Rafael Catalá

The Arabidopsis E3 SUMO Ligase SIZ1 Regulates Plant Growth and Drought Responses

Posttranslational modifications of proteins by small ubiquitin-like modifiers (SUMOs) regulate protein degradation and localization, protein–protein interaction, and transcriptional activity. SUMO E3 ligase functions are executed by SIZ1/SIZ2 and Mms21 in yeast, the PIAS family members RanBP2, and Pc2 in human. The Arabidopsis thaliana genome contains only one gene, SIZ1, that is orthologous to the yeast SIZ1/SIZ2. Here, we show that Arabidopsis SIZ1 is expressed in all plant tissues. Compared with the wild type, the null mutant siz1-3 is smaller in stature because of reduced expression of genes involved in brassinosteroid biosynthesis and signaling. Drought stress induces the accumulation of SUMO-protein conjugates, which is in part dependent on SIZ1 but not on abscisic acid (ABA). Mutant plants of siz1-3 have significantly lower tolerance to drought stress. A genome-wide expression analysis identified ∼1700 Arabidopsis genes that are induced by drought, with SIZ1 mediating the expression of 300 of them by a pathway independent of DREB2A and ABA. SIZ1-dependent, drought-responsive genes include those encoding enzymes of the anthocyanin synthesis pathway and jasmonate response. From these results, we conclude that SIZ1 regulates Arabidopsis growth and that this SUMO E3 ligase plays a role in drought stress response likely through the regulation of gene expression.

The CBFs: Three arabidopsis transcription factors to cold acclimate

Low temperature is one of the adverse environmental factors that most affects plant growth and development. Temperate plants have evolved the capacity to acquire chilling and freezing tolerance after being exposed to low-nonfreezing temperatures. This adaptive response, named cold acclimation, involves many physiological and biochemical changes that mainly rely on reprogramming gene expression. Currently, the best documented genetic pathway leading to gene induction under low temperature conditions is the one mediated by the Arabidopsis C-repeat/dehydration-responsive element binding factors (CBFs), a small family of three transcriptional activators (CBF1-3) that bind to the C-repeat/dehydration-responsive element, which is present in the promoters of many cold-responsive genes, and induce transcription. The CBF genes are themselves induced by cold. Different evidences indicate that the CBF transcriptional network plays a critical role in cold acclimation in Arabidopsis. In this review, recent advances on the regulation and function of CBF factors are provided and discussed.

Mutations in the Ca2+/H+ Transporter CAX1 Increase CBF/DREB1 Expression and the Cold-Acclimation Response in Arabidopsis

Transient increases in cytosolic free calcium concentration ([Ca2+]cyt) are essential for plant responses to a variety of environmental stimuli, including low temperature. Subsequent reestablishment of [Ca2+]cyt to resting levels by Ca2+ pumps and C-REPEAT BINDING FACTOR/DEHYDRATION RESPONSIVE ELEMENT BINDING FACTOR 1 (Ca2+/H+) antiporters is required for the correct transduction of the signal. We have isolated a cDNA from Arabidopsis that corresponds to a new cold-inducible gene, RARE COLD INDUCIBLE4 (RCI4), which was identical to CALCIUM EXCHANGER 1 (CAX1), a gene that encodes a vacuolar Ca2+/H+ antiporter involved in the regulation of intracellular Ca2+ levels. The expression of CAX1 was induced in response to low temperature through an abscisic acid–independent pathway. To determine the function of CAX1 in Arabidopsis stress tolerance, we identified two T-DNA insertion mutants, cax1-3 and cax1-4, that display reduced tonoplast Ca2+/H+ antiport activity. The mutants showed no significant differences with respect to the wild type when analyzed for dehydration, high-salt, chilling, or constitutive freezing tolerance. However, they exhibited increased freezing tolerance after cold acclimation, demonstrating that CAX1 plays an important role in this adaptive response. This phenotype correlates with the enhanced expression of CBF/DREB1 genes and their corresponding targets in response to low temperature. Our results indicate that CAX1 ensures the accurate development of the cold-acclimation response in Arabidopsis by controlling the induction of CBF/DREB1 and downstream genes.

Integration of low temperature and light signaling during cold acclimation response in Arabidopsis

Certain plants increase their freezing tolerance in response to low nonfreezing temperatures, an adaptive process named cold acclimation. Light has been shown to be required for full cold acclimation, although how light and cold signals integrate and cross-talk to enhance freezing tolerance still remains poorly understood. Here, we show that HY5 levels are regulated by low temperature transcriptionally, via a CBF- and ABA-independent pathway, and posttranslationally, via protein stabilization through nuclear depletion of COP1. Furthermore, we demonstrate that HY5 positively regulates cold-induced gene expression through the Z-box and other cis-acting elements, ensuring the complete development of cold acclimation. These findings uncover unexpected functions for HY5, COP1, and the Z-box in Arabidopsis response to low temperature, provide insights on how cold and light signals integrate to optimize plant survival under freezing temperatures, and reveal the complexity of the molecular mechanisms plants have evolved to respond and adapt to their fluctuating natural environment.

