Abdelhak El Amrani

The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance

BackgroundGABA (γ-aminobutyric acid) is a non protein amino acid that has been reported to accumulate in a number of plant species when subjected to high salinity and many other environmental constraints. However, no experimental data are to date available on the molecular function of GABA and the involvement of its metabolism in salt stress tolerance in higher plants. Here, we investigated the regulation of GABA metabolism in Arabidopsis thaliana at the metabolite, enzymatic activity and gene transcription levels upon NaCl stress.ResultsWe identified the GABA transaminase (GABA-T), the first step of GABA catabolism, as the most responsive to NaCl. We further performed a functional analysis of the corresponding gene POP2 and demonstrated that the previously isolated loss-of-function pop2-1 mutant was oversensitive to ionic stress but not to osmotic stress suggesting a specific role in salt tolerance. NaCl oversensitivity was not associated with overaccumulation of Na+ and Cl- but mutant showed a slight decrease in K+. To bring insights into POP2 function, a promoter-reporter gene strategy was used and showed that POP2 was mainly expressed in roots under control conditions and was induced in primary root apex and aerial parts of plants in response to NaCl. Additionally, GC-MS- and UPLC-based metabolite profiling revealed major changes in roots of pop2-1 mutant upon NaCl stress including accumulation of amino acids and decrease in carbohydrates content.ConclusionsGABA metabolism was overall up-regulated in response to NaCl in Arabidopsis. Particularly, GABA-T was found to play a pivotal function and impairment of this step was responsible for a decrease in salt tolerance indicating that GABA catabolism was a determinant of Arabidopsis salt tolerance. GABA-T would act in salt responses in linking N and C metabolisms in roots.

GABA Accumulation Causes Cell Elongation Defects and a Decrease in Expression of Genes Encoding Secreted and Cell Wall-Related Proteins in Arabidopsis thaliana

GABA (γ-aminobutyric acid), a non-protein amino acid, is a signaling factor in many organisms. In plants, GABA is known to accumulate under a variety of stresses. However, the consequence of GABA accumulation, especially in vegetative tissues, remains poorly understood. Moreover, gene expression changes as a consequence of GABA accumulation in plants are largely unknown. The pop2 mutant, which is defective in GABA catabolism and accumulates GABA, is a good model to examine the effects of GABA accumulation on plant development. Here, we show that the pop2 mutants have pollen tube elongation defects in the transmitting tract of pistils. Additionally, we observed growth inhibition of primary root and dark-grown hypocotyl, at least in part due to cell elongation defects, upon exposure to exogenous GABA. Microarray analysis of pop2-1 seedlings grown in GABA-supplemented medium revealed that 60% of genes whose expression decreased encode secreted proteins. Besides, functional classification of genes with decreased expression in the pop2-1 mutant showed that cell wall-related genes were significantly enriched in the microarray data set, consistent with the cell elongation defects observed in pop2 mutants. Our study identifies cell elongation defects caused by GABA accumulation in both reproductive and vegetative tissues. Additionally, our results show that genes that encode secreted and cell wall-related proteins may mediate some of the effects of GABA accumulation. The potential function of GABA as a growth control factor under stressful conditions is discussed.

Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene

A variety of conventional methods allow the expression of multiple foreign proteins in plants by transgene stacking or pyramiding. However, most of these approaches have significant drawbacks. We describe a novel alternative, using a single transgene to coordinate expression of multiple proteins that are encoded as a polyprotein capable of dissociating into component proteins on translation. We demonstrate that this polyprotein system is compatible with the need to target proteins to a variety of subcellular locations, either cotranslationally or posttranslationally. It can also be used to coordinate the expression of selectable marker genes and effect genes or to link genes that are difficult to assay to reporter genes that are easily monitored. The unique features of this polyprotein system are based on the novel activity of the 2A peptide of Foot-and-mouth disease virus (FMDV) that acts cotranslationally to effect a dissociation of the polyprotein while allowing translation to continue. This polyprotein system has many applications both as a research tool and for metabolic engineering and protein factory applications of plant biotechnology.

Genome-wide interacting effects of sucrose and herbicide-mediated stress in Arabidopsis thaliana : novel insights into atrazine toxicity and sucrose-induced tolerance

BackgroundSoluble sugars, which play a central role in plant structure and metabolism, are also involved in the responses to a number of stresses, and act as metabolite signalling molecules that activate specific or hormone-crosstalk transduction pathways. The different roles of exogenous sucrose in the tolerance of Arabidopsis thaliana plantlets to the herbicide atrazine and oxidative stress were studied by a transcriptomic approach using CATMA arrays.ResultsParallel situations of xenobiotic stress and sucrose-induced tolerance in the presence of atrazine, of sucrose, and of sucrose plus atrazine were compared. These approaches revealed that atrazine affected gene expression and therefore seedling physiology at a much larger scale than previously described, with potential impairment of protein translation and of reactive-oxygen-species (ROS) defence mechanisms. Correlatively, sucrose-induced protection against atrazine injury was associated with important modifications of gene expression related to ROS defence mechanisms and repair mechanisms. These protection-related changes of gene expression did not result only from the effects of sucrose itself, but from combined effects of sucrose and atrazine, thus strongly suggesting important interactions of sucrose and xenobiotic signalling or of sucrose and ROS signalling.ConclusionThese interactions resulted in characteristic differential expression of gene families such as ascorbate peroxidases, glutathione-S-transferases and cytochrome P450s, and in the early induction of an original set of transcription factors. These genes used as molecular markers will eventually be of great importance in the context of xenobiotic tolerance and phytoremediation.

