WVD2 is a novel microtubule-associated protein in Arabidopsis thaliana

Arabidopsis WAVE-DAMPENED 2 (WVD2) was identified by forward genetics as an activation-tagged allele that causes plant and organ stockiness and inversion of helical root growth handedness on agar surfaces. Plants with high constitutive expression of WVD2 or other members of the WVD2-LIKE (WDL) gene family have stems and roots that are short and thick, have reduced anisotropic cell elongation, are suppressed in a root-waving phenotype, and have inverted handedness of twisting in hypocotyls and roots compared with wild-type. The wvd2-1 mutant shows aberrantly organized cortical microtubules in peripheral root cap cells as well as reduced branching of trichomes, unicellular leaf structures whose development is regulated by microtubule stability. Orthologs of the WVD2/WDL family are found widely throughout the plant kingdom, but are not similar to non-plant proteins with the exception of a C-terminal domain distantly related to the vertebrate microtubule-associated protein TPX2. in vivo, WVD2 and its closest paralog WDL1 are localized to interphase cortical microtubules in leaves, hypocotyls and roots. Recombinant glutathione-S-transferase:WVD2 or maltose binding protein:WVD2 protein bind to and bundle microtubules in vitro. We speculate that a C-terminal domain of TPX2 has been utilised by the WVD2 family for functions critical to the organization of plant microtubules.     

The ubiquitin pathway is required for innate immunity in Arabidopsis

Plant defences require a multitude of tightly regulated resistance responses. In Arabidopsis, the unique gain-of-function mutant suppressor of npr1-1 constitutive 1 ( snc1 ) carries a point mutation in a Resistance ( R )-gene, resulting in constitutive activation of defence responses without interaction with pathogens. This has allowed us to identify various downstream signalling components essential in multiple defence pathways. One mutant that suppresses snc1 -mediated constitutive resistance is modifier of snc1 5 ( mos5 ), which carries a 15-bp deletion in UBA1 , one of two ubiquitin-activating enzyme genes in Arabidopsis. A mutation in UBA2 does not suppress snc1 , suggesting that these two genes are not equally required in Arabidopsis disease resistance. On the other hand, a mos5 uba2 double mutant is lethal, implying partial redundancy of the two homologues. Apart from affecting snc1 -mediated resistance, mos5 also exhibits enhanced disease susceptibility to a virulent pathogen and is impaired in response to infection with avirulent bacteria carrying the protease elicitor AvrRpt2. The mos5 mutation in the C-terminus of UBA1 might affect binding affinity of the downstream ubiquitin-conjugating enzymes, thus perturbing ubiquitination of target proteins. Furthermore, SGT1b and RAR1, which are necessary for resistance conferred by the SNC1 -related R -genes RPP4 and RPP5 , are dispensable in snc1 -mediated resistance. Our data reveal the definite requirement for the ubiquitination pathway in the activation and downstream signalling of several R-proteins.     

RPN1a, a 26S proteasome subunit, is required for innate immunity in Arabidopsis

Accumulating evidence shows that proper degradation of proteins that affect defense responses in a positive or negative manner is critical in plant immunity. However, the role of plant degradation systems such as the 26S proteasome in plant immunity is not well understood. Loss‐of‐function mutations in EDR2 (ENHANCED DISEASE RESISTANCE 2) lead to increased resistance to the adapted biotrophic powdery mildew pathogen Golovinomyces cichoracearum. To study the molecular interactions between powdery mildew pathogen and Arabidopsis, we performed a screen for suppressors of edr2 and found that mutation in the gene that encodes RPN1a, a subunit of the 26S proteasome, suppressed edr2‐associated disease resistance phenotypes. In addition, RPN1a is required for edr1‐ and pmr4‐mediated powdery mildew resistance and mildew‐induced cell death. Furthermore, we show that rpn1a displayed enhanced susceptibility to the fungal pathogen G. cichoracearum and to virulent and avirulent bacterial Pto DC3000 strains, which indicated that rpn1a has defects in basal defense and resistance (R) protein‐mediated defense. RPN1a–GFP localizes to both the nucleus and cytoplasm. Accumulation of RPN1a is affected by salicylic acid (SA) and the rpn1a mutant has defects in SA accumulation upon Pto DC3000 infection. Further analysis revealed that two other subunits of the 26S proteasome, RPT2a and RPN8a are also involved in edr2‐mediated disease resistance. Based on these results, we conclude that RPN1a is required for basal defense and R protein‐mediated defense. Our data provide evidence that some subunits of the 26S proteasome are involved in innate immunity in Arabidopsis.     

