Antonello Costa

Beyond transcription: RNA-binding proteins as emerging regulators of plant response to environmental constraints.

RNA-binding proteins (RBPs) govern many aspects of RNA metabolism, including pre-mRNA processing, transport, stability/decay and translation. Although relatively few plant RNA-binding proteins have been characterized genetically and biochemically, more than 200 RBP genes have been predicted in Arabidopsis and rice genomes, suggesting that they might serve specific plant functions. Besides their role in normal cellular functions, RBPs are emerging also as an interesting class of proteins involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions. Here, we review the most recent results and evidence on the functional role of RBPs in plant adaptation to various unfavourable environmental conditions and their contribution to enhance plant tolerance to abiotic stresses, with special emphasis on osmotic and temperature stress.

Comparative Analysis of Short- and Long-Term Changes in Gene Expression Caused by Low Water Potential in Potato (Solanum tuberosum) Cell-Suspension Cultures

To dissect the cellular response to water stress and compare changes induced as a generalized response with those involved in tolerance/acclimation mechanisms, we analyzed changes in two-dimensional electrophoretic patterns of in vivo [35S]methionine-labeled polypeptides of cultured potato (Solanum tuberosum) cells after gradual and long exposure to polyethylene glycol (PEG)-mediated low water potential versus those induced in cells abruptly exposed to the same stress intensity. Protein synthesis was not inhibited by gradual stress imposition, and the expression of 17 proteins was induced in adapted cells. Some polypeptides were inducible under mild stress conditions (5% PEG) and accumulated further when cells were exposed to a higher stress intensity (10 and 20% PEG). The synthesis of another set of polypeptides was up-regulated only when more severe water-stress conditions were applied, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly. In contrast, in potato cells abruptly exposed to 20% PEG, protein synthesis was strongly inhibited. Nevertheless, a large set of polypeptides was identified whose expression was increased. Most of these polypeptides were not induced in adapted cells, but many of them were common to those observed in abscisic acid (ABA)-treated cells. These data, along with the finding that cellular ABA content increased in PEG-shocked cells but not in PEG-adapted cells, suggested that this hormone is mainly involved in the rapid response to stress rather than long-term adaptation. A further group of proteins included those induced after long exposure to both water stress and shock. Western blot analysis revealed that osmotin was one protein belonging to this common group. This class may represent induced proteins that accumulate specifically in response to low water potential and that are putatively involved in the maintenance of cellular homeostasis under prolonged stress.

Transcriptomic Changes Drive Physiological Responses to Progressive Drought Stress and Rehydration in Tomato

Tomato is a major crop in the Mediterranean basin, where the cultivation in the open field is often vulnerable to drought. In order to adapt and survive to naturally occurring cycles of drought stress and recovery, plants employ a coordinated array of physiological, biochemical, and molecular responses. Transcriptomic studies on tomato responses to drought and subsequent recovery are few in number. As the search for novel traits to improve the genetic tolerance to drought increases, a better understanding of these responses is required. To address this need we designed a study in which we induced two cycles of prolonged drought stress and a single recovery by rewatering in tomato. In order to dissect the complexity of plant responses to drought, we analyzed the physiological responses (stomatal conductance, CO2 assimilation, and chlorophyll fluorescence), abscisic acid (ABA), and proline contents. In addition to the physiological and metabolite assays, we generated transcriptomes for multiple points during the stress and recovery cycles. Cluster analysis of differentially expressed genes (DEGs) between the conditions has revealed potential novel components in stress response. The observed reduction in leaf gas exchanges and efficiency of the photosystem PSII was concomitant with a general down-regulation of genes belonging to the photosynthesis, light harvesting, and photosystem I and II category induced by drought stress. Gene ontology (GO) categories such as cell proliferation and cell cycle were also significantly enriched in the down-regulated fraction of genes upon drought stress, which may contribute to explain the observed growth reduction. Several histone variants were also repressed during drought stress, indicating that chromatin associated processes are also affected by drought. As expected, ABA accumulated after prolonged water deficit, driving the observed enrichment of stress related GOs in the up-regulated gene fractions, which included transcripts putatively involved in stomatal movements. This transcriptomic study has yielded promising candidate genes that merit further functional studies to confirm their involvement in drought tolerance and recovery. Together, our results contribute to a better understanding of the coordinated responses taking place under drought stress and recovery in adult plants of tomato.

