Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.     

Seeds of Arabidopsis plants expressing dominant-negative AtSKD1 under control of the GL2 promoter show a transparent testa phenotype and a mucilage defect

We have recently shown that overexpression of dominant-negative AtSKD1 versions under control of the trichome and non-root-hair-cell specific GL2 promoter (GL2_pro) blocks trafficking of soluble cargo to the vacuole, resulting in its fragmentation and ultimately cell death. GL2_pro is also active in the Arabidopsis seeds. When we inspected seeds of the dominant-negative AtSKD1 variants we found two phenotypes. The seeds display a transparent testa phenotype caused by a lack of proanthocyanidin (PA) and do not possess seed coat mucilage. Both phenotypes could be connected by cell death induced by the overexpression of dominant-negative AtSKD1.Key words: VPS4, ESCRT, plant, Arabidopsis, SKD1, ATPase, MVB, proanthocyanidin, transparent testa, mucilage, tannin, seed coat, AtSKD1AAA ATPases are important regulators of a plethora of cellular functions such as peroxisome biogenesis, vesicle-mediated transport, control of cell divisions and gene expression. This variety is based on a common mechanism, the energy dependent unfolding, remodeling and disassembly of proteins and protein complexes.¹Mammalian SKD1 and its yeast homolog VPS4 are AAA ATPases involved in the sorting of monoubiquitylated trans-membrane cargo to the lysosome/vacuole by dismantling the members of the endosomal sorting complex required for transport (ESCRT) complexes from the endosomal membrane.² The Arabidopsis SKD1 homolog, AtSKD1, has been characterized recently and has been shown to be an ortholog of SKD1/Vps4.^3,4 Mutations for all three ATPases are known that alone or in combination render them dominant-negative.^3–6 Overexpression of dominant-negative AtSKD1 is, however, lethal for Arabidopsis plants. Therefore, we have used the trichome and non-root-hair-cell-specific GL2_pro promoter to address the function of AtSKD1 in planta. Cells that express the dominant-negative versions show multiple nuclei, fragmented vacuoles and ultimately die. These phenotypes are most likely due to a block in vacuolar trafficking of soluble cargo that is instead secreted.⁴GL2_pro is also active in the coat of developing Arabidopsis seeds. We therefore inspected seeds of lines overexpressing dominant-negative and wild-type AtSKD1.     

Genome-Wide Screening of Salt Tolerant Genes by Activation-Tagging Using Dedifferentiated Calli of Arabidopsis and Its Application to Finding Gene for Myo-Inositol-1-P-Synthase

Salinity represents a major abiotic stress factor that can adversely limit the production, quality and geographical distribution of crops. In this study we focused on dedifferentiated calli with fundamental cell functions, the salt tolerance of which had not been previously examined. The experimental approach was based on activation tagging without regeneration of plants for the identification of salt-tolerant mutants of Arabidopsis. Among 62,000 transformed calli that were screened, 18 potential mutants resistant to 150 mM NaCl were obtained. Thermal asymmetric interlaced (TAIL)-PCR was performed to determine the location of T-DNA integration in the genome. In one line, referred to as salt tolerant callus 1 (stc1), expression of a gene [At4g39800: myo-inositol-1-P-synthase 1 (MIPS1)] was considerably enhanced in calli. Plants regenerated from calli showed tolerance to salt in germination and subsequent growth. Retransformation of wild-type Arabidopsis with MIPS1 conferred salt tolerance, indicating that MIPS1 is the causal gene. The over-expression of MIPS1 increased the content of total inositol. The involvement of MIPS1 in salt tolerance through the fundamental cell growth has been proved in Arabidopsis.     

