MODIFIED VACUOLE PHENOTYPE1 Is an Arabidopsis Myrosinase-Associated Protein Involved in Endomembrane Protein Trafficking

We identified an Arabidopsis (Arabidopsis thaliana) ethyl methanesulfonate mutant, modified vacuole phenotype1-1 (mvp1-1), in a fluorescent confocal microscopy screen for plants with mislocalization of a green fluorescent protein-δ tonoplast intrinsic protein fusion. The mvp1-1 mutant displayed static perinuclear aggregates of the reporter protein. mvp1 mutants also exhibited a number of vacuole-related phenotypes, as demonstrated by defects in growth, utilization of stored carbon, gravitropic response, salt sensitivity, and specific susceptibility to the fungal necrotroph Alternaria brassicicola. Similarly, crosses with other endomembrane marker fusions identified mislocalization to aggregate structures, indicating a general defect in protein trafficking. Map-based cloning showed that the mvp1-1 mutation altered a gene encoding a putative myrosinase-associated protein, and glutathione S-transferase pull-down assays demonstrated that MVP1 interacted specifically with the Arabidopsis myrosinase protein, THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), but not TGG1. Moreover, the mvp1-1 mutant showed increased nitrile production during glucosinolate hydrolysis, suggesting that MVP1 may play a role in modulation of myrosinase activity. We propose that MVP1 is a myrosinase-associated protein that functions, in part, to correctly localize the myrosinase TGG2 and prevent inappropriate glucosinolate hydrolysis that could generate cytotoxic molecules.The plant endomembrane system is a complex network of subcellular compartments that includes the endoplasmic reticulum (ER), Golgi apparatus, vacuole, plasma membrane, secretory vesicles, and numerous intermediary compartments. Protein trafficking through the endomembrane system requires specific cargo recognition and delivery mechanisms that are mediated by a series of highly specific targeting signals (Surpin and Raikhel, 2004), whose proper recognition is critical for the function of numerous downstream processes, such as floral development (Sohn et al., 2007), gravitropism (Kato et al., 2002; Surpin et al., 2003; Yano et al., 2003), abiotic stress tolerance (Zhu et al., 2002), autophagy (Surpin et al., 2003; Bassham., 2007), pathogen defense (Robatzek, 2007), and turgor pressure and growth (De, 2000).The importance of protein trafficking for plant survival was demonstrated by the identification of the essential Arabidopsis (Arabidopsis thaliana) gene VACUOLELESS1 (VCL1; Rojo et al., 2001). VCL1 was identified as a homolog of Saccharomyces cerevisiae VPS16, which is critical for yeast vacuole biogenesis. Knockouts of yeast VPS16 lack discernible vacuoles but survive despite their severe phenotype. The absence of vacuoles in Arabidopsis vcl1-1 mutants results in embryo lethality (Rojo et al., 2001). The essential nature of trafficking in plants was also demonstrated by insertional mutagenesis of syntaxin genes, where lethality was observed after disruption of single genes in families with highly homologous members (Lukowitz et al., 1996; Sanderfoot et al., 2001). Thus, despite large families of endomembrane components with many homologous genes, many are not redundant in Arabidopsis.Although embryo-lethal mutations provide critical data, it is difficult to obtain additional information. Less severe mutations have proven successful for functional genetics studies of endomembrane trafficking proteins. For example, point mutations in the KATAMARI1/MURUS3 (KAM1/MUR3; Tamura et al., 2005) and KATAMARI2/GRAVITROPISM DEFECTIVE2 (KAM2/GRV2; Tamura et al., 2007; Silady et al., 2008) genes lead to disruption of endomembranes, resulting in the formation of perinuclear aggregates containing organelles. Nonlethal trafficking disruptions have also been generated using chemical genomics, where small molecules were used to perturb trafficking of a soluble cargo protein (Zouhar et al., 2004) and localization of endomembrane markers (Surpin et al., 2005; Robert et al., 2008). Such studies have provided valuable clues about these essential cellular processes.In order to obtain less severe, viable mutants with defects in endomembrane protein trafficking, we previously identified point mutants with defects in localization of a tonoplast reporter protein, GFP:δ-TIP (Avila et al., 2003). Two hundred one putative mutants were grouped into four categories based on the nature of their defects. One unique mutant, cell shape phenotype1, was recently characterized as a trehalose-6-phosphate synthase with roles in regulation of plant architecture, epidermal pavement cell shape, and trichome branching (Chary et al., 2008).Here, we describe an endomembrane trafficking mutant categorized by perinuclear aggregates of GFP:δ-TIP fluorescence (Avila et al., 2003). We refer to this mutant as modified vacuole phenotype1-1 (mvp1-1). At least five endomembrane fusion proteins are partially relocalized to these structures. Positional cloning identified MVP1 as a myrosinase-associated protein (MyAP) localized previously to the tonoplast by proteomics (Carter et al., 2004). mvp1-1 mutants showed reduced endomembrane system functionality, as demonstrated by defects in growth, utilization of stored carbon, gravitropic responsiveness, salt sensitivity, and increased susceptibility to a fungal necrotroph. MVP1 interacted specifically with THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), a known myrosinase protein in Arabidopsis, and the mvp1-1 mutation had a significant effect on nitrile production during glucosinolate hydrolysis, suggesting a role in myrosinase function. Furthermore, MVP1 may function in quality control of glucosinolate hydrolysis by contributing to the proper tonoplast localization of TGG2.     