Identification of salt stress-induced transcripts in potato leaves by cDNA-AFLP

Potato (Solanum tuberosum L.) is highly sensitive to salt stress, which is one of the most important factors limiting plant cultivation. The investigation of plant response to high salinity was envisaged in this report using cDNA-amplified fragment length polymorphism (AFLP). This technique was applied to salt-stressed and control potato plants (cv. Nicola). The expression profiles showed approx 5000 bands. Of these, 154 were upregulated and 120 were repressed by salt stress. In this study we have only considered cDNA fragments that seem to be originated from salt-induced mRNA. Eighteen fragments were then reamplified, cloned, and sequenced. Sequence comparison of these cDNA, identified in response to salt stress in potato, revealed that some of them present homologies with proteins in other species that are involved in cell wall structure and turnover such as proline-rich proteins and β-galactosidase. A number of identified clones encoded putative stress response proteins such as NADP-dependant glyceraldehyde dehydrogenase and wound-induced protein. In addition, some of them encode proteins related to hypersensitive response against pathogens such as putative late blight and nematode as well as putative pathogenesis-related proteins. These cDNA seem to be differentially expressed in the presence of salt stress as shown by Northern blot or reverse Northern hybridization experiments.

The Arabidopsis RNA-Binding Protein AtRGGA Regulates Tolerance to Salt and Drought Stress

An Arabidopsis RNA-binding protein contributes to drought and salt stress tolerance. Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

Chloroplast proteome response to drought stress and recovery in tomato (Solanum lycopersicum L.)

BackgroundDrought is a major constraint for plant growth and crop productivity that is receiving an increased attention due to global climate changes. Chloroplasts act as environmental sensors, however, only partial information is available on stress-induced mechanisms within plastids. Here, we investigated the chloroplast response to a severe drought treatment and a subsequent recovery cycle in tomato through physiological, metabolite and proteomic analyses.ResultsUnder stress conditions, tomato plants showed stunted growth, and elevated levels of proline, abscisic acid (ABA) and late embryogenesis abundant gene transcript. Proteomics revealed that water deficit deeply affects chloroplast protein repertoire (31 differentially represented components), mainly involving energy-related functional species. Following the rewatering cycle, physiological parameters and metabolite levels indicated a recovery of tomato plant functions, while proteomics revealed a still ongoing adjustment of the chloroplast protein repertoire, which was even wider than during the drought phase (54 components differentially represented). Changes in gene expression of candidate genes and accumulation of ABA suggested the activation under stress of a specific chloroplast-to-nucleus (retrograde) signaling pathway and interconnection with the ABA-dependent network.ConclusionsOur results give an original overview on the role of chloroplast as enviromental sensor by both coordinating the expression of nuclear-encoded plastid-localised proteins and mediating plant stress response. Although our data suggest the activation of a specific retrograde signaling pathway and interconnection with ABA signaling network in tomato, the involvement and fine regulation of such pathway need to be further investigated through the development and characterization of ad hoc designed plant mutants.