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.     

SNAIL1 is essential for female gametogenesis in Arabidopsis thaliana

Two yeast Brix family members Ssf1 and Ssf2,involved in large ribosomal subunit synthesis, are essential for yeast cell viability and mating efficiency. Their putative homologs exist in the Arabidopsis genome; however, their role in plant development is unknown. Here, we show that Arabidopsis thaliana SNAIL1(At SNAIL1), a protein sharing high sequence identity with yeast Ssf1 and Ssf2, is critical to mitosis progression of female gametophyte development.The snail1 homozygous mutant was nonviable and its heterozygous mutant was semi-sterile with shorter siliques.The mutation in SNAIL1 led to absence of female transmission and reduced male transmission. Further phenotypic analysis showed that the synchronic development of female gametophyte in the snail1 heterozygous mutant was greatly impaired and the snail1 pollen tube growth, in vivo, was also compromised. Furthermore, SNAIL1 was a nucleolarlocalized protein with a putative role in protein synthesis.Our data suggest that SNAIL1 may function in ribosome biogenesis like Ssf1 and Ssf2 and plays an important role during megagametogenesis in Arabidopsis.     

The Arabidopsis thaliana multifunctional protein gene (MFP2) of peroxisomal beta-oxidation is essential for seedling establishment

The multifunctional protein (MFP) of peroxisomal beta-oxidation catalyses four separate reactions, two of which (2-trans enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase) are core activities required for the catabolism of all fatty acids. We have isolated and characterized five Arabidopsis thaliana mutants in the MFP2 gene that is expressed predominantly in germinating seeds. Seedlings of mfp2 require an exogenous supply of sucrose for seedling establishment to occur. Analysis of mfp2-1 seedlings revealed that seed storage lipid was catabolized more slowly, long-chain acyl-CoA substrates accumulated and there was an increase in peroxisome size. Despite a reduction in the rate of beta-oxidation, mfp2 seedlings are not resistant to the herbicide 2,4-dichlorophenoxybutyric acid, which is catabolized to the auxin 2,4-dichlorophenoxyacetic acid by beta-oxidation. Acyl-CoA feeding experiments show that the MFP2 2-trans enoyl-CoA hydratase only exhibits activity against long chain (C18:0) substrates, whereas the MFP2 L-3-hydroxyacyl-CoA dehydrogenase is active on C6:0, C12:0 and C18:0 substrates. A mutation in the abnormal inflorescence meristem gene AIM1, the only homologue of MFP2, results in an abnormal inflorescence meristem phenotype in mature plants (Richmond and Bleecker, Plant Cell 11, 1999, 1911) demonstrating that the role of these genes is very different. The mfp2-1 aim1double mutant aborted during the early stages of embryo development showing that these two proteins share a common function that is essential for this key stage in the life cycle.     

FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs

FPA is a gene that regulates flowering time in Arabidopsis via a pathway that is independent of daylength (the autonomous pathway). Mutations in FPA result in extremely delayed flowering. FPA was identified by means of positional cloning. The predicted FPA protein contains three RNA recognition motifs in the N-terminal region. FPA is expressed most strongly in developing tissues, similar to the expression of FCA and LUMINIDEPENDENS, two components of the autonomous pathway previously identified. Overexpression of FPA in Arabidopsis causes early flowering in noninductive short days and creates plants that exhibit a more day-neutral flowering behavior.     

