Artur Conde

Membrane Transport, Sensing and Signaling in Plant Adaptation to Environmental Stress

Plants are generally well adapted to a wide range of environmental conditions. Even though they have notably prospered in our planet, stressful conditions such as salinity, drought and cold or heat, which are increasingly being observed worldwide in the context of the ongoing climate changes, limit their growth and productivity. Behind the remarkable ability of plants to cope with these stresses and still thrive, sophisticated and efficient mechanisms to re-establish and maintain ion and cellular homeostasis are involved. Among the plant arsenal to maintain homeostasis are efficient stress sensing and signaling mechanisms, plant cell detoxification systems, compatible solute and osmoprotectant accumulation and a vital rearrangement of solute transport and compartmentation. The key role of solute transport systems and signaling proteins in cellular homeostasis is addressed in the present work. The full understanding of the plant cell complex defense mechanisms under stress may allow for the engineering of more tolerant plants or the optimization of cultivation practices to improve yield and productivity, which is crucial at the present time as food resources are progressively scarce.

Mannitol transport and mannitol dehydrogenase activities are coordinated in olea europaea under salt and osmotic stresses

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 μmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.

Transporters, channels, or simple diffusion? Dogmas, atypical roles and complexity in transport systems.

The recent breakthrough discoveries of transport systems assigned with atypical functions provide evidence for complexity in membrane transport biochemistry. Some channels are far from being simple pores creating hydrophilic passages for solutes and can, unexpectedly, act as enzymes, or mediate high-affinity uptake, and some transporters are surprisingly able to function as sensors, channels or even enzymes. Furthermore, numerous transport studies have demonstrated complex multiphasic uptake kinetics for organic and mineral nutrients. The biphasic kinetics of glucose uptake in Saccharomyces cerevisiae, a result of several genetically distinct uptake systems operating simultaneously, is a classical example that is a subject of continuous debate. In contrast, some transporters display biphasic kinetics, being bona fidae dual-affinity transporters, their kinetic properties often modulated by post-translational regulation. Also, aquaporins have recently been reported to exhibit diverse transport properties and can behave as highly adapted, multifunctional channels, transporting solutes such as CO(2), hydrogen peroxide, urea, ammonia, glycerol, polyols, carbamides, purines and pyrimidines, metalloids, glycine, and lactic acid, rather than being simple water pores. The present review provides an overview on some atypical functions displayed by transporter proteins and discusses how this novel knowledge on cellular uptake systems may be related to complex multiphasic uptake kinetics often seen in a wide variety of living organisms and the intriguing diffusive uptake of sugars and other solutes.

Polyols in grape berry: transport and metabolic adjustments as a physiological strategy for water-deficit stress tolerance in grapevine

Polyols are important metabolites that often function as carbon and energy sources and/or osmoprotective solutes in some plants. In grapevine, and in the grape berry in particular, the molecular aspects of polyol transport and metabolism and their physiological relevance are virtually unknown to date. Here, the biochemical function of a grapevine fruit mesocarp polyol transporter (VvPLT1) was characterized after its heterologous expression in yeast. This H(+)-dependent plasma membrane carrier transports mannitol (K m=5.4mM) and sorbitol (K m=9.5mM) over a broad range of polyols and monosaccharides. Water-deficit stress triggered an increase in the expression of VvPLT1 at the fully mature stage, allowing increased polyol uptake into pulp cells. Plant polyol dehydrogenases are oxireductases that reversibly oxidize polyols into monosaccharides. Mannitol catabolism in grape cells (K m=30.1mM mannitol) and mature berry mesocarps (K m=79mM) was, like sorbitol dehydrogenase activity, strongly inhibited (50-75%) by water-deficit stress. Simultaneously, fructose reduction into polyols via mannitol and sorbitol dehydrogenases was stimulated, contributing to their higher intracellular concentrations in water-deficit stress. Accordingly, the concentrations of mannitol, sorbitol, galactinol, myo-inositol, and dulcitol were significantly higher in berry mesocarps from water-deficit-stressed Tempranillo grapevines. Metabolomic profiling of the berry pulp by GC-TOF-MS also revealed many other changes in its composition induced by water deficit. The impact of polyols on grape berry composition and plant response to water deficit stress, via modifications in polyol transport and metabolism, was analysed by integrating metabolomics with transcriptional analysis and biochemical approaches.

Kaolin exogenous application boosts antioxidant capacity and phenolic content in berries and leaves of grapevine under summer stress

Heat waves, high light intensities and water deficit are becoming important threats in many important viticultural areas worldwide, so the implementation of efficient and cost-effective mitigation strategies is crucial for the production of premium wines while maintaining productivity. In this context, the foliar application of kaolin, a chemically inert mineral with excellent reflective properties, is being developed and experimented as a strategy to reduce the impact of heat and drought in Douro vineyards (Northern Portugal), already revealing promising results. In the present study we investigated if an improved antioxidant capacity is part of the beneficial effects of kaolin, by studying changes in the enzymatic and nonenzymatic antioxidant system in leaves and berries (cv Touriga Nacional). Results showed that mature grape berries contained higher amounts of total phenols (40%), flavonoids (24%), anthocyanins (32%) and vitamin C (12%) than fruits from control vines, and important changes were also measured in leaves. In parallel, kaolin application improved the antioxidant capacity in berries, which was correlated with the observed increased content in secondary metabolites. Kaolin application also regulated secondary metabolism at the transcriptional level through the increase in the transcript abundance of genes encoding phenylalanine ammonia lyase and chalcone synthase.

