| Cell proliferation,expansion,and differentiation,which lay the foundation of plant development,are regulated by the cell wall.Pectins are a major component of primary cell walls in eudicots.Pectin biosynthesis,modification,and degradation influence the flexibility and extensibility of the cell walls.Therefore,studying pectin metabolism will advance our understanding of how cell wall dynamics function in plant development,and guide breeding strategies to improve economic traits and control crop fertility.Polygalacturonases(PGs)are a crucial class of pectin hydrolases,which function in pectin degradation.Genes encode PGs underwent rapid expansion during plant evolution,resulting in a large multigene family.Recent studies proposed retention mechanisms of the duplicated PG genes based on molecular evolution investigations and gene expression studies of the PG family.However,to validate such mechanisms,the biological functions of duplicated PG genes need to be investigated.Up to date,only a few PGs have been functionally characterized during plant development.These studies demonstrate that PGs function in multiple developmental processes,such as leaf expansion,fruit ripening,and organ adhesion,and are essential for cell expansion and separation.However,little is known about the role of PGs in organ morphogenesis,especially how they act at the cellular level,and how they affect pectin degradation and oligogalacturonides accumulation in plant tissues.Moreover,pectin degradation is a well-known process in pollen development.Studies have shown that PGs are required for pollen intine development,but little is known about their roles in pollen exine development.In previous studies,we identified a PG gene in Brassica campestris ssp.chinensis Makino,syn.B.rapa ssp.chinensis,which functions in pollen wall development and pollen germination.A comprehensive follow-up search in the recently published B.rapa genome data revealed four extra duplicated copies of this gene.We designated these five genes as BrPG45-1 to BrPG45-5.Transcriptome analysis of B.rapa genic male sterile AB lines revealed that three copies of BrPG45 were differentially expressed in stamen development.To investigate the roles of these five copies during stamen development and use it as a case to study the retention mechanism,we first analyzed the origin of PG45 in land plants and its evolutionary history in Brassica species,followed with gene expression analyses and biological functions characterization of BrPG45-1 to BrPG45-5.The retention mechanism of BrPG45 duplicated genes was discussed based on gene expression patterns and functions.Arabidopsis thaliana,a model organism closely related to B.rapa,only contain one orthologous copy of PG45,AtPG45.To facilitate our understanding of the functions of BrPG45,we examined the expression pattern and biological roles of AtPG45.We also tested the PG activity of AtPG45 and investigate its mechanism of action in cell dynamics.Main results and conclusions are summarized as follows:(1)Based on the PG gene family phylogeny from 31 represented plant species inferred by maximum likelihood algorithm,we found that PG45 is originated in the common ancestor of eudicots.The gene duplication event in the common ancestor of Brassica resulted in the multiple copies of PG45 in the Brassica species investigated in this research.By performing q RT-PCR analyses,we found that BrPG45 copies are highly expressed in stamens during late developmental stages and all these five copies are differentially expressed in the stamens of B.rapa genic male sterile AB lines.AtPG45 is highly expressed in inflorescences,but it is also expressed in other tissues like rosette leaves.GUS staining results of promoter-GUS lines revealed that BrPG45 and AtPG45 are expressed in the tapetum at uninucleate and binucleate stages,and in pollen grains at binucleate and trinucleate stages.BrPG45-1 to BrPG45-5 share the same expression pattern during pollen development.GUS signals from AtPG45 promoter is also detected in meristematic tissues like shoot apical meristem and rosette leaf axils.Subcellular localization was analyzed in the epidermal cells of onion and Arabidopsis root,respectively.Signals of BrPG45 and AtPG45 could be observed outside the plasma membrane.