A mixture of peptides and sugars derived from plant cell walls increases plant defense responses to stress and attenuates ageing-associated molecular changes in cultured skin cells.

Small peptides and aminoacid derivatives have been extensively studied for their effect of inducing plant defense responses, and thus increasing plant tolerance to a wide range of abiotic stresses. Similarly to plants, these compounds can activate different signaling pathways in mammalian skin cells as well, leading to the up-regulation of anti-aging specific genes. This suggests the existence of analogous defense response mechanisms, well conserved both in plants and animal cells. In this article, we describe the preparation of a new mixture of peptides and sugars derived from the chemical and enzymatic digestion of plant cell wall glycoproteins. We investigate the multiple roles of this product as potential "biostimulator" to protect plants from abiotic stresses, and also as potential cosmeceutical. In particular, the molecular effects of the peptide/sugar mixture of inducing plant defense responsive genes and protecting cultured skin cells from oxidative burst damages were deeply evaluated.

Drought, abscisic acid and transpiration rate effects on the regulation of PIP aquaporin gene expression and abundance in Phaseolus vulgaris plants.

BACKGROUND AND AIMS: Drought causes a decline of root hydraulic conductance, which aside from embolisms, is governed ultimately by aquaporins. Multiple factors probably regulate aquaporin expression, abundance and activity in leaf and root tissues during drought; among these are the leaf transpiration rate, leaf water status, abscisic acid (ABA) and soil water content. Here a study is made of how these factors could influence the response of aquaporin to drought. METHODS: Three plasma membrane intrinsic proteins (PIPs) or aquaporins were cloned from Phaseolus vulgaris plants and their expression was analysed after 4 d of water deprivation and also 1 d after re-watering. The effects of ABA and of methotrexate (MTX), an inhibitor of stomatal opening, on gene expression and protein abundance were also analysed. Protein abundance was examined using antibodies against PIP1 and PIP2 aquaporins. At the same time, root hydraulic conductance (L), transpiration rate, leaf water status and ABA tissue concentration were measured. KEY RESULTS: None of the treatments (drought, ABA or MTX) changed the leaf water status or tissue ABA concentration. The three treatments caused a decline in the transpiration rate and raised PVPIP2;1 gene expression and PIP1 protein abundance in the leaves. In the roots, only the drought treatment raised the expression of the three PIP genes examined, while at the same time diminishing PIP2 protein abundance and L. On the other hand, ABA raised both root PIP1 protein abundance and L. CONCLUSIONS: The rise of PvPIP2;1 gene expression and PIP1 protein abundance in the leaves of P. vulgaris plants subjected to drought was correlated with a decline in the transpiration rate. At the same time, the increase in the expression of the three PIP genes examined caused by drought and the decline of PIP2 protein abundance in the root tissues were not correlated with any of the parameters measured.

The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots.

When chilling-sensitive plants are chilled, root hydraulic conductance (L(o)) declines precipitously; L(o) also declines in chilling-tolerant plants, but it subsequently recovers, whereas in chilling-sensitive plants it does not. As a result, the chilling-sensitive plants dry out and may die. Using a chilling-sensitive and a chilling-tolerant maize genotype we investigated the effect of chilling on L(o), and its relationship to osmotic water permeability of isolated root cortex protoplasts, aquaporin gene expression, aquaporin abundance, and aquaporin phosphorylation, hydrogen peroxide (H(2)O(2)) accumulation in the roots and electrolyte leakage from the roots. Because chilling can cause H(2)O(2) accumulation we also determined the effects of a short H(2)O(2) treatment of the roots and examined the same parameters. We conclude from these studies that the recovery of L(o) during chilling in the chilling-tolerant genotype is made possible by avoiding or repairing membrane damage and by a greater abundance and/or activity of aquaporins. The same changes in aquaporins take place in the chilling-sensitive genotype, but we postulate that membrane damage prevents the L(o) recovery. It appears that the aquaporin response is necessary but not sufficient to respond to chilling injury. The plant must also be able to avoid the oxidative damage that accompanies chilling.

Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes.

We analyzed the breadth of the unfolded protein response (UPR) in Arabidopsis using gene expression analysis with Affymetrix GeneChips. With tunicamycin and DTT as endoplasmic reticulum (ER) stress-inducing agents, we identified sets of UPR genes that were induced or repressed by both stresses. The proteins encoded by most of the upregulated genes function as part of the secretory system and comprise chaperones, vesicle transport proteins, and ER-associated degradation proteins. Most of the downregulated genes encode extracellular proteins. Therefore, the UPR may constitute a triple effort by the cell: to improve protein folding and transport, to degrade unwanted proteins, and to allow fewer secretory proteins to enter the ER. No single consensus response element was found in the promoters of the 53 UPR upregulated genes, but half of the genes contained response elements also found in mammalian UPR regulated genes. These elements are enriched from 4.5- to 15-fold in this upregulated gene set.

Origin of plant glycerol transporters by horizontal gene transfer and functional recruitment.

