Proteome Analysis of the ROF-FKBP Mutants Reveals Functional Relations among Heat Stress Responses, Plant Development, and Protein Quality Control during Heat Acclimation in Arabidopsis thaliana.

In the present study, a differential screening following heat stress acclimation was performed in Arabidopsis thaliana WT and ROF-FKBP mutated plants using mass spectrometry, and the results were used to understand and analyze the effect of the ROF PPIases during thermotolerance acquisition in plants. Our data highlight the central role of these two PPIases in heat stress and point to their direct or indirect effect on other proteins participating in cellular functions such as protein folding and quality control, cell division, photosynthesis, and other metabolic and signaling processes. Specifically, the heat stress response, protein folding, and protein ER processing pathways are enhanced following a 37 °C acclimation period independent of the mutation state. However, at 37 °C, and in the double-mutated rof1-/2- plants, a higher accumulation of proteins belonging to the above pathways is observed compared with all other conditions (WT, single mutants, control, and heat-acclimated plants). Furthermore, the proteasomal pathway, involving the common member of both the protasomal and the lysosomal degradation pathway, CDC48, is over-represented in the extracts of both the untreated and heat-stressed rof1-/2- mutants compared with the other extracts. In contrast, in the single rof1- mutation, the heat acclimation pathway is suppressed at 37 °C when compared to the WT. Protein accumulation related to the heat stress and the protein quality control pathways points to a differential but also synergistic role of the two proteins. Protein complexes of other biochemical and developmental mechanisms, such as the light-harvesting complex of the photosynthetic pathway and the phosphoinositide binding proteins involved in membrane-trafficking events during cell plate formation and cytokinesis (patellin 1, 2, and 4), are negatively regulated in the rof1-/2- mutant. Our results suggest that ROF1 and ROF2 FKBPs regulate stress response, and developmental and metabolic pathways via a complex feedback mechanism involving partners that ensure protein quality control and plant survival during heat stress.

Study of the Involvement of Phosphatidic Acid Formation in the Expression of Wound-Responsive Genes in Cotton.

Plants use phospholipase D (PLD, EC 3.1.4.4)/phosphatidic acid (PtdOH) for the transduction of environmental signals including those coming from wounding. Based on our previous findings suggesting that wound-induced PLDα-derived PtdOH can act as a local signaling molecule in cotton (Gossypium hirsutum), we show that wounding immediately increases local NADPH oxidase (NADPHox) and cellulose synthase A (CeSA) gene expression. After developing a novel fluorimetric assay for the investigation of n-butanol inhibitory effect on PLD activity, we show that only NADPHox gene upregulation is reduced when n-butanol is applied prior to wounding. This suggests that NADPHox is a possible downstream target of PLD function, while a different CeSA-involving response system may exist in cotton. Overall, this study provides new knowledge on signal-transduction mechanisms following wounding of cotton leaves.

Phospholipases Dα and δ are involved in local and systemic wound responses of cotton (G. hirsutum).

Phospholipases D (PLDs) catabolize structural phospholipids to produce phosphatidic acid (PtdOH), a lipid playing central role in signalling pathways in animal, yeast and plant cells. In animal cells two PLD genes have been studied while in model plant Arabidopsis twelve genes exist, classified in six classes (α-ζ). This underlines the role of these enzymes in plant responses to environmental stresses. However, information concerning the PLD involvement in the widely cultivated and economically important cotton plant responses is very limited. The aim of this report was to study the activity of conventional cotton PLD and its participation in plant responses to mechanical wounding, which resembles both biotic and abiotic stresses. PLDα activity was identified and further characterized by transphosphatidylation reaction. Upon wounding, cotton leaf responses consist of an acute in vitro increase of PLDα activity in both wounded and systemic tissue. However, determination of the in vivo PtdOH levels under the same wounding conditions revealed a rapid PtdOH formation only in wounded leaves and a late response of a PtdOH increase in both tissues. Εxpression analysis of PLDα and PLDδ isoforms showed mRNA accumulation of both isoforms in the wounded tissue, but only PLDδ exerts a high and sustainable expression in systemic leaves, indicating that this isoform is mainly responsible for the systemic wound-induced PtdOH production. Therefore, our data suggest that PLDα and PLDδ isoforms are involved in different steps in cotton wound signalling.