Overexpression of soybean <Emphasis Type="Italic">GmERF9</Emphasis> enhances the tolerance to drought and cold in the transgenic tobacco

Ethylene response factors (ERFs) are widespread in plants, which are widely involved in plant response to biotic and abiotic stress. In this research, a soybean gene, GmERF9, was identified and the function was characterized. The results showed that GmERF9 contained a typical AP2/ERF binding domain and a putative nuclear localization signal sequence. The real-time fluorescence quantitative PCR (qPCR) revealed that the expression of GmERF9 could be induced by ethylene (ET), abscisic acid (ABA), drought, salt and cold stresses. GmERF9 protein could specifically bind to the GCC-box and activate the expression of the reporter gene in the yeast cells and tobacco leaves. Overexpression of GmERF9 enhanced the expression of pathogenesis-related (PR) genes, including PR1, PR2, Osmotin (PR5), and SAR8.2. Also, the overexpression of GmERF9 increased the accumulation of proline and soluble carbohydrate, and decreased the accumulation of malondialdehyde under drought and cold stresses in the transgenic tobacco compared to the wild type (WT) tobacco, which indicated that GmERF9 enhanced the tolerance to drought and cold stresses in the transgenic tobacco. In summary, the function of GmERF9 is involved in the response to environmental stresses for plants, which can be used as a candidate gene for genetic engineering of crops.     

The garlic NF-YC gene, <Emphasis Type="Italic">AsNF-YC8</Emphasis>, positively regulates non-ionic hyperosmotic stress tolerance in tobacco

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.     

A Small GTPase Activator Protein Interacts with Cytoplasmic Phytochromes in Regulating Root Development

Phytochromes enable plants to sense light information and regulate developmental responses. Phytochromes interact with partner proteins to transmit light signals to downstream components for plant development. PIRF1 (phytochrome-interacting ROP guanine-nucleotide exchange factor (RopGEF 1)) functions as a light-signaling switch regulating root development through the activation of ROPs (Rho-like GTPase of plant) in the cytoplasm. In vitro pulldown and yeast two-hybrid assays confirmed the interaction between PIRF1 and phytochromes. PIRF1 interacted with the N-terminal domain of phytochromes through its conserved PRONE (plant-specific ROP nucleotide exchanger) region. PIRF1 also interacted with ROPs and activated them in a phytochrome-dependent manner. The Pr form of phytochrome A enhanced the RopGEF activity of PIRF1, whereas the Pfr form inhibited it. A bimolecular fluorescence complementation analysis demonstrated that PIRF1 was localized in the cytoplasm and bound to the phytochromes in darkness but not in light. PIRF1 loss of function mutants (pirf1) of Arabidopsis thaliana showed a longer root phenotype in the dark. In addition, both PIRF1 overexpression mutants (PIRF1-OX) and phytochrome-null mutants (phyA-211 and phyB-9) showed retarded root elongation and irregular root hair formation, suggesting that PIRF1 is a negative regulator of phytochrome-mediated primary root development. We propose that phytochrome and ROP signaling are interconnected through PIRF1 in regulating the root growth and development in Arabidopsis.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol

Introgression and functional expression of either the PcINO1 (l-myo-inositol 1-phosphate synthase or MIPS coding gene from the wild halophytic rice, Porteresia coarctata) or McIMTI (inositol methyl transferase, IMTI coding gene from common ice plant Mesembryanthemum crystallinum) has earlier been shown to confer salt tolerance to transgenic tobacco plants (Sheveleva et al., Plant Physiol 115:1211–1219, 1997; Majee et al., J Biol Chem 279:28539–28552, 2004). In this communication, we show that transgenic tobacco plants co-expressing PcINO1 and McIMT1 gene either in cytosol or in chloroplasts accumulate higher amount of total inositol (free and methyl inositol) compared to non-transgenic plants. These transgenic plants were more competent in terms of growth potential and photosynthetic activity and were less prone to oxidative stress under salt stress. A positive correlation between the elevated level of total inositol and methylated inositol and the capability of the double transgenic plants to withstand a higher degree of salt stress compared to the plants expressing either PcINO1 or McIMT1 alone is inferred.