Phosphatidylglycerol is essential for the development of thylakoid membranes in Arabidopsis thaliana

Phosphatidylglycerol is a ubiquitous phospholipid in the biological membranes of many organisms. In plants, phosphatidylglycerol is mainly present in thylakoid membranes and has been suggested to play specific roles in photosynthesis. Here, we have isolated two T-DNA tagged lines of Arabidopsis thaliana that have a T-DNA insertion in the PGP1 gene encoding a phosphatidylglycerolphosphate synthase involved in the biosynthesis of phosphatidylglycerol. In homozygous plants of the T-DNA tagged lines, the PGP1 gene was completely disrupted. The growth of these knockout mutants was dependent on the presence of sucrose in the growth medium, and these plants had pale yellow-green leaves. The leaves of the mutants had remarkably large intercellular spaces due to the reduction in the number of mesophyll cells. The development of chloroplasts in the leaf cells was severely arrested in the mutants. Mesophyll cells with chloroplast particles are only found around vascular structures, whereas epidermal cells are enlarged but largely conserved. The content of phosphatidylglycerol in the mutants was reduced to 12% of that of the wild type. These results demonstrate that PGP1 plays a major role in the biosynthesis of phosphatidylglycerol in chloroplasts, and that phosphatidylglycerol is essential for the development of thylakoid membranes in A. thaliana.     

Vacuolar processing enzyme is essential for mycotoxin-induced cell death in Arabidopsis thaliana

Some compatible pathogens secrete toxins to induce host cell death and promote their growth. The toxin-induced cell death is a pathogen strategy for infection. To clarify the executioner of the toxin-induced cell death, we examined a fungal toxin (fumonisin B1 (FB1))-induced cell death of Arabidopsis plants. FB1-induced cell death was accompanied with disruption of vacuolar membrane followed by lesion formation. The features of FB1-induced cell death were completely abolished in the Arabidopsis vacuolar processing enzyme (VPE)-null mutant, which lacks all four VPE genes of the genome. Interestingly, an inhibitor of caspase-1 abolished FB1-induced lesion formation, as did a VPE inhibitor. The VPE-null mutant had no detectable activities of caspase-1 or VPE in the FB1-treated leaves, although wild-type leaves had the caspase-1 and VPE activities, both of which were inhibited by a caspase-1 inhibitor. gammaVPE is the most essential among the four VPE homologues for FB1-induced cell death in Arabidopsis leaves. Recombinant gammaVPE recognized a VPE substrate with Km = 30.3 microm and a caspase-1 substrate with Km = 44.2 microm, which is comparable with the values for mammalian caspase-1. The gammaVPE precursor was self-catalytically converted into the mature form exhibiting caspase-1 activity. These in vivo and in vitro analyses demonstrate that gammaVPE is the proteinase that exhibits a caspase-1 activity. We show that VPE exhibiting a caspase-1 activity is a key molecule in toxin-induced cell death. Our findings suggest that a susceptible response of toxin-induced cell death is caused by the VPE-mediated vacuolar mechanism similar to a resistance response of hypersensitive cell death (Hatsugai, N., Kuroyanagi, M., Yamada, K., Meshi, T., Tsuda, S., Kondo, M., Nishimura, M., and Hara-Nishimura, I. (2004) Science 305, 855-858).     

Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana

In plants and other eukaryotes, long-chain acyl-CoAs are assumed to be imported into peroxisomes for beta-oxidation by an ATP binding cassette (ABC) transporter. However, two genes in Arabidopsis thaliana, LACS6 and LACS7, encode peroxisomal long-chain acyl-CoA synthetase (LACS) isozymes. To investigate the biochemical and biological roles of peroxisomal LACS, we identified T-DNA knockout mutants for both genes. The single-mutant lines, lacs6-1 and lacs7-1, were indistinguishable from the wild type in germination, growth, and reproductive development. By contrast, the lacs6-1 lacs7-1 double mutant was specifically defective in seed lipid mobilization and required exogenous sucrose for seedling establishment. This phenotype is similar to the A. thaliana pxa1 mutants deficient in the peroxisomal ABC transporter and other mutants deficient in beta-oxidation. Our results demonstrate that peroxisomal LACS activity and the PXA1 transporter are essential for early seedling growth. The peroxisomal LACS activity would be necessary if the PXA1 transporter delivered unesterified fatty acids into the peroxisomal matrix. Alternatively, PXA1 and LACS6/LACS7 may act in parallel pathways that are both required to ensure adequate delivery of acyl-CoA substrates for beta-oxidation and successful seedling establishment.     

Increased cysteine availability is essential for cadmium tolerance and accumulation in Arabidopsis thaliana

Employing genetic transformation using an Atcys-3A cDNA construct expressing the cytosolic O-acetylserine(thiol)lyase (OASTL), we obtained two Arabidopsis lines with different capabilities for supplying cysteine under metal stress conditions. Lines 1-2 and 10-10, grown under standard conditions, showed similar levels of cysteine and glutathione (GSH) to those of the wild-type. However, in the presence of cadmium, line 10-10 showed significantly higher levels. The increased thiol content allowed line 10-10 to survive under severe heavy metal stress conditions (up to 400 microm of cadmium in the growth medium), and resulted in an accumulation of cadmium in the leaves to a level similar to that of metal hyperaccumulator plants. Investigation of the epidermal leaf surface clearly showed that most of the cadmium had accumulated in the trichomes. Furthermore, line 10-10 was able to accumulate more cadmium in its trichomes than the wild-type, whereas line 1-2 showed a reduced capacity for cadmium accumulation. Our results suggest that an increased rate of cysteine biosynthesis is responsible for the enhanced cadmium tolerance and accumulation in trichome leaves. Thus, molecular engineering of the cysteine biosynthesis pathway, together with modification of the number of leaf trichomes, may have considerable potential in increasing heavy metal accumulation for phytoremediation purposes.     

Arabidopsis thaliana squalene epoxidase 1 is essential for root and seed development

Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1-3 and sqe1-4 plants have diminished stature and produce inviable seeds. The sqe1-3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1.     

The Arabidopsis dynamin-related protein2 family is essential for gametophyte development

Clathrin-mediated membrane trafficking is critical for multiple stages of plant growth and development. One key component of clathrin-mediated trafficking in animals is dynamin, a polymerizing GTPase that plays both regulatory and mechanical roles. Other eukaryotes use various dynamin-related proteins (DRP) in clathrin-mediated trafficking. Plants are unique in the apparent involvement of both a family of classical dynamins (DRP2) and a family of dynamin-related proteins (DRP1) in clathrin-mediated membrane trafficking. Our analysis of drp2 insertional mutants demonstrates that, similar to the DRP1 family, the DRP2 family is essential for Arabidopsis thaliana development. Gametophytes lacking both DRP2A and DRP2B were inviable, arresting prior to the first mitotic division in both male and female gametogenesis. Mutant pollen displayed a variety of defects, including branched or irregular cell plates, altered Golgi morphology and ectopic callose deposition. Ectopic callose deposition was also visible in the pollen-lethal drp1c-1 mutant and appears to be a specific feature of pollen-defective mutants with impaired membrane trafficking. However, drp2ab pollen arrested at earlier stages in development than drp1c-1 pollen and did not accumulate excess plasma membrane or display other gross defects in plasma membrane morphology. Therefore, the DRP2 family, but not DRP1C, is necessary for cell cycle progression during early gametophyte development. This suggests a possible role for DRP2-dependent clathrin-mediated trafficking in the transduction of developmental signals in the gametophyte.     