Dual Localized AtHscB Involved in Iron Sulfur Protein Biogenesis in Arabidopsis

Background

Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.

Methodology/Principal Findings

In this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.

Conclusions

Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.     

MET1 Is a Thylakoid-Associated TPR Protein Involved in Photosystem II Supercomplex Formation and Repair in Arabidopsis

Photosystem II (PSII) requires constant disassembly and reassembly to accommodate replacement of the D1 protein. Here, we characterize Arabidopsis thaliana MET1, a PSII assembly factor with PDZ and TPR domains. The maize (Zea mays) MET1 homolog is enriched in mesophyll chloroplasts compared with bundle sheath chloroplasts, and MET1 mRNA and protein levels increase during leaf development concomitant with the thylakoid machinery. MET1 is conserved in C3 and C4 plants and green algae but is not found in prokaryotes. Arabidopsis MET1 is a peripheral thylakoid protein enriched in stroma lamellae and is also present in grana. Split-ubiquitin assays and coimmunoprecipitations showed interaction of MET1 with stromal loops of PSII core components CP43 and CP47. From native gels, we inferred that MET1 associates with PSII subcomplexes formed during the PSII repair cycle. When grown under fluctuating light intensities, the Arabidopsis MET1 null mutant (met1) showed conditional reduced growth, near complete blockage in PSII supercomplex formation, and concomitant increase of unassembled CP43. Growth of met1 in high light resulted in loss of PSII supercomplexes and accelerated D1 degradation. We propose that MET1 functions as a CP43/CP47 chaperone on the stromal side of the membrane during PSII assembly and repair. This function is consistent with the observed differential MET1 accumulation across dimorphic maize chloroplasts.     

拟南芥钙依赖蛋白激酶参与植物激素信号转导

在植物信号通路中,涉及到钙应答的蛋白激酶大多是钙依赖蛋白激酶。钙依赖蛋白激酶作为钙信号转导因子,参与了包括激素信号转导途径在内的很多传递过程。本工作在前人研究的基础上,对拟南芥AtCPK30基因的功能进行了深入的研究。RT-PCR分析结果表明:AtCPK30在植物根中的表达量很高,其在幼苗中的转录水平分别受ABA、IAA、2,4-D、GA_3和6-BA等激素的诱导调节。AtCPK30基因过表达的转基因株系幼苗的主根比野生型的长,同时发现转基因植株幼苗的根在缺钙的MS培养基上生长较野生型植株长,表明缺钙对转基因幼苗影响较小。用ABA、IAA、GA_3和BA处理时,转基因植株幼苗的根对激素更敏感。当野生型和转基因植株生长在含有生长素抑制剂NPA的MS培养基上时,NPA对转基因植株侧根的抑制比对野生型弱。GFP-CPK30融合蛋白的亚细胞定位研究结果表明:CPK30蛋白定位在细胞壁和细胞膜上。这些研究结果说明了AtCPK30作为钙信号转导因子,参与了多种激素调节植物根生长的过程。     

Differential Radiosensitivity Phenotypes of DNA-PKcs Mutations Affecting NHEJ and HRR Systems following Irradiation with Gamma-Rays or Very Low Fluences of Alpha Particles

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G₁ than in S/G₂ phase. In G₁-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G₂ phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.     

