Nodulin 22, a novel small heat-shock protein of the endoplasmic reticulum, is linked to the unfolded protein response in common bean.

The importance of plant small heat shock proteins (sHsp) in multiple cellular processes has been evidenced by their unusual abundance and diversity; however, little is known about their biological role. Here, we characterized the in vitro chaperone activity and subcellular localization of nodulin 22 of Phaseolus vulgaris (PvNod22; common bean) and explored its cellular function through a virus-induced gene silencing-based reverse genetics approach. We established that PvNod22 facilitated the refolding of a model substrate in vitro, suggesting that it acts as a molecular chaperone in the cell. Through microscopy analyses of PvNod22, we determined its localization in the endoplasmic reticulum (ER). Furthermore, we found that silencing of PvNod22 resulted in necrotic lesions in the aerial organs of P. vulgaris plants cultivated under optimal conditions and that downregulation of PvNod22 activated the ER-unfolded protein response (UPR) and cell death. We also established that PvNod22 expression in wild-type bean plants was modulated by abiotic stress but not by chemicals that trigger the UPR, indicating PvNod22 is not under UPR control. Our results suggest that the ability of PvNod22 to suppress protein aggregation contributes to the maintenance of ER homeostasis, thus preventing the induction of cell death via UPR in response to oxidative stress during plant-microbe interactions.

Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket.

Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.

Unique N-Terminal Interactions Connect F-BOX STRESS INDUCED (FBS) Proteins to a WD40 Repeat-like Protein Pathway in Arabidopsis.

SCF-type E3 ubiquitin ligases provide specificity to numerous selective protein degradation events in plants, including those that enable survival under environmental stress. SCF complexes use F-box (FBX) proteins as interchangeable substrate adaptors to recruit protein targets for ubiquitylation. FBX proteins almost universally have structure with two domains: A conserved N-terminal F-box domain interacts with a SKP protein and connects the FBX protein to the core SCF complex, while a C-terminal domain interacts with the protein target and facilitates recruitment. The F-BOX STRESS INDUCED (FBS) subfamily of plant FBX proteins has an atypical structure, however, with a centrally located F-box domain and additional conserved regions at both the N- and C-termini. FBS proteins have been linked to environmental stress networks, but no ubiquitylation target(s) or biological function has been established for this subfamily. We have identified two WD40 repeat-like proteins in Arabidopsis that are highly conserved in plants and interact with FBS proteins, which we have named FBS INTERACTING PROTEINs (FBIPs). FBIPs interact exclusively with the N-terminus of FBS proteins, and this interaction occurs in the nucleus. FBS1 destabilizes FBIP1, consistent with FBIPs being ubiquitylation targets SCFFBS1 complexes. This work indicates that FBS proteins may function in stress-responsive nuclear events, and it identifies two WD40 repeat-like proteins as new tools with which to probe how an atypical SCF complex, SCFFBS, functions via FBX protein N-terminal interaction events.

The Arabidopsis F-box protein AtFBS1 interacts with 14-3-3 proteins.

AtFBS1 is an F-box protein whose transcript accumulates in response to biotic and abiotic stresses. Previous evidence suggests that a postranscriptional event regulates AtFBS1 expression [1]. We now found that AtFBS1 interacts with 14-3-3 proteins through its amino-terminus and the F-box motif. Deletion of any of these regions abolishes the interaction between AtFBS1 and 14-3-3 proteins. On the other hand, the treatment with the proteasome inhibitor MG132 or the deletion of the F-box from AtFBS1 increases ß-glucuronidase (GUS) activity in plants containing a translational fusion of AtFBS1 with the GUS reporter gene, indicating that AtFBS1 is degraded by the 26S proteasome. MG132 treatment of seedlings containing a gene fusion between AtFBS1 and the TAP (Tandem Affinity Purification) cassette causes an increase in the half-life of the protein. In an attempt to understand the role of 14-3-3 interactions, we treated Arabidopsis seedlings with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (AICAR), an inhibitor of 14-3-3 client interactions. We observed an increase in AtFBS1-TAP stability as a consequence of AICAR treatment. Based on these data we propose that 14-3-3 proteins promote the dimerization of SCF(AtFBS1). This also may enhance the AtFBS1 autoubiquitination activity and its degradation by the 26S proteasome. AICAR also affects Cullin1 (CUL1) modification by RUB1, which would provide an additional element to the effect of this compound on AtFBS1 stability.

Characterization of novel F-box proteins in plants induced by biotic and abiotic stress.

Plants protect against pathogen infections by a combination of constitutive and induced strategies. The induction of plant defense involves the recognition of compounds derived from the pathogen or the plant itself, called elicitors. Looking for new genes involved in plant defense responses, we isolated a cDNA clone corresponding to an elicitor-induced mRNA from Phaseolus vulgaris cell suspension cultures. This clone, PvFBS1, encodes a protein with an F-box, therefore a putative component of an SCF ubiquitin ligase complex. PvFBS1 mRNA accumulates in leaves of whole plants in response to wounding or osmotic stress, as well as, following the application of methyl jasmonate (MeJA). salicylic acid (SA) or abscisic acid (ABA). Several sequences related to PvFBS1 were found in the GenBank. In Arabidopsis thaliana there are 4 genomic sequences coding for proteins with similarity to PvFBS1. One of them, AtFBS1, displays a pattern of induction analogous to the one observed for PvFBS1. A yeast two-hybrid assay proved that AtFBS1 was able to interact with ASK1, the component of the SCF complex that binds the F-box. A deletion of the F-box in AtFBS1 abolishes the ability of this protein to interact with ASK1. This demonstrates the functionality of the F-box contained in AtFBS1. Gene fusions to the GUS reporter gene revealed a complex regulation for AtFBS1 expression.

