM related genes being identified between LT2 and LT3,some of which showing significant increase after 1 hour's recovery.Moreover,15 genes encoding intracellular vesicular trafficking proteins were found to be exclusively differential expressed at recovery stage.It suggested that the intracellular vesicle trafficking might be essential for PM recovery through PM turnover.Furthermore,transcriptome analyses on imbibed embryos under normal condition(25°C)were also made as a contrast.A total of 651 DEGs were identified to mainly involved in protein metabolism,transcriptional regulation,signaling and energy productions.Overall,the RNA-Seq results provided us a deep knowledge of imbibitional chilling injury on plasma membrane and a new view on PM repaired mechanism during early seed imbibition at transcriptional level.The DEGs identified in this work would be useful references in future seed germination research.In conclusion,two annexins ZmAnn33 and ZmAnn35 are important genes for regulating the maize seed germination.They function in promoting the repair of embryo membrane damage during germination,especially at chilling imbibition process.It is the first research focusing on the function of annexin genes in seed biology,suggesting that annexin could be involved in the repair of membrane injury in plant cells.Results in this study will be beneficial to understand the molecular mechanisms,the repair mechanism in particular,of seed germination.They also exhibit theoretical and practical significance to improve seed vigor and low temperature resistance in crop genetic engineering.