[Show abstract][Hide abstract] ABSTRACT: Plant male gametogenesis involves complex and dynamic changes in gene expression. At present, little is known about the transcription factors involved in this process and how their activities are regulated. Here, we show that a pollen-specific transcription factor, WRKY34, and its close homolog, WRKY2, are required for male gametogenesis in Arabidopsis thaliana. When overexpressed using LAT52, a strong pollen-specific promoter, epitope-tagged WRKY34 is temporally phosphorylated by MPK3 and MPK6, two mitogen-activated protein kinases (MAPKs, or MPKs), at early stages in pollen development. During pollen maturation, WRKY34 is dephosphorylated and degraded. Native promoter-driven WRKY34-YFP fusion also follows the same expression pattern at the protein level. WRKY34 functions redundantly with WRKY2 in pollen development, germination, and pollen tube growth. Loss of MPK3/MPK6 phosphorylation sites in WRKY34 compromises the function of WRKY34 in vivo. Epistasis interaction analysis confirmed that MPK6 belongs to the same genetic pathway of WRKY34 and WRKY2. Our study demonstrates the importance of temporal post-translational regulation of WRKY transcription factors in the control of developmental phase transitions in plants.
[Show abstract][Hide abstract] ABSTRACT: The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%-48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%-43%), total GS activity (39%-45%) and soluble protein concentration (23%-24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency.
[Show abstract][Hide abstract] ABSTRACT: A novel glutamine synthetase (GS) gene DvGS1 showing highest amino acid sequence identity of 78 % with the other homologous GS proteins from green algae, was isolated and characterized from Dunaliella viridis. Phylogenetic analysis revealed that DvGS1 occupied an independent phylogenetic position which was different with the GSs from higher plants, animals and microbes. Functional complement in E. coli mutant confirmed that the DvGS1 encoded functional GS enzyme. Real-time PCR analysis of DvGS1 in D. viridis cells under nitrogen starvation revealed that the mRNA level of DvGS1 was positively up-regulated in 12 h. The DvGS1 levels at the points of 12 and 24 h were separately twofold and fourfold of the level before nitrogen starvation. In order to investigate the potential application of DvGS1 in higher plants, the transgenic study of DvGS1 in Arabidopsis thaliana was carried out. Phenotype identification demonstrated that all three transgenic lines of T3 generation showed obviously enhanced root length (26 %), fresh weight (22-46 % at two concentrations of nitrate supplies), stem length (26 %), leaf size (29 %) and silique number (30 %) compared with the wild-type Arabidopsis. Biochemical analysis confirmed that all three transgenic lines had higher total nitrogen content, soluble protein concentration, total amino acid content and the leaf GS activity than the wild type plants. The free NH4 (+) and NO3 (-) concentration in fresh leaves of three transgenic lines were reduced by 17-26 % and 14-15 % separately (at two concentrations of nitrate supplies) compared with those of the wild types. All the results indicated that over-expression of DvGS1 in Arabidopsis significantly results in the improvement of growth phenotype and the host's nitrogen use efficiency.
