Identification of key residues for pH dependent activation of violaxanthin de-epoxidase from Arabidopsis thaliana

Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE) plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pK(a) values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206) caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121-Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role.     

Vacuolar digestion of entire damaged chloroplasts in Arabidopsis thaliana is accomplished by chlorophagy

In yeast and mammals, selective vacuolar delivery and degradation of whole mitochondria, or mitophagy, represents an important quality control system and is achieved by a cargo recognition mechanism enabling selective elimination of dysfunctional mitochondria. As photosynthetic organelles that need light for energy production, plant chloroplasts accumulate sunlight-induced damage. Plants have evolved multiple mechanisms to avoid, relieve, or repair chloroplast photodamage. Our recent study showed that vacuolar degradation of entire chloroplasts, termed chlorophagy, is induced to degrade chloroplasts that are collapsed due to photodamage. Our results underscore the involvement of autophagy in the quality control of endosymbiotic, energy-converting organelles in eukaryotes.     

Raphanusanin-mediated resistance to pathogens is light dependent in radish and Arabidopsis thaliana

Raphanusanin (Ra) is a light-induced inhibitor of hypocotyl growth that responds to unilateral blue light illumination in radish seedlings. We have previously shown that Ra regulates genes that are involved in common defense mechanisms. Many genes that are induced by Ra are also positively regulated by early blue light. To extend the understanding of the role of Ra in pathogen defense, we evaluated the effects of Ra on radish and Arabidopsis thaliana (A. thaliana) infected with the necrotrophic pathogen Botrytis cinerea (B. cinerea) and biotrophic pathogen Pseudomonas syringae (P. syringae). Radish and A. thaliana were found to be resistant to both pathogens when treated with Ra, depending on the concentration used. Interestingly, Ra-mediated resistance to P. syringae is dependent on light because Ra-treated seedlings exhibited enhanced susceptibility to P. syringae infection when grown in the dark. In addition to regulating the biotic defense response, Ra inhibited seed germination and root elongation and enhanced the growth of root hairs in the presence of light in radish and A. thaliana. Our data suggest that Ra regulates the expression of a set of genes involved in defense signaling pathways and plays a role in pathogen defense and plant development. Our results show that light may be generally required not only for the accumulation of Ra but also for its activation during the pathogen defense response.     

Aphid-induced accumulation of trehalose in Arabidopsis thaliana is systemic and dependent upon aphid density

Trehalose is a disaccharide sugar that is now considered to be widely distributed among higher plants. Trehalose has been attributed a number of roles, including control of basic plant processes, such as photosynthesis, and conferring tolerance to abiotic stresses, such as desiccation and high salinity. Trehalose is also a common storage sugar used by insects. In this study, we used laboratory investigations to examine various aspects of trehalose dynamics in an aphid–host plant system (Arabidopsis and the peach potato aphid, Myzus persicae). Trehalose concentrations were measured by [1-H]-NMR. Myzus persicae reared on Arabidopsis, but not on black mustard or spring cabbage, contained considerable quantities of trehalose (5 % w/w dry matter). In Arabidopsis foliage, feeding by aphids induced a density-dependent accumulation of trehalose up to 5 mg g^?1 dry weight. Leaves that were not challenged directly by aphids also exhibited increased trehalose concentrations, indicating that this accumulation was systemic. Trehalose was measured at high concentrations in the phloem sap of plants challenged by aphids, suggesting that aphid feeding induced the plant to produce significant quantities of trehalose, which moved through the plant and into the aphids via the phloem sap. Trehalose was also excreted in the aphid honeydew. Further work is required to clarify whether this trehalose accumulation in Arabidopsis has a direct role or a signalling function in plant tolerance of, or resistance to, aphid feeding, and if a similar accumulation of this sugar occurs when other species or genotypes of aphids are reared on this host plant.     