COMMD1: A Novel Protein Involved in the Proteolysis of Proteins

COMMD1 is a protein which is associated with multiple cellular pathways, including NFκB signaling, copper homeostasis and sodium transport. Recently we found that COMMD1 is also essential for normal mouse embryogenesis. Embryos deficient for Commd1 are retarded and die between 9.5 and 10.5 dpc. Increased HIF-1 activity and elevated HIF-1α protein expression were observed in 9.5 dpc Commd1-deficient embryos. In line with these in vivo data, in vitro studies showed that reduced COMMD1 expression caused increased HIF-1α protein stability and HIF-1 activity. Functional characterization of COMMD1 in NFκB signaling and ATP7B-dependent biliary copper excretion suggested that COMMD1 also has a role in regulating the protein degradation of RelA (p65) and ATP7B. The exact function of COMMD1 in these pathways remains elusive but these recent studies suggest that COMMD1 is associated with the ubiquitin-proteasomal system for regulating protein stability.     

Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1

Calcium (Ca²⁺) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K_d ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca²⁺/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.     

GRUSP,an Universal Stress Protein,Is Involved in Gibberellin-dependent Induction of Flowering in Arabidopsis thaliana

Doklady Biochemistry and Biophysics - The effect of T-DNA insertion in the 3'-UTR region of Arabidopsis thaliana At3g58450 gene encoding the Germination-Related Universal Stress Protein (GRUSP)...     

The Cytosolic DnaJ-like Protein Djp1p Is Involved Specifically in Peroxisomal Protein Import

The Saccharomyces cerevisiae DJP1 gene encodes a cytosolic protein homologous to Escherichia coli DnaJ. DnaJ homologues act in conjunction with molecular chaperones of the Hsp70 protein family in a variety of cellular processes. Cells with a DJP1 gene deletion are viable and exhibit a novel phenotype among cytosolic J-protein mutants in that they have a specific impairment of only one organelle, the peroxisome. The phenotype was also unique among peroxisome assembly mutants: peroxisomal matrix proteins were mislocalized to the cytoplasm to a varying extent, and peroxisomal structures failed to grow to full size and exhibited a broad range of buoyant densities. Import of marker proteins for the endoplasmic reticulum, nucleus, and mitochondria was normal. Furthermore, the metabolic adaptation to a change in carbon source, a complex multistep process, was unaffected in a DJP1 gene deletion mutant. We conclude that Djp1p is specifically required for peroxisomal protein import.     

An Ipomoea batatas Iron-Sulfur Cluster Scaffold Protein Gene,IbNFU1, Is Involved in Salt Tolerance

Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.     

PIF3 Is Involved in the Primary Root Growth Inhibition of Arabidopsis Induced by Nitric Oxide in the Light

PHYTOCHROME INTERACTING FACTOR3 （PIF3） is an important component in the phytochrome signaling pathway and mediates plant responses to various environmental conditions. We found that PIF3 is involved in the inhibition of root growth of Arabidopsis thaliana seedlings induced by nitric oxide （NO） in light. Overexpression of PIF3 partially alleviated the inhibitory effect of NO on root growth, whereas the pif3-1 mutant displayed enhanced sensitivity to NO in terms of root growth. During phytochrome signaling, the photoreceptor PHYB mediates the degradation of PIF3. We found that the phyB-9 mutant had a similar phenotype to that of PIF3ox in terms of responsiveness to NO. Furthermore, NO treatment promoted the accumulation of PHYB, and thus reduced PIF3 content. Our results further show that the activity of PIF3 is regulated by the DELLA protein RGL3[RGA （repressor of gal-3） LIKE 3]. Therefore, we speculate that PIF3 lies downstream of PHYB and RGL3, and plays an important role in the inhibitory effect of NO on root growth of Arabidopsis seedlings in light.     

Arabidopsis NRTI.1 Is a Bidirectional Transporter Involved in Root-to-Shoot Nitrate Translocation

Dear Editor, In most plants, nitrogen （N） is acquired by roots in the form of nitrate （NO3-）. In many species, NO3- is not assimi- lated in the roots, but is secreted into the xylem sap for translocation to the shoot, where it enters the cells to be metabolized and/or stored in the vacuoles. Several plasma membrane transporters involved in NO3- influx into the cell have been identified in Arabidopsis （Wang et ai., 2012）, especially in the roots where members of the NPF （NRTI/PTR Family, L~ran et al., 2013） and NRT2 transporter families are predominantiy implicated. Concerning efflux to the xylem sap, only one transporter, NPF7.3/NRT1.5, has been shown to be involved. However, physiological characterization of npf7.31nrtl.5 knockout mutant plants demonstrated that other transporter（s） is （are） also contributing to xylem Ioad- inq of NO~- （Lin et al., 2008）.