Functional analysis of polyphenol oxidases by antisense/sense technology

Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting that PPO may have a role in the development of plant water stress and potential for photoinhibition and photooxidative damage that may be unrelated to any effects on the Mehler reaction. These results substantiate the defensive role of PPO and suggest that manipulation of PPO activity in specific tissues has the potential to provide broad-spectrum resistance simultaneously to both disease and insect pests, however, effects of PPO on postharvest quality as well as water stress physiology should also be considered. In addition to the functional analysis of tomato PPO, the application of antisense/sense technology to decipher the functions of PPO in other plant species as well as for commercial uses are discussed.     

Sterol Glycosyltransferases—The Enzymes That Modify Sterols

Sterols are important components of cell membranes, hormones, signalling molecules and defense-related biotic and abiotic chemicals. Sterol glycosyltransferases (SGTs) are enzymes involved in sterol modifications and play an important role in metabolic plasticity during adaptive responses. The enzymes are classified as a subset of family 1 glycosyltransferases due to the presence of a signature motif in their primary sequence. These enzymes follow a compulsory order sequential mechanism forming a ternary complex. The diverse applications of sterol glycosides, like cytotoxic and apoptotic activity, anticancer activity, medicinal values, anti-stress roles and anti-insect and antibacterial properties, draws attention towards their synthesis mechanisms. Many secondary metabolites are derived from sterol pathways, which are important in defense mechanisms against pathogens. SGTs in plants are involved in changed sensitivity to stress hormones and their agrochemical analogs and changed tolerance to biotic and abiotic stresses. SGTs that glycosylate steroidal hormones, such as brassinosteroids, function as growth and development regulators in plants. In terms of metabolic roles, it can be said that SGTs occupy important position in plant metabolism and may offer future tools for crop improvement.     

Peroxidative reactions attenuate oxygen effect on spectroscopic properties of isolated chloroplasts.

Steady-state absorption and fluorescence excitation spectra have been measured at 25 degrees C in order to elucidate the differences between isolated chloroplasts from pea (chilling-sensitive plant) and bean (chilling-tolerant plant) and their response to oxygen treatment. A weaker light harvesting in bean in comparison with pea chloroplasts is related to higher free fatty acids level and extended peroxidation activities of bean chloroplasts. Peroxidation of free fatty acids in bean chloroplasts results in an accumulation of oxygenated forms of fatty acids demonstrated by a large negative band around 400 nm in absorption difference spectra, while the excitation spectra are not significantly altered. Similar changes have been observed in the lipase-treated pea chloroplasts. In contrast, in both pea and bean chloroplasts exhibiting no peroxidation due to antimycin A treatment, oxygen induces a pronounced absorbance increase in the regions around 435, 470 and 674 nm indicating the chloroplast swelling. A decline of chlorophyll fluorescence excitation caused by oxygen, may result from a decrease in energy transfer from antennae complexes to chlorophyll species emitting at both 680 and 740 nm. The oxygen-induced changes are partially reversed upon restoration of anaerobic conditions. The presented data show for the first time, that in contrast to pea chloroplasts the peroxidation abolishes an oxygen-induced decrease in light harvesting in bean chloroplasts, i.e., a chilling-sensitive plant.     

An Overview of the Genetics of Plant Response to Salt Stress: Present Status and the Way Forward

Salinity is one of the major threats faced by the modern agriculture today. It causes multidimensional effects on plants. These effects depend upon the plant growth stage, intensity, and duration of the stress. All these lead to stunted growth and reduced yield, ultimately inducing economic loss to the farming community in particular and to the country in general. The soil conditions of agricultural land are deteriorating at an alarming rate. Plants assess the stress conditions, transmit the specific stress signals, and then initiate the response against that stress. A more complete understanding of plant response mechanisms and their practical incorporation in crop improvement is an essential step towards achieving the goal of sustainable agricultural development. Literature survey shows that investigations of plant stresses response mechanism are the focus area of research for plant scientists. Although these efforts lead to reveal different plant response mechanisms against salt stress, yet many questions still need to be answered to get a clear picture of plant strategy to cope with salt stress. Moreover, these studies have indicated the presence of a complicated network of different integrated pathways. In order to work in a progressive way, a review of current knowledge is critical. Therefore, this review aims to provide an overview of our understanding of plant response to salt stress and to indicate some important yet unexplored dynamics to improve our knowledge that could ultimately lead towards crop improvement.     