[Show abstract][Hide abstract] ABSTRACT: Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules downstream of receptors/sensors that transduce extracellular stimuli into intracellular responses in eukaryotes. Plant MAPK cascades play pivotal roles in signaling plant defense against pathogen attack. In this review, we summarize recent advances in the identification of upstream receptors/sensors and downstream MAPK substrates. These findings revealed the molecular mechanisms underlying MAPK functions in plant disease resistance. MAPK cascades have also emerged as battlegrounds of plant-pathogen interactions. Activation of MAPKs is one of the earliest signaling events after plant sensing of pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) and pathogen effectors. They are involved in signaling multiple defense responses, including the biosynthesis/signaling of plant stress/defense hormones, reactive oxygen species (ROS) generation, stomatal closure, defense gene activation, phytoalexin biosynthesis, cell wall strengthening, and hypersensitive response (HR) cell death. Pathogens, however, employ effectors to suppress plant MAPK activation and downstream defense responses to promote pathogenesis. Expected final online publication date for the Annual Review of Phytopathology Volume 51 is August 04, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Annual Review of Phytopathology 05/2013; · 10.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arabidopsis thaliana MPK3 and MPK6, two mitogen-activated protein kinases (MAPKs or MPKs), play critical roles in plant disease resistance by regulating multiple defense responses. Previously, we characterized the regulation of phytoalexin biosynthesis by Arabidopsis MPK3/MPK6 cascade and its downstream WRKY33 transcription factor. Here, we report another substrate of MPK3/MPK6, ETHYLENE RESPONSE FACTOR6 (ERF6), in regulating Arabidopsis defense gene expression and resistance to the necrotrophic fungal pathogen Botrytis cinerea. Phosphorylation of ERF6 by MPK3/MPK6 in either the gain-of-function transgenic plants or in response to B. cinerea infection increases ERF6 protein stability in vivo. Phospho-mimicking ERF6 is able to constitutively activate defense-related genes, especially those related to fungal resistance, including PDF1.1 and PDF1.2, and confers enhanced resistance to B. cinerea. By contrast, expression of ERF6-EAR, in which ERF6 was fused to the ERF-associated amphiphilic repression (EAR) motif, strongly suppresses B. cinerea-induced defense gene expression, leading to hypersusceptibility of the ERF6-EAR transgenic plants to B. cinerea. Different from ERF1, the regulation and function of ERF6 in defensin gene activation is independent of ethylene. Based on these data, we conclude that ERF6, another substrate of MPK3 and MPK6, plays important roles downstream of the MPK3/MPK6 cascade in regulating plant defense against fungal pathogens.
[Show abstract][Hide abstract] ABSTRACT: Spatiotemporal-specific cell proliferation and cell differentiation are critical to the formation of normal tissues, organs, and organisms. The highly coordinated cell differentiation and proliferation events illustrate the importance of cell-cell communication during growth and development. In Arabidopsis thaliana, ERECTA (ER), a receptor-like protein kinase, plays important roles in promoting localized cell proliferation, which determines inflorescence architecture, organ shape, and size. However, the downstream signaling components remain unidentified. Here, we report a mitogen-activated protein kinase (MAPK; or MPK) cascade that functions downstream of ER in regulating localized cell proliferation. Similar to an er mutant, loss of function of MPK3/MPK6 or their upstream MAPK kinases (MAPKKs; or MKKs), MKK4/MKK5, resulted in shortened pedicels and clustered inflorescences. Epistasis analysis demonstrated that the gain of function of MKK4 and MKK5 transgenes could rescue the loss-of-function er mutant phenotype at both morphological and cellular levels, suggesting that the MPK3/MPK6 cascade functions downstream of the ER receptor. Furthermore, YODA (YDA), a MAPKK kinase, was shown to be upstream of MKK4/MKK5 and downstream of ER in regulating inflorescence architecture based on both gain- and loss-of-function data. Taken together, these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the ER receptor in regulating localized cell proliferation, which further shapes the morphology of plant organs.
[Show abstract][Hide abstract] ABSTRACT: Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea-induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea-induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction.
[Show abstract][Hide abstract] ABSTRACT: Plant sensing of invading pathogens triggers massive metabolic reprogramming, including the induction of secondary antimicrobial compounds known as phytoalexins. We recently reported that MPK3 and MPK6, two pathogen-responsive mitogen-activated protein kinases, play essential roles in the induction of camalexin, the major phytoalexin in Arabidopsis thaliana. In search of the transcription factors downstream of MPK3/MPK6, we found that WRKY33 is required for MPK3/MPK6-induced camalexin biosynthesis. In wrky33 mutants, both gain-of-function MPK3/MPK6- and pathogen-induced camalexin production are compromised, which is associated with the loss of camalexin biosynthetic gene activation. WRKY33 is a pathogen-inducible transcription factor, whose expression is regulated by the MPK3/MPK6 cascade. Chromatin immunoprecipitation assays reveal that WRKY33 binds to its own promoter in vivo, suggesting a potential positive feedback regulatory loop. Furthermore, WRKY33 is a substrate of MPK3/MPK6. Mutation of MPK3/MPK6 phosphorylation sites in WRKY33 compromises its ability to complement the camalexin induction in the wrky33 mutant. Using a phospho-protein mobility shift assay, we demonstrate that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to Botrytis cinerea infection. Based on these data, we conclude that WRKY33 functions downstream of MPK3/MPK6 in reprogramming the expression of camalexin biosynthetic genes, which drives the metabolic flow to camalexin production in Arabidopsis challenged by pathogens.