The inhibition of protein translation mediated by AtGCN1 is essential for cold tolerance in Arabidopsis thaliana

In yeast, the interaction of General Control Non‐derepressible 1 (GCN1) with GCN2 enables GCN2 to phosphorylate eIF2α (the alpha subunit of eukaryotic translation initiation factor 2) under a variety of stresses. Here, we cloned AtGCN1, an Arabidopsis homologue of GCN1. We show that AtGCN1 directly interacts with GCN2 and is essential for the phosphorylation of eIF2α under salicylic acid (SA), ultraviolet (UV), cold stress and amino acid deprivation conditions. Two mutant alleles, atgcn1‐1 and atgcn1‐2, which are defective in the phosphorylation of eIF2α, showed increased sensitivity to cold stress, compared with the wild type. Ribosome‐bound RNA profiles showed that the translational state of mRNA was higher in atgcn1‐1 than in the wild type. Our result also showed that cold treatment reduced the tendency of the tor mutant seedlings to produce purple hypocotyls. In addition, the kinase activity of TOR was transiently inhibited when plants were exposed to cold stress, suggesting that the inhibition of TOR is another pathway important for plants to respond to cold stress. In conclusion, our results indicate that the AtGCN1‐mediated phosphorylation of eIF2α, which is required for inhibiting the initiation of protein translation, is essential for cold tolerance in Arabidopsis.     

The HCF136 protein is essential for assembly of the photosystem II reaction center in Arabidopsis thaliana

Hcf136 encodes a hydrophilic protein localized in the lumen of stroma thylakoids. Its mutational inactivation in Arabidopsis thaliana results in a photosystem II (PHII)-less phenotype. Under standard illumination, PSII is not detectable and the amount of photosystem I (PSI) is reduced, which implies that HCF136p may be required for photosystem biogenesis in general. However, at low light, a comparison of mutants with defects in PSII, PSI, and the cytochrome b(6)f complex reveals that HCF136p regulates selectively biogenesis of PSII. We demonstrate by in vivo radiolabeling of hcf136 that biogenesis of the reaction center (RC) of PSII is blocked. Gel blot analysis and affinity chromatography of solubilized thylakoid membranes suggest that HCF136p associates with a PSII precomplex containing at least D2 and cytochrome b(559). We conclude that HCF136p is essential for assembly of the RC of PSII and discuss its function as a chaperone-like assembly factor.     

The Drosophila inhibitor of apoptosis protein DIAP2 functions in innate immunity and is essential to resist gram-negative bacterial infection

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes.     

Ribosomal protein L27a is required for growth and patterning in Arabidopsis thaliana

Ribosomal proteins are integral to ribosome biogenesis, and function in protein synthesis. In higher eukaryotes, loss of cytoplasmic ribosomal proteins results in a reduced growth rate as well as developmental defects. To what extent and how ribosomal proteins affect development is currently not known. Here we describe a semi-dominant mutation in the cytoplasmic ribosomal protein gene RPL27aC that affects multiple aspects of plant shoot development, including leaf patterning, inflorescence and floral meristem function, and seed set. In the embryo, RPL27aC is required to maintain the growth rate and for the transition from radial to bilateral symmetry associated with initiation of cotyledons. rpl27ac-1d embryos undergo stereotypical patterning to establish a globular embryo. However, a temporal delay in initiation and outgrowth of cotyledon primordia leads to development of an enlarged globular embryo prior to apical domain patterning. Defects in embryo development are coincident with tissue-specific ectopic expression of the shoot meristem genes SHOOT MERISTEMLESS (STM) and CUP-SHAPED COTYLEDON2 (CUC2), in addition to delayed expression of the abaxial gene FILAMENTOUS FLOWER (FIL) and mis-regulation of the auxin efflux effector PIN-FORMED1 (PIN1). Genetic interactions with other ribosomal protein mutants indicate that RPL27aC is a component of the ribosome. We propose that RPL27aC regulates discrete developmental events by controlling spatial and temporal expression of developmental patterning genes via an as yet undefined process involving the ribosome.