R. M. Tavares

Pathways of Glucose Regulation of Monosaccharide Transport in Grape Cells

Grape (Vitis vinifera) heterotrophic suspension-cultured cells were used as a model system to study glucose (Glc) transport and its regulation. Cells transported d-[14C]Glc according to simple Michaelis-Menten kinetics superimposed on first-order kinetics. The saturating component is a high-affinity, broad-specificity H+-dependent transport system (Km = 0.05 mm). Glc concentration in the medium tightly regulated the transcription of VvHT1 (Vitis vinifera hexose transporter 1), a monosaccharide transporter previously characterized in grape berry, as well as VvHT1 protein amount and monosaccharide transport activity. All the remaining putative monosaccharide transporters identified so far in grape were poorly expressed and responded weakly to Glc. VvHT1 transcription was strongly repressed by Glc and 2-deoxy-d-Glc, but not by 3-O-methyl-d-Glc or Glc plus mannoheptulose, indicating the involvement of a hexokinase-dependent repression. 3-O-Methyl-d-Glc, which cannot be phosphorylated, and Glc plus mannoheptulose induced a decrease of transport activity caused by the reduction of VvHT1 protein in the plasma membrane without affecting VvHT1 transcript levels. This demonstrates hexokinase-independent posttranscriptional regulation. High Glc down-regulated VvHT1 transcription and Glc uptake, whereas low Glc increased those parameters. Present data provide an example showing control of plant sugar transporters by their own substrate both at transcriptional and posttranscriptional levels. VvHT1 protein has an important role in the massive import of monosaccharides into mesocarp cells of young grape berries because it was localized in plasma membranes of the early developing fruit. Protein amount decreased abruptly throughout fruit development as sugar content increases, consistent with the regulating role of Glc on VvHT1 expression found in suspension-cultured cells.

An improved method for high-quality RNA isolation from needles of adult maritime pine trees

When conventional RNA isolation methods optimized for pine seedlings are applied to needles of adult pine trees, poor-quality RNA results. Here we describe a modified procedure to isolate high-quality RNA from needles of 30-year-old maritime pines, exhibiting high levels of phenolics, polysaccharides, and RNases. Major changes are the inclusion of proteinase K in the extraction medium followed by incubation at 42°C. Integrity and purity were evaluated by using denaturing gel electrophoresis and spectrophotometry (A260/A230 and A260/A280). The total RNA could be successfully used for poly(A)+-RNA isolation and cDNA library construction.

SUMO, a heavyweight player in plant abiotic stress responses

Protein post-translational modifications diversify the proteome and install new regulatory levels that are crucial for the maintenance of cellular homeostasis. Over the last decade, the ubiquitin-like modifying peptide small ubiquitin-like modifier (SUMO) has been shown to regulate various nuclear processes, including transcriptional control. In plants, the sumoylation pathway has been significantly implicated in the response to environmental stimuli, including heat, cold, drought, and salt stresses, modulation of abscisic acid and other hormones, and nutrient homeostasis. This review focuses on the emerging importance of SUMO in the abiotic stress response, summarizing the molecular implications of sumoylation and emphasizing how high-throughput approaches aimed at identifying the full set of SUMO targets will greatly enhance our understanding of the SUMO–abiotic stress association.

Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between Castanea sativa and Pisolithus tinctorius

Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal (ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H2O2 and O2·− during the early stages of fungal contact. Three peaks of H2O2 production were detected, the first two coinciding with O2·− bursts. The first H2O2 production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H2O2 scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H2O2 production profile with a delay was detected. The result suggests that during the early stages of ECM establishment, H2O2 results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment.

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

In contrast with animals, little is known about the regulation of HMGR, the rate-limiting enzyme of isoprenoid biosynthesis, in plants. Through the identification of second-site suppressors of the Arabidopsis dry2/sqe1-5 mutant, we found that the putative E3 ubiquitin ligase SUD1, likely involved in endoplasmic reticulum–associated degradation, is a regulator of HMGR activity. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

Oak root response to ectomycorrhizal symbiosis establishment: RNA-Seq derived transcript identification and expression profiling.