Tolerance to abiotic stresses in potato plants: a molecular approach

SummaryContinuing study of the potato plants response to stressful conditions has led to the identification of a large number of plant genes whose expression, is regulated by external stimuli. Stress-induced genes can be broadly divided into functional or regulatory genes. To the first category belong genes encoding proteins or enzymes of plant metabolic pathway, of molecules involved in repairing cellular damages and/or indispensable for restoring a new cellular homeostasis compatible with the external conditions. The other class includes genes primarily involved in the perception and/or intracellular transduction of the stress signal, such as kinases, phosphatases or transcription, factors. The research objectives in the field of plant stress tolerance has recently evolved from a mere cloning and description of stress-induced genes to the design of the best strategy of producing transgenic plants tolerant to environmental constraints. It is well known that stress tolerance is a complex trait, requiring the coordinated regulation of a network of genes that act synergistically and additively. At best, manipulation of one single down-stream gene may contribute only partially to the tolerance of the transgenic plants. Recent studies have shown that it is feasible to regulate the level of expression of many down-stream stress-induced genes in a coordinated fashion by regulating the expression of genes encoding transcription factors able to bind DNA motifs in the promoter of stress-induced genes. However, the constitutive high level of expression of transcription factors often causes detrimental phenotypic effects. This drawback could be bypassed by putting genes for transcription factors under the control of inducible promoters. In this way, endogenous tolerance genes are activated only when the stress event occurs, minimizing the negative pleiotropic effect. Novel technology (reverse genetics, DNA microarrays, mRNA differential display, T-DNA tagging, complementation and over-expression of plant cDNA in yeast as model for cellular stress tolerance), improvement of genetic transformation techniques (multiple gene transfer, gene targeting by homologous recombination) as well as a better efficiency of foreign gene expression (discovery of plant promoters with cell-specific, tissue-specific, developmental stage-specific, and/or inducible patterns of expression) will give a tremendous impulse to produce stress tolerant commercial cultivars of the main crops through genetic engineering.

Genetics of Drought Stress Tolerance in Crop Plants

Drought is the single abiotic stress with the biggest impact on global crop yields, thus improving crop performance under water-limiting conditions is essential to global food security. This chapter is intended to cover a broad overview of traits and mechanisms known to play a role in drought tolerance in crop plants. Although traditional breeding has largely focused on improving yield under stress-free conditions, a number of mechanisms exist that may be exploited to improve drought tolerance; these include the ABA signaling network for regulation of stomatal movements and root architecture modifications. This chapter reviews the biochemical and molecular modifications induced by water deficit. Stress-induced regulatory genes, specifically involved in marshaling survival responses in metabolism and development, are covered. Traits and genes from tolerant varieties, landraces, and wild relatives, known to contribute to drought tolerance from major crops are also addressed. As our understanding of signaling pathways improves based on studies in models, new traits and opportunities for genetic improvement emerge. Developing varieties that have high yields and are yield-stable in dry environments requires progress in both understanding and applying of genetic and physiological processes. This chapter covers the key traits and genes that have the greatest potential for improving drought tolerance in crop species.

Isolation, characterization and expression of an elongation factor 1α gene in potato (Solanum tuberosum) cell cultures

Abstract Using a differential display (DDRT‐PCR) approach to screen genes involved in response to osmotic stress, we isolated six cDNA clones from potato cell cultures. One of these, clone #54, is a member of the gene family encoding the α‐subunit of translation elongation factor 1 (EF‐1α). After sequencing, this clone showed a deduced amino acid sequence of 447 residues. Southern blot analysis revealed that this EF‐1α is a member of a family consisting of at least nine genes. The expression of EF‐1α increased upon osmotic stress mediated by polyethylene glycol (PEG) but was unaffected by abscisic acid (ABA) treatment. Salt also increased EF‐1α expression but with a different kinetics. These results indicate that we cloned an EF‐1α in potato which is ABA responsive, independent of osmotic stress. Abbreviations: DDRT‐PCR, differential display reverse transcriptase‐polymerase chain reaction; PEG, polyethylene glycol; RACE, rapid amplification of cDNA ends; RT‐PCR, reverse transcriptase‐polymerase chain reaction

Regulation of Gene Expression during Cellular Adaptation to Water Stress

Gradual adaptation to water stress was accomplished by a set of metabolic adjustments, including proline accumulation, synthesis ofde novo proteins, and changes in gene expression. The ability of the adapted cells to maintain the cellular and subcellular membrane integrity under conditions of severe water stress was found to be associated with a reduced level of unsaturation of fatty acids of membrane lipids. By cloning two different desaturase genes (Δ9-stearoyl-ACP-desaturase and a Δ12-oleoyl-desaturase gene, respectively) it was possible to establish that the reduced level of unsaturation observed in the adapted cells was related to the down-regulation of this class of gene.