| Cystatin is a group of small proteins and known to inhibit the activities of cysteine proteases in the papain C1A and legumain C13 peptidase families in plants.These three gene families play key roles in plant developmental processes,such as seed germination,pollen development,programmed cell death,plant defense against pathogens and response to insect attack and abiotic environmental stresses.Genome-wide identification and characterization are powerful tools to obtain valuable insights into the relationship of the proteinases and their proteinaceous inhibitors,and could aid to elucidate issues concerning the function of these proteins.However,these three gene families have not been well characterized in plants,especially in Oryza sativa,a model plant for cereal biology study.Here we searched for three families' genes in rice genome,made a bioinformatics analysis in an all-round way,and at the same time,carried out comprehensive expression profile analysis of these genes in both normal growth conditions and diverse stressed environments.The analysis suggests multiple roles of cysteine proteases and cystatin in both seed development and plant response to environmental variations.The main results are as follows:1.Totally,11 cystatin genes,33 papain-like cysteine protease genes and 5 legumain-like peptidase genes were identified and cloned through a genome-wide survey in rice genome.Gene structure,chromosomal localization,duplication analysis,protein sequence and phylogenetic analysis were carried out,revealing special characteristics of three family genes in rice:1).several typical motifs for three family proteins have been identified;2).the majority of these proteins contain a signal peptide and can enter into the endomembrane system;3).The duplication events exisit and these genes are evolutionarily conserved.2.Comprehensive expression profile analysis,especially in the processes of seed development and germination was performed in the present study,which reveals that these three family genes in rice display diverse expression patterns in normal conditions.The majority of these genes exhibit a rather broad expression profile and two genes(OsCP3 and OsCP12)are abundantly expressed in anther.During the process of seed production and germination,these three family genes are temporally regulated at different developmental stages and it's found that OsVPE2 is predominantly expressed in the process of seed germination,speculating that its function is closely related to this process.Our experiments also reveal that the majority of them are responsive to plant hormones,but not sensitive to gibberellin.Meanwhile,they could be regulated by different environmental cues including cold,drought and other abiotic stresses,while some others are very stable under different stresses,indicating their fundamental roles in normal plant development.Besides this,the investigation of caspase-like proteases in plants was also done.Caspases are a family of cysteine proteases and are important components of animal apoptosis,which can cleave amino acid residues only after aspartate.They initiate and execute programmed cell death in metazoans.In plants,PCD occurs throughout the whole life from embryogenesis to the death,for example deletion of the embryonic suspensor,anther dehiscence,tapetum degradation,xylogenesis and leaf senescence.In addition,it can also be induced in response to severe environment.In the process plants do display caspase-like activities and the activity has been shown to be required for PCD.Although the entire genome of many species has been sequenced and screened,to date no clear homologs for caspases have been found in plant and meanwhile potential candidates which operate as caspase-like proteases are largely unknown.In this study,tobacco ovules were used as experiment material to search for caspase-like proteases and two candidates(NtTPE8,NtCP20)were found to possess caspase-like activity.Subsequently,gene structure,protein sequence,phylogenetic analysis and expression pattern of NtTPE8 were characterized.RNAi and CRISPR/Cas9-based genome editing technology were used to explore the function of NtTPE8 on seed development.The main results are as follows:1.Using the affinity chromatography and mass spectrometry four candidates were got and they were NtTPE8,NtPB28,NtCP20,NtSCP1.All of them belong to protease.2.Prokaryotic expression in vitro was used to identify the caspase-like activity of four candidates.Except NtPB28,the rest were successfully expressed and purified.Through enzyme activity detection,NtTPE8 and NtCP20 have caspase-like activity.The former mainly possesses caspase-1 activity and the latter can cleave DEVD-AMC in vitro.3.Through NCBI sequence alignment and PCR,the full-length gene of NtTPE8 was obtained.NtTPE8 encodes a protein of the legumain C13 peptidase family with 455 amino acid residues.In total,6 homologs were found in tobacco genome,phylogenetic analysis indicated that NtTPE8 and At?VPE have a close relationship,which was specifically and transiently expressed in two cell layers of the seed coat(ii2 and ii3)at an early stage of Arabidopsis thaliana seed development.4.NtTPE8 was exclusively and abundantly expressed in seed by semi-quantitative PCR and RT PCR.To get more accurate subcellular localization,promoter analysis showed that NtTPE8 is detected in a circle of cells around embryo sac,also called integument tapetum.5.Down-regulation of NtTPE8 was successful but there was no obvious phenotype.And surprisingly the progress of tobacco seed coat PCD was not destroyed or delayed.Considering the limited degree of down-regulation,NtTPE8 was knocked out using the CRISPR/Cas9-based genome editing technology,unfortunately the progress of tobacco seed coat PCD was not yet influenced.Perhaps redundant genes exist.Although the phenotype was not observed in RNAi and knock-out lines,we speculate that the function of NtTPE8 is related to the currently overlooked process in PCD.Up to date,little is known about the protease possessing caspase-like activity in plants as compared to that in animals.In this study,we identified two caspase-like proteases in tobacco,one is for caspase 1,and the other is for caspase 3,providing novel clues for further research of molecular mechanism in plant PCD. |
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