Effects of water deficit on photosystem II photochemistry and photoprotection during acclimation of lyreleaf sage (Salvia lyrata L.) plants to high light

Acclimation of photosynthetic light reactions to high light requires adjustments in photosystem II (PSII) photochemistry and may be affected by environmental stresses, such as water deficit. In this study, we examined the effects of this stress on PSII photochemistry and photoprotection, with an emphasis on the role of carotenoids and tocopherols, during acclimation of lyreleaf sage (Salvia lyrata L.) plants to high light. Violaxanthin was rapidly converted to zeaxanthin under high light, the de-epoxidation state of the xanthophyll cycle reaching maximum levels of 0.97 after 10 days of high light exposure. Under a higher photoprotective demand caused by water deficit, plants showed significant decreases in beta-carotene and enhanced oxidation of alpha-tocopherol to alpha-tocopherol quinone, which was followed by decreases in the F(v)/F(m) ratio. The levels of beta-carotene decreased more in water-stressed than irrigated plants during acclimation to high light, being particularly degraded (up to 73%) after 14 days of water deficit. Tocopherol levels increased significantly during acclimation to high light, particularly under water deficit, which caused 6.6- and 10-fold increases in alpha-tocopherol and alpha-tocopherol quinone, respectively. We conclude that when xanthophyll cycle-dependent excess energy dissipation could not afford further protection during high light acclimation and the photoprotective demand increased in lyreleaf sage plants by water deficit, enhanced oxidation of alpha-tocopherol and beta-carotene occurred. As stress persisted, enhanced formation of reactive oxygen species might ultimately damage the PSII, as indicated by the reductions in the F(v)/F(m) ratio.     

Strigolactones,from Plants to Human Health: Achievements and Challenges

Strigolactones (SLs) are a class of sesquiterpenoid plant hormones that play a role in the response of plants to various biotic and abiotic stresses. When released into the rhizosphere, they are perceived by both beneficial symbiotic mycorrhizal fungi and parasitic plants. Due to their multiple roles, SLs are potentially interesting agricultural targets. Indeed, the use of SLs as agrochemicals can favor sustainable agriculture via multiple mechanisms, including shaping root architecture, promoting ideal branching, stimulating nutrient assimilation, controlling parasitic weeds, mitigating drought and enhancing mycorrhization. Moreover, over the last few years, a number of studies have shed light onto the effects exerted by SLs on human cells and on their possible applications in medicine. For example, SLs have been demonstrated to play a key role in the control of pathways related to apoptosis and inflammation. The elucidation of the molecular mechanisms behind their action has inspired further investigations into their effects on human cells and their possible uses as anti-cancer and antimicrobial agents.     

Plant volatiles: production, function and pharmacology

Plant volatiles typically occur as a complex mixture of low-molecular weight lipophilic compounds derived from different biosynthetic pathways, and are seemingly produced as part of a defense strategy against biotic and abiotic stress, as well as contributing to various physiological functions of the producer organism. The biochemistry and molecular biology of plant volatiles is complex, and involves the interplay of several biochemical pathways and hundreds of genes. All plants are able to store and emit volatile organic compounds (VOCs), but the process shows remarkable genotypic variation and phenotypic plasticity. From a physiological standpoint, plant volatiles are involved in three critical processes, namely plant–plant interaction, the signaling between symbiotic organisms, and the attraction of pollinating insects. Their role in these ‘‘housekeeping’’ activities underlies agricultural applications that range from the search for sustainable methods for pest control to the production of flavors and fragrances. On the other hand, there is also growing evidence that VOCs are endowed with a range of biological activities in mammals, and that they represent a substantially under-exploited and still largely untapped source of novel drugs and drug leads. This review summarizes recent major developments in the study of biosynthesis, ecological functions and medicinal applications of plant VOCs.     