Ectomycorrhizal symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with ectomycorrhizal root tips are more tolerant to environmental stresses, such as drought, and biotic stresses such as root pathogens. Detailed information on these molecular processes is essential for the understanding of symbiotic tissue development in order to optimize the benefits of this natural phenomenon. Next generation sequencing tools allow the analysis of non model ectomycorrhizal plant-fungal interactions that can contribute to find the “symbiosis toolkits” and better define the role of each partner in the mutualistic interaction. By using 454 pyrosequencing we compared ectomycorrhizal cork oak roots with non-symbiotic roots. From the two cDNA libraries sequenced, over 2 million reads were obtained that generated 19552 cork oak root unique transcripts. A total of 2238 transcripts were found to be differentially expressed when ECM roots were compared with non-symbiotic roots. Identification of up- and down-regulated gens in ectomycorrhizal roots lead to a number of insights into the molecular mechanisms governing this important symbiosis. In cork oak roots, ectomycorrhizal colonization resulted in extensive cell wall remodelling, activation of the secretory pathway, alterations in flavonoid biosynthesis, and expression of genes involved in the recognition of fungal effectors. In addition, we identified genes with putative roles in symbiotic processes such as nutrient exchange with the fungal partner, lateral root formation or root hair decay. These findings provide a global overview of the transcriptome of an ectomycorrhizal host root, and constitute a foundation for future studies on the molecular events controlling this important symbiosis.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

BackgroundCork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management.ResultsWe generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org.ConclusionsThis genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

SIZ1-Dependent Post-Translational Modification by SUMO Modulates Sugar Signaling and Metabolism in Arabidopsis thaliana.

Post-translational modification mechanisms function as switches that mediate the balance between optimum growth and the response to environmental stimuli, by regulating the activity of key proteins. SUMO (small ubiquitin-like modifier) attachment, or sumoylation, is a post-translational modification that is essential for the plant stress response, also modulating hormonal circuits to co-ordinate developmental processes. The Arabidopsis SUMO E3 ligase SAP and Miz 1 (SIZ1) is the major SUMO conjugation enhancer in response to stress, and is implicated in several aspects of plant development. Here we report that known SUMO targets are over-represented in multiple carbohydrate-related proteins, suggesting a functional link between sumoylation and sugar metabolism and signaling in plants. We subsequently observed that SUMO-conjugated proteins accumulate in response to high doses of sugar in a SIZ1-dependent manner, and that the null siz1 mutant displays increased expression of sucrose and starch catabolic genes and shows reduced starch levels. We demonstrated that SIZ1 controls germination time and post-germination growth via osmotic and sugar-dependent signaling, respectively. Glucose was specifically linked to SUMO-sugar interplay, with high levels inducing root growth inhibition and aberrant root hair morphology in siz1. The use of sugar analogs and sugar marker gene expression analysis allowed us to implicate SIZ1 in a signaling pathway dependent on glucose metabolism, probably involving modulation of SNF1-related kinase 1 (SnRK1) activity.

Phenotypic analysis of the Arabidopsis heat stress response during germination and early seedling development.

BackgroundPhenotypic characterization of Arabidopsis thaliana gain- and loss-of-function mutants is a delicate and meticulous task that often involves the analysis of multiple parameters. Arabidopsis heat tolerance has been evaluated based on direct assessments that include seed germination, seedling survival, hypocotyl and root elongation, or indirect measurements such as chlorophyll content or ion leakage.ResultsIn an attempt to simplify the detection of heat stress-associated phenotypes, a collection of protocols for analysis of seed germination and seedling survival to heat treatment is proposed. Temperatures and lengths of heat treatments were combined into several heat tolerance assays, to be used as a primary approach for the search and characterization of basal and acquired heat tolerance-associated phenotypes at early developmental stages. The usefulness of this methodology was illustrated through the characterization of heat-related phenotypes in different Arabidopsis ecotypes as well as in gain- and loss-of-function mutants.ConclusionsThe use of standardized experimental protocols designed to detect temperature-related phenotypes is proposed. The suggested plate-based assays provide an appropriate framework of experimental conditions for detection of variability amongst natural accessions or mutants lines. Functional studies could be facilitated by using this inexpensive and undemanding approach.

Thylakoid membrane reorganization during Zantedeschia aethiopica spathe regreening: Consequence of the absence of Δ3-trans-hexadecenoic acid in photochemical activity

Abstract Soon after its formation the fruiting spathe of Zantedeschia aethiopica undergoes senescence which is characterized by the loss of its photosynthetic activity. During fruiting, spathe senescence is inhibited and regreening takes place. The amyloplasts present in the white spathe turn to functional chloroplasts and the spathe acquires photosynthetic capacity. In this work, thylakoid membranes were isolated from three distinct stages of spathe development (flora bud, white and regreened spathes) and used for the study of pigment-protein complexes, as well as for the determination of PSI and PSII activities and photophosphorylation rates. Our results indicate that, besides their ultrastructural similarity to leaf chloroplasts, regreened spathe chloroplasts showed values of photochemical and photophosphorylation rates lower than those found in leaf chloroplasts. These results are discussed in relation to the absence of the fatty acid Δ 3 - trans -hexadecenoic acid from regreened spathe thylakoid membranes, and we suggest that this fatty acid could be an important factor for optimal photochemical and photophosforylation activities.