γ‐Aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell wall defects during salt stress in Arabidopsis roots

Environmental constraints challenge cell homeostasis and thus require a tight regulation of metabolic activity. We have previously reported that the γ-aminobutyric acid (GABA) metabolism is crucial for Arabidopsis salt tolerance as revealed by the NaCl hypersensitivity of the GABA transaminase (GABA-T, At3g22200) gaba-t/pop2-1 mutant. In this study, we demonstrate that GABA-T deficiency during salt stress causes root and hypocotyl developmental defects and alterations of cell wall composition. A comparative genome-wide transcriptional analysis revealed that expression levels of genes involved in carbon metabolism, particularly sucrose and starch catabolism, were found to increase upon the loss of GABA-T function under salt stress conditions. Consistent with the altered mutant cell wall composition, a number of cell wall-related genes were also found differentially expressed. A targeted quantitative analysis of primary metabolites revealed that glutamate (GABA precursor) accumulated while succinate (the final product of GABA metabolism) significantly decreased in mutant roots after 1 d of NaCl treatment. Furthermore, sugar concentration was twofold reduced in gaba-t/pop2-1 mutant roots compared with wild type. Together, our results provide strong evidence that GABA metabolism is a major route for succinate production in roots and identify GABA as a major player of central carbon adjustment during salt stress.

Sugar-induced tolerance to the herbicide atrazine in Arabidopsis seedlings involves activation of oxidative and xenobiotic stress responses

Exogenous sucrose confers to Arabidopsis seedlings a very high level of tolerance to the herbicide atrazine that cannot be ascribed to photoheterotrophic growth. Important differences of atrazine tolerance between sucrose and glucose treatments showed that activation of chloroplast biogenesis per se could not account for induced tolerance. Sucrose-induced acquisition of defence mechanisms was shown by the gene expression pattern of a chloroplastic iron superoxide dismutase and by enhancement of whole-cell glucose-6-phosphate dehydrogenase activity. Activation of these defence mechanisms depended on both soluble sugar and atrazine. Moreover, acquisition of sucrose protection was shown to unmask atrazine-induced gene expression, such as that of a cytosolic glutathione-S-transferase, which remained otherwise cryptic because of the lethal effects of atrazine in the absence of soluble sugars.

Abiotic stressors and stress responses: What commonalities appear between species across biological organization levels?

Organisms are regularly subjected to abiotic stressors related to increasing anthropogenic activities, including chemicals and climatic changes that induce major stresses. Based on various key taxa involved in ecosystem functioning (photosynthetic microorganisms, plants, invertebrates), we review how organisms respond and adapt to chemical- and temperature-induced stresses from molecular to population level. Using field-realistic studies, our integrative analysis aims to compare i) how molecular and physiological mechanisms related to protection, repair and energy allocation can impact life history traits of stressed organisms, and ii) to what extent trait responses influence individual and population responses. Common response mechanisms are evident at molecular and cellular scales but become rather difficult to define at higher levels due to evolutionary distance and environmental complexity. We provide new insights into the understanding of the impact of molecular and cellular responses on individual and population dynamics and assess the potential related effects on communities and ecosystem functioning.

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings' Identification of Etiolation-Related Plastidial Aminopeptidase Activities

We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris L.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greening capacity. However, this situation of efficient greening capacity was short-lived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation.

Domain organization within repeated DNA sequences: application to the study of a family of transposable elements

MOTIVATION The analysis of repeated elements in genomes is a fascinating domain of research that is lacking relevant tools for transposable elements (TEs), the most complex ones. The dynamics of TEs, which provides the main mechanism of mutation in some genomes, is an essential component of genome evolution. In this study we introduce a new concept of domain, a segmentation unit useful for describing the architecture of different copies of TEs. Our method extracts occurrences of a terminus-defined family of TEs, aligns the sequences, finds the domains in the alignment and searches the distribution of each domain in sequences. After a classification step relative to the presence or the absence of domains, the method results in a graphical view of sequences segmented into domains. RESULTS Analysis of the new non-autonomous TE AtREP21 in the model plant Arabidopsis thaliana reveals copies of very different sizes and various combinations of domains which show the potential of our method. AVAILABILITY DomainOrganizer web page is available at www.irisa.fr/symbiose/DomainOrganizer/.

Purification and Characterization of a Novel Aminopeptidase, Plastidial Alanine-Aminopeptidase, from the Cotyledons of Etiolated Sugar Beet Seedlings

During prolonged dark growth of sugar beet (Beta vulgaris L.) seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications, protein loss, and the decrease of carotenoid and chlorophyll accumulation upon illumination. Two plastidial aminopeptidase activities were identified as early markers of this degenerative process (A. El Amrani, I. Couee, J.-P. Carde, J.-P. Gaudillere, P. Raymond [1994] Plant Physiology 106: 1555–1565). The present study focuses on one of these markers and describes the purification to homogeneity and characterization of plastidial alanine-aminopeptidase. This novel aminopeptidase was found to be a metallo-type naphthylamidase particularly active with alanyl, arginyl, and leucyl substrates. Its plastidial location was confirmed by immunofluorescence with polyclonal antibodies against the purified enzyme. Its physico-chemical and enzymic properties are discussed with respect to other higher plant aminopeptidases and to its potential functions during prolonged dark growth.