High-yield expression of a viral peptide vaccine in transgenic plants

A high‐yield production of a peptide vaccine in transgenic plants is described here. A 21‐mer peptide, which confers protection to dogs against challenge with virulent canine parvovirus, has been expressed in transgenic plants as an amino‐terminal translational fusion with the GUS gene. Transformants were selected on the basis of their GUS activities, showing expression levels of the recombinant protein up to 3% of the total leaf soluble protein, a production yield comparable to that obtained with the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant‐derived peptide was demonstrated in mice immunized either intraperitoneally or orally with transgenic plant extracts, providing the suitability of the GUS fusions approach for low‐cost production of peptide vaccines.

The Arabidopsis 14-3-3 Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate Freezing Tolerance and Cold Acclimation

The Arabidopsis 14-3-3 RCI1A protein plays a critical role in freezing tolerance, partially through an ethylene-dependent signaling pathway. RCI1A interacts with different ACS isoforms to regulate the levels of ethylene that are necessary to promote accurate cold-induced gene expression and freezing tolerance under both control and low-temperature conditions. In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis.

Identification of SUMO Targets by a Novel Proteomic Approach in PlantsF

Post-translational modifications (PTMs) chemically and physically alter the properties of proteins, including their folding, subcellular localization, stability, activity, and consequently their function. In spite of their relevance, studies on PTMs in plants are still limited. Small Ubiquitin-like Modifier (SUMO) modification regulates several biological processes by affecting protein-protein interactions, or changing the subcellular localizations of the target proteins. Here, we describe a novel proteomic approach to identify SUMO targets that combines 2-D liquid chromatography, immunodetection, and mass spectrometry (MS) analyses. We have applied this approach to identify nuclear SUMO targets in response to heat shock. Using a bacterial SUMOylation system, we validated that some of the targets identified here are, in fact, labeled with SUMO1. Interestingly, we found that GIGANTEA (GI), a photoperiodic-pathway protein, is modified with SUMO in response to heat shock both in vitro and in vivo.

The LSM1-7 Complex Differentially Regulates Arabidopsis Tolerance to Abiotic Stress Conditions by Promoting Selective mRNA Decapping

A decapping activator complex ensures the adequacy and specificity of the response of Arabidopsis to different abiotic stresses by promoting selective mRNA degradation. In eukaryotes, the decapping machinery is highly conserved and plays an essential role in controlling mRNA stability, a key step in the regulation of gene expression. Yet, the role of mRNA decapping in shaping gene expression profiles in response to environmental cues and the operating molecular mechanisms are poorly understood. Here, we provide genetic and molecular evidence that a component of the decapping machinery, the LSM1-7 complex, plays a critical role in plant tolerance to abiotic stresses. Our results demonstrate that, depending on the stress, the complex from Arabidopsis thaliana interacts with different selected stress-inducible transcripts targeting them for decapping and subsequent degradation. This interaction ensures the correct turnover of the target transcripts and, consequently, the appropriate patterns of downstream stress-responsive gene expression that are required for plant adaptation. Remarkably, among the selected target transcripts of the LSM1-7 complex are those encoding NCED3 and NCED5, two key enzymes in abscisic acid (ABA) biosynthesis. We demonstrate that the complex modulates ABA levels in Arabidopsis exposed to cold and high salt by differentially controlling NCED3 and NCED5 mRNA turnover, which represents a new layer of regulation in ABA biosynthesis in response to abiotic stress. Our findings uncover an unanticipated functional plasticity of the mRNA decapping machinery to modulate the relationship between plants and their environment.

Molecular responses to extreme temperatures

Publisher Summary From the temperature change sensing to the temperature-responsive gene expression, with the signal transduction pathways in between, there are many levels at which cold and heat responses overlap. This chapter presents a general view of the current knowledge of how plants perceive and transduce the cold signal, and how low temperature response is regulated. It also discusses what has been reported on how plant responds to low and high temperatures, interact, and integrate, and on the potential common mechanisms and components that can mediate such an integration. Temperatures above the optimum are also highly stressful for plants, disturbing cellular homeostasis and leading to severe retardation in growth and development, and even death. Exposure to low temperature initiates numerous physiological perturbations; the first includes changes in cell membrane fluidity state and fatty acid composition. The content of sugar is also altered in plants in response to low temperature. The chapter also provides a general overview of how the cold signal is perceived, decoded, and transduced, and about the regulatory mechanisms controlling plant response to low temperature. Further, it is to be noted that some intermediates have been identified that seem to be particularly relevant and would constitute important nodes of integration between low- and high-temperature responses.

The Arabidopsis ethylene overproducer mutant eto1-3 displays enhanced freezing tolerance

Low temperature is one of the most important environmental stresses constraining plant development and distribution. Plants have evolved complex adaptive mechanisms to face and survive freezing temperatures. Different signaling pathways regulating plant response to cold have been described, and some of them are mediated by hormones. Recently, we reported that ethylene (ET) acts as a positive regulator of plant freezing tolerance through the activation of cold-induced gene expression, including the CBF-regulon. Here, we present data demonstrating that the Arabidopsis ET overproducer mutant eto1-3 has enhanced freezing tolerance. Moreover, we also show that this mutant exhibits increased accumulation of CBF1, 2 and 3 transcripts, which should account for its tolerant phenotype. All these results constitute new genetic evidence supporting an important role for ET in plant response to low temperature by mediating the CBF-dependent signaling pathway.