The Plant Cell 04/2011; 23(4):1639-53. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peroxiredoxins (Prxs), a group of antioxidant enzymes, are an important component of the oxidative defense system and have been demonstrated to function as peroxidases, sensors of H(2)O(2)-mediated signaling and/or chaperones. In this study, a cDNA library was constructed from a halotolerant alga, Dunaliella viridis, and was used in a functional complementation screen for antioxidative genes in an oxidative sensitive yeast mutant. Two Prx genes, DvPrx1 and DvPrx2, were obtained from this screen. These two genes were classified as type II Prx and 2-Cys Prx based on amino acid sequence and phylogenetic analysis. When over-expressed in yeast cells, both Prx genes were able to confer better oxidative tolerance and decrease the level of reactive oxygen species (ROS). Subcellular localization experiments in tobacco cells revealed that both DvPrx1 and DvPrx2 were localized in the cytosol. The transcription of DvPrx1 and DvPrx2 can be induced by hypersalinity shock, but is not obviously affected by treatment with high levels of oxidant. Our results shed light on the function and regulation of Prx genes from Dunaliella and their potential roles in salt tolerance.
[Show abstract][Hide abstract] ABSTRACT: The halotolerant alga Dunaliella adapts to exceptionally high salinity and possesses efficient mechanisms for regulating intracellular Na(+). In plants, sequestration of Na(+) into the vacuole is driven by the electrochemical H(+) gradient generated by H(+) pumps, and this Na(+) sequestration is one mechanism that confers salt tolerance to plants. To investigate the role of vacuolar H(+) pumps in the salt tolerance of Dunaliella, we isolated the cDNA of the vacuolar proton-translocating inorganic pyrophosphatase (V-H(+)-PPase) from Dunaliella viridis. The DvVP cDNA is 2,984 bp in length, codes for a polypeptide of 762 amino acids and has 15 transmembrane domains. The DvVP protein is highly similar to V-H(+)-PPases from other green algae and higher plant species, in terms of its amino acid sequence and its transmembrane model. A phylogenetic analysis of V-H(+)-PPases revealed the close relationship of Dunaliella to green algal species of Charophyceae and land plants. The heterologous expression of DvVP in the yeast mutant G19 (Δena1-4) suppressed Na(+) hypersensitivity, and a GFP-fusion of DvVP localized to the vacuole membranes in yeast, indicating that DvVP encodes a functional V-H(+)-PPase. A northern blot analysis showed a decrease in the transcript abundance of DvVP at higher salinity in D. viridis cells, which is in contrast to the salt-induced upregulation of V-H(+)-PPase in some plants, suggesting that the expression of DvVP under salt stress may be regulated by different mechanisms in Dunaliella. This study not only enriched our knowledge about the biological functions of V-H(+)-PPases in different organisms but also improved our understanding of the molecular mechanism of salt tolerance in Dunaliella.
[Show abstract][Hide abstract] ABSTRACT: Tandemly arrayed genes (TAGs) account for about one-third of the duplicated genes in eukaryotic genomes. They provide raw genetic material for biological evolution, and play important roles in genome evolution. The 22-kDa prolamin genes in cereal genomes represent typical TAG organization, and provide the good material to investigate gene amplification of TAGs in closely related grass genomes. Here, we isolated and sequenced the Coix 22-kDa prolamin (coixin) gene cluster (283 kb), and carried out a comparative analysis with orthologous 22-kDa prolamin gene clusters from maize and sorghum. The 22-kDa prolamin gene clusters descended from orthologous ancestor genes, but underwent independent gene amplification paths after the separation of these species, therefore varied dramatically in sequence and organization. Our analysis indicated that the gene amplification model of 22-kDa prolamin gene clusters can be divided into three major stages. In the first stage, rare gene duplications occurred from the ancestor gene copy accidentally. In the second stage, rounds of gene amplification occurred by unequal crossing over to form tandem gene array(s). In the third stage, gene array was further diverged by other genomic activities, such as transposon insertions, segmental rearrangements, etc. Unlike their highly conserved sequences, the amplified 22-kDa prolamin genes diverged rapidly at their expression capacities and expression levels. Such processes had no apparent correlation to age or order of amplified genes within TAG cluster, suggesting a fast evolving nature of TAGs after gene amplification. These results provided insights into the amplification and evolution of TAG families in grasses.