Kaolin foliar application has a stimulatory effect on phenylpropanoid and flavonoid pathways in grape berries

Drought, elevated air temperature, and high evaporative demand are increasingly frequent during summer in grape growing areas like the Mediterranean basin, limiting grapevine productivity and berry quality. The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven effective in mitigating the negative impacts of these abiotic stresses in grapevine and other fruit crops, however, little is known about its influence on the composition of the grape berry and on key molecular mechanisms and metabolic pathways notably important for grape berry quality parameters. Here, we performed a thorough molecular and biochemical analysis to assess how foliar application of kaolin influences major secondary metabolism pathways associated with berry quality-traits, leading to biosynthesis of phenolics and anthocyanins, with a focus on the phenylpropanoid, flavonoid (both flavonol- and anthocyanin-biosynthetic) and stilbenoid pathways. In grape berries from different ripening stages, targeted transcriptional analysis by qPCR revealed that several genes involved in these pathways—VvPAL1, VvC4H1, VvSTSs, VvCHS1, VvFLS1, VvDFR, and VvUFGT—were more expressed in response to the foliar kaolin treatment, particularly in the latter maturation phases. In agreement, enzymatic activities of phenylalanine ammonia lyase (PAL), flavonol synthase (FLS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were about two-fold higher in mature or fully mature berries from kaolin-treated plants, suggesting regulation also at a transcriptional level. The expression of the glutathione S-transferase VvGST4, and of the tonoplast anthocyanin transporters VvMATE1 and VvABCC1 were also all significantly increased at véraison and in mature berries, thus, when anthocyanins start to accumulate in the vacuole, in agreement with previously observed higher total concentrations of phenolics and anthocyanins in berries from kaolin-treated plants, especially at full maturity stage. Metabolomic analysis by reverse phase LC-QTOF-MS confirmed several kaolin-induced modifications including a significant increase in the quantities of several secondary metabolites including flavonoids and anthocyanins in the latter ripening stages, probably resulting from the general stimulation of the phenylpropanoid and flavonoid pathways.

Kaolin particle film application stimulates photoassimilate synthesis and modifies the primary metabolome of grape leaves

Water scarcity is associated with extreme temperatures and high irradiance, and significantly and increasingly affects grapevine yield and quality. In this context, the foliar application of kaolin, a chemically inert mineral that greatly reflects ultraviolet and infrared radiations, as well as, in part, photosynthetically active radiation, has recently been shown to decrease photoinhibition in mature leaves. Here, the influence of this particle film on grapevine leaf metabolome and carbohydrate metabolism was evaluated. Molecular mechanisms underlying photoassimilate synthesis, metabolism and transport capacity were assessed by targeted transcriptional analyses and enzymatic activity assays. Kaolin application increased sucrose concentration in leaves and sucrose transport/phloem loading capacity, as suggested by the stimulation of the transcription of sucrose transporters VvSUC12 and VvSUC27 in these source organs. While the biosynthesis of sucrose increased, as evidenced by higher sucrose content and sucrose phosphate synthase (SPS) activity in leaves, the concentration of transitory starch before the dark period remained unaltered, despite a higher total amylolytic activity in the leaves of kaolin-treated plants. Metabolomic analysis by GC-TOF-MS showed that the application of kaolin enhanced the amounts of simple sugars, including fructose, maltose, xylulose, xylose, sophorose, ribose and erythrose; sugars-phosphate, like mannose-6-Pi, hexose-6-Pi, glucose-6-Pi, glucose-1-Pi, glycerol-α-Pi and fructose-6-Pi; polyols, like xylitol, maltitol, lactitol, glycerol, galactinol and erythritol; organic acids and amino acids.

Postharvest dehydration induces variable changes in the primary metabolism of grape berries

Postharvest dehydration causes changes in texture, color, taste and nutritional value of food due to the high temperatures and long drying times required. In grape berries, a gradual dehydration process is normally utilized for raisin production and for making special wines. Here we applied a raisin industry-mimicking dehydration process for eleven days at 50°C to intact berry clusters from cv. Sémillon plants, and a set of molecular, cellular and biochemical analyses were performed to study the impact of postharvest dehydration in the primary metabolism. Transcriptional analyses by real time qPCR showed that several aquaporins (VvTIP1;2 and VvSIP1) and sugar transporters (VvHT1, VvSWEET11, VvSWEET15, VvTMT1, VvSUC12) genes were strongly upregulated. Moreover, the study of key enzymes of osmolytes metabolism, including mannitol dehydrogenase (VvMTD) and sorbitol dehydrogenase (VvSDH), at gene expression and protein activity level, together with the transcriptional analysis of the polyol transporter gene VvPLT1, showed an enhanced polyol biosynthesis capacity, which was supported by the detection of sorbitol in dehydrated grapes only. The metabolism of organic acids was also modulated, by the induction of transcriptional and biochemical activity modifications in malate dehydrogenases and malic enzymes that led to organic acid degradation, as demonstrated by HPLC analysis. Taken together, this study showed that primary metabolism of harvested berries was severely influenced in response to dehydration treatments towards lower organic acid and higher sorbitol concentrations, while sugar transporter and aquaporin genes were significantly upregulated.