The study of fkbp and ubiquitin reveals interesting aspects of Artemia stress history.

Research on stress responses in animals has increased greatly during the last decades. Though most studies focus on the cellular and molecular bases of the stress response mechanisms, the ecological and evolutionary aspects of stress responses gain more and more interest. Here, we use species and parthenogenetic strains of the genus Artemia, an extremophile model organism, to study, for the first time, a protein well known for its chaperone activity and its involvement in stress responses. More specifically, transcription and protein accumulation of an FK506-Binding Protein (FKBP) homologue were investigated under heat and salt stresses. Additionally, the mRNA levels of ubiquitin, a heat-inducible protein related to the proteasomal pathway, were quantitated under these conditions. Biochemical and phylogenetic analyses showed that the studied FKBP orthologue is a typical representative of the family that clusters with other crustacean sequences. The expression was increased in both fkbp and ubiquitin genes after salt and heat stresses. However, our results in combination with the fact that Artemia species and parthenogenetic strains, selected for this study, exhibit different heat or salt tolerance provide useful hints about the evolutionary significance of FKBP and ubiquitin. Regarding FKBP, mRNA expression and protein accumulation seem to depend on the environmental conditions and the evolutionary history of each Artemia population while ubiquitin has a clear and more conserved role under heat shock.

Jasmonate-dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors.

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell-wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell-wall composition to reinforce this defensive barrier remains unknown. The enzyme 13-allene oxide synthase (13-AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13-AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13-AOS enzymes. Indeed, transgenic potato plants lacking both St13-AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound-responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild-type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell-wall pectin composition between wild-type and CoAOS1/2 plants. Importantly, wild-type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.

Differential isolation and identification of PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana using an agarose-phosphatidylinositol-phosphate affinity chromatography.

A phosphatidylinositol-phosphate affinity chromatographic approach combined with mass spectrometry was used in order to identify novel PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana suspension cell extracts. Most of the phosphatidylinositol-phosphate interacting candidates identified from this differential screening are characterized by lysine/arginine rich patches. Direct phosphoinositide binding was identified for important membrane trafficking regulators as well as protein quality control proteins such as the ATG18p orthologue involved in autophagosome formation and the lipid Sec14p like transfer protein. A pentatricopeptide repeat (PPR) containing protein was shown to directly bind to PI(3,5)P2 but not to PI(3)P. PIP chromatography performed using extracts obtained from high salt (0.4M and 1M NaCl) pretreated suspensions showed that the association of an S5-1 40S ribosomal protein with both PI(3)P and PI(3,5)P2 was abolished under salt stress whereas salinity stress induced an increase in the phosphoinositide association of the DUF538 domain containing protein SVB, associated with trichome size. Additional interacting candidates were co-purified with the phosphoinositide bound proteins. Binding of the COP9 signalosome, the heat shock proteins, and the identified 26S proteasomal subunits, is suggested as an indirect effect of their interaction with other proteins directly bound to the PI(3)P and the PI(3,5)P2 phosphoinositides. BIOLOGICAL SIGNIFICANCE: PI(3,5)P2 is of special interest because of its low abundance. Furthermore, no endogenous levels have yet been detected in A. thaliana (although there is evidence for its existence in plants). Therefore the isolation of novel interacting candidates in vitro would be of a particular importance since the future study and localization of the respective endogenous proteins may indicate possible targeted compartments or tissues where PI(3,5)P2 could be enriched and thereafter identified. In addition, PI(3,5)P2 is a phosphoinositide extensively studied in mammalian and yeast systems. However, our knowledge of its role in plants as well as a list of its effectors from plants is very limited.

The Arabidopsis thaliana immunophilin ROF1 directly interacts with PI(3)P and PI(3,5)P2 and affects germination under osmotic stress.