[Show abstract][Hide abstract] ABSTRACT: Coix lacryma-jobi L. (Coix) is a close relative of maize and is considered a valuable genetic resource for crop improvement. Here we report the construction of the first Coix bacterial artificial chromosome (BAC) library using accession PI 324059. This BAC library contains about 230 400 clones with an average insert size of 113 kb, has low organellar DNA contamination, and provides 16.3-fold coverage of the genome. The library was stored in 12 × 96 pools that could be screened with a PCR protocol. Library screening was performed for the 22 kDa α-coixin gene family. A total of 57 positive pools were identified, and single clones were isolated from 19 of these pools. Based on DNA fingerprinting and Southern blot analysis, these 19 BAC clones form a single contig of about 340 kb in length, indicating that the 22 kDa α-coixin genes occur in a cluster. These results demonstrated the suitability of this BAC library for gene isolation and comparative genomics studies of the Coix genome.
[Show abstract][Hide abstract] ABSTRACT: Dunaliella is a group of green algae with exceptional stress tolerance capability, and is considered as an important model organism for stress tolerance study. Here we cloned a TPS (trehalose-6-phosphate synthase) gene from Dunaliella viridis and designated it as DvTPS (D. viridis trehalose-6-phosphate synthase/phosphatase).The DvTPS cDNA contained an ORF of 2793 bp encoding 930 aa. DvTPS had both TPS and TPP domain and belonged to the Group II TPS/TPP fusion gene family. Southern blots showed it has a single copy in the genome. Genome sequence analysis revealed that it has 18 exons and 17 introns. DvTPS had a constitutive high expression level under various NaCl culture conditions, however, could be induced by salt shock. Promoter analysis indicated there were ten STREs (stress response element) in its promoter region, giving a possible explanation of its inducible expression pattern upon salt shock. Yeast functional complementation analysis showed that DvTPS had neither TPS nor TPP activity. However, DvTPS could improve the salt tolerance of yeast salt sensitive mutant G19. Our results indicated that despite DvTPS showed significant similarity with TPS/TPP, its real biological function is still remained to be revealed.
[Show abstract][Hide abstract] ABSTRACT: Coix lacryma-jobi L. (Coix) is a close relative of maize and is considered a valuable genetic resource for crop improvement. Here we report the construction of the first Coix bacterial artificial chromosome (BAC) library using accession PI 324059. This BAC library contains about 230 400 clones with an average insert size of 113 kb, has low organellar DNA contamination, and provides 16.3-fold coverage of the genome. The library was stored in 12 × 96 pools that could be screened with a PCR protocol. Library screening was performed for the 22 kDa α-coixin gene family. A total of 57 positive pools were identified, and single clones were isolated from 19 of these pools. Based on DNA fingerprinting and Southern blot analysis, these 19 BAC clones form a single contig of about 340 kb in length, indicating that the 22 kDa α-coixin genes occur in a cluster. These results demonstrated the suitability of this BAC library for gene isolation and comparative genomics studies of the Coix genome.La larme-de-Job (Coix lacryma-jobi L.) est un proche parent du maïs et elle constitue un germoplasme utile pour l'amélioration génétique. Dans ce travail, les auteurs rapportent la production de la première banque de clones BAC (chromosomes bactériens artificiels) à partir de l'accession PI 324059. Cette banque compte environ 230 400 clones dont la taille moyenne des inserts est de 113 kb, elle est peu contaminée avec de l'ADN provenant des organites et présente une couverture équivalant à 16,3 génomes. La banque a été répartie en 12 × 96 « pools », lesquels peuvent faire l'objet d'un criblage par PCR. Le criblage de la banque a été réalisé pour la famille multigénique codant pour l'α-coixine, une protéine de réserve majeure de 22 kDa. Au total, 57 « pools » positifs ont été identifiés et des clones individuels ont été isolés pour 19 de ces « pools ». Sur la base de l'analyse d'empreintes génétiques et d'hybridations Southern, ces 19 clones BAC forment un seul contig d'environ 340 kb, ce qui suggère que les gènes qui codent pour l'α-coixine de 22 kDa sont regroupés au sein d'un amas de gènes. Ces résultats démontrent l'utilité de cette banque de BAC pour l'isolement de gènes et des analyses génomiques comparées du génome de la larme-de-Job.