Insights into the Clp/HSP100 chaperone system from chloroplasts of Arabidopsis thaliana

HSP100 proteins are molecular chaperones involved in protein quality control. They assist in protein (un)folding, prevent aggregation, and are thought to participate in precursor translocation across membranes. Caseinolytic proteins ClpC and ClpD from plant chloroplasts belong to the HSP100 family. Their role has hitherto been investigated by means of physiological studies and reverse genetics. In the present work, we employed an in vitro approach to delve into the structural and functional characteristics of ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). They were expressed in Escherichia coli and purified to near-homogeneity. The proteins were detected mainly as dimers in solution, and, upon addition of ATP, the formation of hexamers was observed. Both proteins exhibited basal ATPase activity (K(m), 1.42 mm, V(max), 0.62 nmol/(min × μg) for AtClpC2 and K(m) ～19.80 mm, V(max) ～0.19 nmol/(min × μg) for AtClpD). They were able to reactivate the activity of heat-denatured luciferase (～40% for AtClpC2 and ～20% for AtClpD). The Clp proteins tightly bound a fusion protein containing a model transit peptide. This interaction was detected by binding assays, where the chaperones were selectively trapped by the transit peptide-containing fusion, immobilized on glutathione-agarose beads. Association of HSP100 proteins to import complexes with a bound transit peptide-containing fusion was also observed in intact chloroplasts. The presented data are useful to understand protein quality control and protein import into chloroplasts in plants.     

Anion channel activation and proton pumping inhibition involved in the plasma membrane depolarization induced by ABA in Arabidopsis thaliana suspension cells are both ROS dependent

In Arabidopsis thaliana suspension cells, ABA was previously shown to promote the activation of anion channels and the reduction of proton pumping that both contribute to the plasma membrane depolarization. These two ABA responses were shown to induce two successive [Ca(2+)](cyt) spikes. As reactive oxygen species (ROS) have emerged as components of ABA signaling pathways especially by promoting [Ca(2+)](cyt) variations, we studied whether ROS were involved in the regulation of anion channels and proton pumps activities. Here we demonstrated that ABA induced ROS production which triggered the second of the two [Ca(2+)](cyt) increases observed in response to ABA. Blocking ROS generation using diphenyleneiodonium (DPI) impaired the proton pumping reduction, the anion channel activation and the RD29A gene expression in response to ABA. Furthermore, H(2)O(2) was shown to activate anion channels and to inhibit plasma membrane proton pumping, as did ABA. However, ROS partially mimicked ABA's effects since H(2)O(2) treatment elicited anion channel activation but not the subsequent expression of the RD29A gene as did ABA. This suggests that expression of the RD29A gene in response to ABA results from the activation of multiple concomitant signaling pathways: blocking of one of them would impair gene expression whereas stimulating only one would not. We conclude that ROS are a central messenger of ABA in the signaling pathways leading to the plasma membrane depolarization induced by ABA.     

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase(NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Lossof-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion,our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.     

Low levels of polymorphism in genes that control the activation of defense response in Arabidopsis thaliana

Plants use signaling pathways involving salicylic acid, jasmonic acid, and ethylene to defend against pathogen and herbivore attack. Many defense response genes involved in these signaling pathways have been characterized, but little is known about the selective pressures they experience. A representative set of 27 defense response genes were resequenced in a worldwide set of 96 Arabidopsis thaliana accessions, and patterns of single nucleotide polymorphisms (SNPs) were evaluated in relation to an empirical distribution of SNPs generated from either 876 fragments or 236 fragments with >400 bp coding sequence (this latter set was selected for comparisons with coding sequences) distributed across the genomes of the same set of accessions. Defense response genes have significantly fewer protein variants, display lower levels of nonsynonymous nucleotide diversity, and have fewer nonsynonymous segregating sites. The majority of defense response genes appear to be experiencing purifying selection, given the dearth of protein variation in this set of genes. Eight genes exhibit some evidence of partial selective sweeps or transient balancing selection. These results therefore provide a strong contrast to the high levels of balancing selection exhibited by genes at the upstream positions in these signaling pathways.     