A direct interaction of the Arabidopsis thaliana immunophilin ROF1 with phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate was identified using a phosphatidylinositol-phosphate affinity chromatography of cell suspension extracts, combined with a mass spectrometry (nano LC ESI-MS/MS) analysis. The first FK506 binding domain was shown sufficient to bind to both phosphatidylinositol-phosphate stereoisomers. GFP-tagged ROF1 under the control of a 35S promoter was localised in the cytoplasm and the cell periphery of Nicotiana tabacum leaf explants. Immunofluorescence microscopy of Arabidopsis thaliana root tips verified its cytoplasmic localization and membrane association and showed ROF1 localization in the elongation zone which was expanded to the meristematic zone in plants grown on high salt media. Endogenous ROF1 was shown to accumulate in response to high salt treatment in Arabidopsis thaliana young leaves as well as in seedlings germinated on high salt media (0.15 and 0.2 M NaCl) at both an mRNA and protein level. Plants over-expressing ROF1, (WSROF1OE), exhibited enhanced germination under salinity stress which was significantly reduced in the rof1(-) knock out mutants and abolished in the double mutants of ROF1 and of its interacting homologue ROF2 (WSrof1(-)/2(-)). Our results show that ROF1 plays an important role in the osmotic/salt stress responses of germinating Arabidopsis thaliana seedlings and suggest its involvement in salinity stress responses through a phosphatidylinositol-phosphate related protein quality control pathway.

Cold acclimation and low temperature resistance in cotton: Gossypium hirsutum phospholipase Dalpha isoforms are differentially regulated by temperature and light.

Phospholipase Dalpha (PLDalpha) was isolated from cultivated cotton (Gossypium hirsutum) and characterized. Two PLDalpha genes were identified in the allotetraploid genome of G. hirsutum, derived from its diploid progenitors, G. raimondii and G. arboreum. The genes contained three exons and two introns. The translated products shared a 98.6% homology and were designated as GrPLDalpha and GaPLDalpha. Their ORFs encoded a polypeptide of 807 amino acids with a predicted molecular mass of 91.6 kDa sharing an 81-82% homology with PLDalpha1 and PLDalpha2 from A. thaliana. A possible alternative splicing event was detected at the 5' untranslated region which, however, did not result in alternative ORFs. Cold stress (10 degrees C or less) resulted in gene induction which was suppressed below control levels (25 degrees C or 22 degrees C growth temperature) when plants were acclimated at 17 degrees C before applying the cold treatment. Differences in the expression levels of the isoforms were recorded under cold acclimation, and cold stress temperatures. Expression was light regulated under growth, acclimation, and cold stress temperatures. Characterization of the products of lipid hydrolysis by the endogenous PLDalpha indicated alterations in lipid species and a variation in levels of the signalling molecule phosphatidic acid (PA) following acclimation or cold stress.

Sharp phylogeographic breaks and patterns of genealogical concordance in the brine shrimp Artemia franciscana.

Genealogical concordance is a critical overlay of all phylogenetic analyses, irrespective of taxonomic level. To assess such patterns of congruence we have compiled and derived sequence data for two mitochondrial (16S rRNA, COI) and two nuclear (ITS1, p26) markers in 14 American populations of the hypersaline branchiopod Artemia franciscana. Cladistic analysis revealed three reciprocally monophyletic mitochondrial clades. For nuclear DNA, incomplete lineage sorting was evident presumably as a result of slower coalescence or male-mediated dispersal. Our findings capture the genealogical interval between gene splitting and population divergence. In this sense, strong indications are provided in favour of a superspecies status and ongoing speciation in A. franciscana.

Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum).

Lipid modifying enzymes play a key role in the development of cold stress tolerance in cold-resistant plants such as cereals. However, little is known about the role of the endogenous enzymes in cold-sensitive species such as cotton. Delta 12 fatty acid desaturases (FAD2), known to participate in adaptation to low temperatures through acyl chain modifications were used in gene expression studies in order to identify parameters of plant response to low temperatures. The induction of microsomal delta 12 fatty acid desaturases at an mRNA level under cold stress in plants is shown here for first time. Quantitative PCR showed that though both delta 12 omega 6 fatty acid desaturase genes FAD2-3 and FAD2-4 identified in cotton are induced under cold stress, FAD2-4 induction is significantly higher than FAD2-3. The induction of both isoforms was light regulated, in contrast a third isoform FAD2-2 was not affected by cold or light. Stress tolerance and light regulatory elements were identified in the predicted promoters of both FAD2-3 and FAD2-4 genes. Di-unsaturated fatty acid species rapidly increased in the microsomal fraction isolated from cotton leaves, following cold stress. Expression analysis patterns were correlated with the observed increase in both total and microsomal fatty acid unsaturation levels suggesting the direct role of the FAD2 genes in membrane adaptation to cold stress.

Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts.

The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.