Gene-family evolution mostly relies on gene duplication coupled with functional diversification of gene products. However, other evolutionary mechanisms may also be important in generating protein diversity. The ubiquitous membrane intrinsic protein (MIP) gene family is an excellent model system to search for such alternative evolutionary mechanisms. MIPs are proteins that transport water, glycerol, and small solutes across cell membranes in all living organisms. We reconstructed the molecular phylogeny of MIPs based on amino acid sequence data by using neighbor-joining, maximum-likelihood, and Bayesian methods of phylogenetic inference. The recovered trees show an early and distinct separation of water and glycerol transporters, i.e., aquaporins (AQPs), and aquaglyceroporins. The latter are absent from plants. As expected, gene duplication and functional diversification account for most of the diversity of animal and plant members of the family. However, in contrast to this model, we find that the sister group of plant glycerol transporters are bacterial AQPs. This relationship suggests first that plant glycerol transporters may resulted from a single event of horizontal gene transfer from bacteria, which we have estimated to have occurred approximately 1,200 million years ago, at the origin of plants, and second that bacterial AQPs were likely recruited to transport glycerol in plants because of their absence of aquaglyceroporins. This striking example of adaptive evolution at the molecular level was demonstrated further by finding convergent or parallel replacements at particular amino acid positions related to water- and glycerol-transporting specificity.

The G-protein-coupled receptor GCR1 regulates DNA synthesis through activation of phosphatidylinositol-specific phospholipase C.

Different lines of evidence suggest that specific events during the cell cycle may be mediated by a heterotrimeric G-protein activated by a cognate G-protein coupled receptor. However, coupling between the only known Galpha-subunit of the heterotrimeric G-protein (GPA1) and the only putative G-protein coupled receptor (GCR1) of plants has never been shown. Using a variety of approaches, we show here that GCR1-enhanced thymidine incorporation into DNA depends on an increase in phosphatidylinositol-specific phospholipase C activity and an elevation of inositol 1,4,5-trisphosphate levels in the cells. Tobacco (Nicotiana tabacum) cells that overexpress either Arabidopsis GCR1 or GPA1 display this phenomenon. We suggest on the basis of these results that GCR1-controlled events during the cell cycle involve phosphatidylinositol-specific phospholipase C as an effector of GCR1 and inositol 1,4,5-trisphosphate as a second messenger, and that GCR1 and GPA1 are both involved in this particular signaling pathway.

GCR1, the putative Arabidopsis G protein-coupled receptor gene is cell cycle-regulated, and its overexpression abolishes seed dormancy and shortens time to flowering.

Although signaling through heterotrimeric G proteins has been extensively studied in eukaryotes, there is little information about this important signaling pathway in plants. We observed that expression of GCR1, the gene encoding the only known (but still putative) G protein-coupled receptor of Arabidopsis thaliana, is modulated during the cell cycle and during plant development. Overexpression of GCR1 in tobacco (Nicotiana tabacum) BY-2 cells caused an increase in thymidine incorporation and in the mitotic index of aphidicolin synchronized cells. Overexpression of GCR1 in Arabidopsis caused two remarkable phenotypes: seed dormancy was abolished and time to flowering was reduced. Molecular markers of these two developmental processes (phosphatase PP2A and MYB65 in germination; LFY during flowering) were up-regulated in GCR1 overexpressors. These data are consistent with the hypothesis that GCR1 may be a regulator of the cell cycle and that this regulation underlies the developmental changes observed in the GCR1 transformants.

Plasma membrane aquaporins play a significant role during recovery from water deficit.

The role of plasma membrane aquaporins (PIPs) in water relations of Arabidopsis was studied by examining plants with reduced expression of PIP1 and PIP2 aquaporins, produced by crossing two different antisense lines. Compared with controls, the double antisense (dAS) plants had reduced amounts of PIP1 and PIP2 aquaporins, and the osmotic hydraulic conductivity of isolated root and leaf protoplasts was reduced 5- to 30-fold. The dAS plants had a 3-fold decrease in the root hydraulic conductivity expressed on a root dry mass basis, but a compensating 2.5-fold increase in the root to leaf dry mass ratio. The leaf hydraulic conductance expressed on a leaf area basis was similar for the dAS compared with the control plants. As a result, the hydraulic conductance of the whole plant was unchanged. Under sufficient and under water-deficient conditions, stomatal conductance, transpiration rate, plant hydraulic conductance, leaf water potential, osmotic pressure, and turgor pressure were similar for the dAS compared with the control plants. However, after 4 d of rewatering following 8 d of drying, the control plants recovered their hydraulic conductance and their transpiration rates faster than the dAS plants. Moreover, after rewatering, the leaf water potential was significantly higher for the control than for the dAS plants. From these results, we conclude that the PIPs play an important role in the recovery of Arabidopsis from the water-deficient condition.

Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree.

In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.

Signal transduction networks and the biology of plant cells

The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, adn researches focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are the se molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction pathway usually involves multiple organelles of cellular structures Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense response against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of ...