Molecular and genetic analyses of Tic20 homologues in Arabidopsis thaliana chloroplasts

The Tic20 protein was identified in pea (Pisum sativum) as a component of the chloroplast protein import apparatus. In Arabidopsis, there are four Tic20 homologues, termed atTic20‐I, atTic20‐IV, atTic20‐II and atTic20‐V, all with predicted topological similarity to the pea protein (psTic20). Analysis of Tic20 sequences from many species indicated that they are phylogenetically unrelated to mitochondrial Tim17‐22‐23 proteins, and that they form two evolutionarily conserved subgroups [characterized by psTic20/atTic20‐I/IV (Group 1) and atTic20‐II/V (Group 2)]. Like psTic20, all four Arabidopsis proteins have a predicted transit peptide consistent with targeting to the inner envelope. Envelope localization of each one was confirmed by analysis of YFP fusions. RT‐PCR and microarray data revealed that the four genes are expressed throughout development. To assess the functional significance of the genes, T‐DNA mutants were identified. Homozygous tic20‐I plants had an albino phenotype that correlated with abnormal chloroplast development and reduced levels of chloroplast proteins. However, knockouts for the other three genes were indistinguishable from the wild type. To test for redundancy, double and triple mutants were studied; apart from those involving tic20‐I, none was distinguishable from the wild type. The tic20‐I tic20‐II and tic20‐I tic20‐V double mutants were albino, like the corresponding tic20‐I parent. In contrast, tic20‐I tic20‐IV double homozygotes could not be identified, due to gametophytic and embryonic lethality. Redundancy between atTic20‐I and atTic20‐IV was confirmed by complementation analysis. Thus, atTic20‐I and atTic20‐IV are the major functional Tic20 isoforms in Arabidopsis, with partially overlapping roles. While the Group 2 proteins have been conserved over approximately 1.2 billion (1.2 × 10⁹) years, they are not essential for normal development.     

Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent

Dark-grown Arabidopsis seedlings develop an apical hook by differential elongation and division of hypocotyl cells. This allows the curved hypocotyl to gently drag the apex, which is protected by the cotyledons, upwards through the soil. Several plant hormones are known to be involved in hook development, including ethylene, which causes exaggeration of the hook. We show that gibberellins (GAs) are also involved in this process. Inhibition of GA biosynthesis with paclobutrazol (PAC) prevented hook formation in wild-type (WT) seedlings and in constitutive ethylene response (ctr)1-1, a mutant that exhibits a constitutive ethylene response. In addition, a GA-deficient mutant (ga1-3) did not form an apical hook in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). Analysis of transgenic Arabidopsis seedlings expressing a green fluorescent protein (GFP)-repressor of ga1-3 (RGA) fusion protein suggested that ACC inhibits cell elongation in the apical hook by inhibition of GA signaling. A decreased feedback of GA possibly causes an induction of GA biosynthesis based upon the expression of genes encoding copalyl diphosphate synthase (CPS; GA1) and GA 2-oxidase (AtGA2ox1). Furthermore, expression of GASA1, a GA-response gene, suggests that differential cell elongation in the apical hook might be a result of differential GA-sensitivity.     

Translocons on the inner and outer envelopes of chloroplasts share similar evolutionary origin in Arabidopsis thaliana

The process of importing nuclear encoded proteins into chloroplasts is mediated by the T ranslocons on the O uter/I nner Envelope of C hloroplasts (TOC and TIC complex). The ancestor of the TOC complex was formed by pre‐existing proteins from the cyanobacterial ancestor; other proteins recruited from the host cell or cyanobacterial ancestor were subsequently integrated into the complex. However, little is known about the origin of the TIC complex. In this work, the origin of the TIC complex was investigated through one of its channel proteins, AtTic21. Phylogenetic analysis suggested that AtTic21 is conserved in photosynthetic organisms. AtTic21 showed 33% sequence identity to a Synechocystis protein SynTic21. The successful genetic complementation of an AtTic21 knockout mutant by SynTic21 plus the transit peptide coding sequence of AtTic21 suggested that SynTic21 is an ortholog of AtTic21. The sequence and functional conservation between SynTic21 and AtTic21 suggested that the TIC complex shares a similar evolutionary origin to the TOC complex.