[Show abstract][Hide abstract] ABSTRACT: Green algae of the genus Dunaliella can adapt to hypersaline environments and are considered model organisms for salinity tolerance. In an EST analysis in Dunaliella viridis under salt stress, we isolated a salt-inducible cDNA coding for the 26S proteasome subunit RPN10, designated DvRPN10. The DvRPN10 cDNA is 1472 bp and encodes a polypeptide of 377 amino acids. The DvRPN10 protein shares a high similarity to orthologs from other species. The function of DvRPN10 was confirmed by complementation of the yeast Deltarpn10 mutant. Q-PCR analysis of D. viridis cells grown in different salinities revealed that the transcript level of DvRPN10 increased in proportion to the external salinity within a range of 0.5-3 M NaCl, but decreased significantly at extremely high salinities (4-5 M NaCl). When a salinity shock of 1-3 M NaCl was applied to D. viridis cells, DvRPN10 mRNA levels remained steady during the first 36 h, and then gradually elevated to the level observed at 3 M NaCl. The gene structure of DvRPN10 was revealed by sequencing of a BAC clone containing this gene. Possible transcription factor binding sites related to stress tolerance were found in the promoter region of DvRPN10. The expression of DvRPN10 in response to the external salinity suggests that RPN10-mediated protein degradation plays a role in the salinity tolerance of D. viridis.
Journal of plant physiology 08/2010; 167(12):1003-8. · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dunaliella, a unicellular green alga, has the unusual ability to survive dramatic osmotic stress by accumulating high concentrations of intracellular glycerol as a compatible solute. The chloroplastic glycerol-3-phosphate dehydrogenase (GPDH) has been considered to be the key enzyme that produces glycerol for osmoregulation in Dunaliella. In this study, we cloned the two most prominent GPDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively. Unlike higher plant GPDHs, both proteins contained extra phosphoserine phosphatase (SerB) domains at their N-termini in addition to C-terminal GPDH domains. Such bi-domain GPDHs represent a novel type of GPDH and are found exclusively in the chlorophyte lineage. Transient expression of EGFP fusion proteins in tobacco leaf cells demonstrated that both DvGPDH1 and DvGPDH2 are localized in the chloroplast. Overexpression of DvGPDH1 or DvGPDH2 could complement a yeast GPDH mutant (gpd1Delta), but not a yeast SerB mutant (ser2Delta). In vitro assays with purified DvGPDH1 and DvGPDH2 also showed apparent GPDH activity for both, but no SerB activity was detected. Surprisingly, unlike chloroplastic GPDHs from plants, DvGPDH1 and DvGPDH2 could utilize both NADH and NADPH as coenzymes and exhibited significantly higher GPDH activities when NADH was used as the coenzyme. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression. These results shed light on the regulation of glycerol synthesis during salt stress in Dunaliella.
[Show abstract][Hide abstract] ABSTRACT: The sodium-dependent phosphate transporter gene from unicellular green algae Dunaliella viridis, DvSPT1, shares similarity with members of Pi transporter family. Sequencing analysis of D. viridis BAC clone containing the DvSPT1 gene revealed two inverted duplicated copies of this gene (DvSPT1 and DvSPT1-2 respectively). The duplication covered most of both genes except for their 3' downstream region. The duplicated genomic sequences exhibited 97.9% identity with a synonymous divergence of Ks=0.0126 in the coding region. This data indicated very recent gene duplication in D. viridis genome, providing an excellent opportunity to investigate sequence and expression divergence of duplicated genes at an early stage. Scattered point mutations and length polymorphism of simple sequence repeats (SSRs) were predominant among the sequence divergence soon after gene duplication. Due to sequence divergence in the 5' regulatory regions and a swap of the entire 3' downstream regions (3'-UTR), DvSPT1 and DvSPT1-2 showed expression divergence in response to extra-cellular NaCl concentration changes. According to their expression patterns, the two diverged gene copies would provide better adaptation to a broader range of extra-cellular NaCl concentration. Furthermore, Southern blot analysis indicated that there might be a large phosphate transporter gene family in D. viridis.