Wounding and pathogen infection induce a chloroplast-targeted lipoxygenase in the common bean (Phaseolus vulgaris L.).

Chloroplastic LOXs are implicated in the biosynthesis of oxylipins like jasmonic acid and C6 volatiles among others. In this study, we isolated the cDNA of a novel chloroplast-targeted Phaseolus vulgaris LOX, (PvLOX6). This gene is highly induced after wounding, non-host pathogen infection, and by signaling molecules as H2O2, SA, ethylene and MeJA. The phylogenetic analysis of PvLOX6 showed that it is closely related to chloroplast-targeted LOX from potato (H1) and tomato (TomLOXC); both of them are implicated in the biosynthesis of C6 volatiles. Induction of PvLOX6 mRNA by wounding ethylene and jasmonic acid on the one side, and non-host pathogen, salicylic acid on the other indicates that common bean uses the same LOX to synthesize oxylipins in response to different stresses.

AtMCP1b, a chloroplast-localised metacaspase, is induced in vascular tissue after wounding or pathogen infection.

cDNA corresponding to the Arabidopsis type I metacaspase AtMCP1b was isolated from plants infected with Pseudomonas syringae. A positive correlation between AtMCP1b expression and cell death was observed in the presence of staurosporine, a protein kinase inhibitor that induces programmed cell death. The tissue localisation of an AtMCP1b promoter-GUS fusion was observed in the vascular tissue of transgenic plants. GUS activity increased in response to an incompatible DC3000 (avrRpm1) or a compatible DC3000 P. syringae infection, or to wounding. Confocal and immunohistochemical analysis of Arabidopsis thaliana (L.) leaves showed that an AtMCP1b-GFP fusion protein was localised in the chloroplasts. Our data support a positive correlation between AtMCP1b gene expression and cell death in response to wounding or pathogenic interactions. Moreover, the localisation of AtMCP1b gene expression within vascular tissue and cells of abscission regions strongly supports a role for AtMCP1b in programmed cell dismantling events in response to environmental and developmental triggers. The AtMCP1b-GFP subcellular localisation infers a role for the plastid organelles in PCD and, thus, in responses to pathogen attack and development.

Hormonal and stress induction of the gene encoding common bean acetyl-coenzyme A carboxylase.

Regulation of the cytosolic acetyl-coenzyme A carboxylase (ACCase) gene promoter from common bean (Phaseolus vulgaris) was studied in transgenic Arabidopsis (Arabidopsis thaliana) plants using a beta-glucuronidase (GUS) reporter gene fusion (PvACCase::GUS). Under normal growth conditions, GUS was expressed in hydathodes, stipules, trichome bases, flowers, pollen, and embryos. In roots, expression was observed in the tip, elongation zone, hypocotyl-root transition zone, and lateral root primordia. The PvACCase promoter was induced by wounding, Pseudomonas syringae infection, hydrogen peroxide, jasmonic acid (JA), ethylene, or auxin treatment. Analysis of PvACCase::GUS expression in JA and ethylene mutants (coronatine insensitive1-1 [coi1-1], ethylene resistant1-1 [etr1-1], coi1-1/etr1-1) suggests that neither JA nor ethylene perception participates in the activation of this gene in response to wounding, although each of these independent signaling pathways is sufficient for pathogen or hydrogen peroxide-induced PvACCase gene expression. We propose a model involving different pathways of PvACCase gene activation in response to stress.

A red beet (Beta vulgaris) UDP-glucosyltransferase gene induced by wounding, bacterial infiltration and oxidative stress.

Mechanical wounding, infiltration with P. syringae or A. tumefaciens, and exposure to an H(2)O(2)-generating system (Glc/Glc oxidase) induce betacyanin synthesis in red beet (Beta vulgaris) leaves. These conditions also induced the expression of BvGT, a gene encoding a glucosyltransferase (GT) from Beta vulgaris. BvGT has a high similarity to Dorotheanthus bellidiformis betanidin-5 GT involved in betacyanin synthesis. Furthermore, the transient expression of a BvGT antisense construct resulted in the reduction of BvGT transcript accumulation and betanin synthesis, suggesting a role for this gene product in betacyanin glucosylation. In addition, the NADPH oxidase inhibitor, diphenylene iodonium (DPI), inhibited the accumulation of the BvGT transcript in response to infiltration with Agrobacterium tumefaciens. Hence, this result suggests that ROS produced by a plasma membrane NADPH oxidase may act as a signal to induce BvGT expression, necessary for betanin synthesis after wounding and bacterial infiltration.