(2)By using a TYMV derived virus-induced gene silencing system,we knocked down all BrPG45 copies in B.rapa.In BrPG45 knockdown lines,26.1% mature pollen showed adherent phenotype,and pollen germination rate in vitro was decreased to 44.4% of control lines.During vegetative development or in flower structures,no obvious difference was observed between BrPG45 knockdown lines and control plants.The ultrastructure of pollen grains was examined using transmission electron microscopy,and the callose component in the pollen wall was analyzed by staining pollen grains with aniline blue dye.These results suggest that impaired structure of the tectum and bacula could result from the disrupted deposition of sporopollenin.Overaccumulated tryphine at the pollen grain surface might explain the adherent pollen and decreased pollen germination rate.No defect was found during callose wall degradation.We also transformed BrPG45-1,BrPG45-3,and BrPG45-5 into A.thaliana,respectively.Transgenic lines for all the three genes shared similar phenotype during pollen development,including adherent pollen and reduced pollen viability.These results indicated that gene dosage could be the retention mechanism for the duplicated copies of BrPG45.(3)Phenotypes of atpg45 mutants and AtPG45 overexpression lines were examined during plant growth.Altered expression of AtPG45 leads to reduced hypocotyl length and plant height.81.0% and 16.8% of flowers showed variable stamen numbers in atpg45 mutants and AtPG45 overexpression lines,respectively.Complementation assay was performed and the mutant phenotypes in the atpg45 plants could be rescued by transforming the complementation construct into mutant background.During lateral organ development,we observed undulating leaves in both atpg45 mutants and AtPG45 overexpression lines.Compared with Col lines,longitudinal leaf curvature index was 2-fold higher in atpg45 mutants and AtPG45 overexpression lines.In addition,we found branch number was 3.3-fold higher in AtPG45 knockout lines than Col plants.We also observed abnormal floral organ structures as a result of impaired structure of floral meristem and floral organ meristem.Enlarged cell size and reduced leaf length to width ratio were found in AtPG45 overexpression lines,which resulted from excess growth in leaf width.The phenotypes we observed in atpg45 mutants and AtPG45 overexpression lines demonstrated that AtPG45 functions in organ elongation and expansion,and lateral organ morphogenesis,which is different from the function of BrPG45.(4)We expressed His-SUMO tagged AtPG45 in E.coli and tested its protein activity.The results confirmed that AtPG45 encodes a bona fide PG.Amino acid alignment showed that BrPG45 and AtPG45 share same sequences in conserved PG functional domain,indicating that BrPG45 could also function in pectin degradation.We then characterized AtPG45 activity in vivo.A significantly lower level of PG activity and pectin content was found in atpg45 leaves.To investigate the functions of AtPG45 in regulating leaf flatness,we analyzed cell arrangement and shape in palisade mesophyll and spongy mesophyll.Altered palisade mesophyll cell shape were found in leaves of atpg45 and AtPG45 plants,which further resulted in the disrupted leaf adaxial-abaxial polarity.By imaging and analyzing the GFP-MAP4 signals in epidermal layers in Col and atpg45 plants transformed with the GFP-MAP4 vector,we studied cell proliferation and expansion rate in the adaxial and abaxial epidermis of developing leaves.We found that knocking-out AtPG45 is related to imbalanced cell proliferation duration between epidermis.Compared with the abaxial epidermis,cell proliferation duration was 48.0% shorter in the adaxial epidermis,but no difference was found for the relative cell expansion rate.Immunolabeling were performed by probing pectins in mesophyll cell walls,but no difference was found for the localization of methylesterified and demethylesterified pectin in leaf sections.These results suggested that how AtPG45 functions in leaf polarity is not related to pectin distribution.Highperformance size-exclusion chromatography-mass spectrometry-based method was used to analyze endogenous oligogalacturonide profiling from Arabidopsis leaves.For oligogalacturonides with degree of polymerization(DP)between 2-7,the mutation of AtPG45 led to more oligogalacturonides with higher DPs,whereas the overexpression of AtPG45 resulted in overaccumulation of oligogalacturonides with smaller DPs.In addition,reduced proportions of oligogalacturonides with DP 8-15 was observed in both atpg45 mutants and AtPG45 overexpression leaves.These results suggested that the role of AtPG45 in leaf morphogenesis might be related to the altered oligogalacturonide profiling.Together,these results highlight a previously undiscovered function for PGs in determining leaf flatness and regulating cell proliferation and cell shape by influencing pectin degradation in plant tissues. |
PDF Full Text Request