Expression Profiling of Flavonoid Biosynthesis Genes and Secondary Metabolites Accumulation in Populus under Drought Stress

Flavonoids are key secondary metabolites that are biologically active and perform diverse functions in plants such as stress defense against abiotic and biotic stress. In addition to its importance, no comprehensive information has been available about the secondary metabolic response of Populus tree, especially the genes that encode key enzymes involved in flavonoid biosynthesis under drought stress. In this study, the quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the expression of flavonoid biosynthesis genes (PtPAL, Pt4-CL, PtCHS, PtFLS-1, PtF3H, PtDFR, and PtANS) gradually increased in the leaves of hybrid poplar (P. tremula × P. alba), corresponding to the drought stress duration. In addition, the activity and capacity of antioxidants have also increased, which is positively correlated with the increment of phenolic, flavonoid, anthocyanin, and carotenoid compounds under drought stress. As the drought stress prolonged, the level of reactive oxygen species such as hydrogen peroxide (H₂O₂) and singlet oxygen (O₂⁻) too increased. The concentration of phytohormone salicylic acid (SA) also increased significantly in the stressed poplar leaves. Our research concluded that drought stress significantly induced the expression of flavonoid biosynthesis genes in hybrid poplar plants and enhanced the accumulation of phenolic and flavonoid compounds with resilient antioxidant activity.     

Multicolor fluorescence imaging of leaves--a useful tool for visualizing systemic viral infections in plants

Abstract Multicolor fluorescence induced by UV light is a sensitive and specific tool that may be used to provide information about the primary and secondary metabolism of plants by monitoring signals of the chlorophyll fluorescence (Chl-F) and blue-green fluorescence (BGF), respectively. We have followed the systemic infection of Nicotiana benthamiana plants with the Pepper mild mottle virus (PMMoV) by means of a multicolor fluorescence-imaging system, to detect differences between two strains of PMMoV during the infection process and to establish a correlation between the virulence and changes induced in the host plant. Changes in both BGF and Chl-F were monitored. BGF increased mainly in the abaxial side of the leaf during pathogenesis and the corresponding images showed a clear vein-associated pattern in leaves of infected plants. HPLC analysis of leaf extracts was carried out to identify compounds emitting BGF, and determined that chlorogenic acid was one of the main contributors. BGF imaging was able to detect viral-induced changes in asymptomatic (AS) leaves before detection of the virus itself. Chl-F images confirmed our previous results of alterations in the photosynthetic apparatus of AS leaves from infected plants that were detected with other imaging techniques. Fluorescence ratios F440/F690 and F440/F740, which increase during pathogenesis, were excellent indicators of biotic stress.     

Structural biology of plant sulfur metabolism: From assimilation to biosynthesis

Covering: 1966 to 2012Sulfur is an essential element that must be assimilated by all organisms; however, the metabolic pathways for this task vary significantly, even among individual genera of bacteria, and especially so among eukaryotes. While all organisms require sulfurous amino acids, plants require specialized sulfur-containing metabolites, such as glucosinolates and allylsulfur compounds, for protection from herbivory and microbial infection; and the synthesis of specialized peptides (i.e., glutathione and phytochelatins) for protection against reactive oxygen species and exposure to transition metals, such as cadmium. In order to provide the complex array of sulfur-containing metabolites essential to plant viability, flux through the sulfur assimilatory pathway must be tightly regulated by controlling enzymatic activity. The X-ray crystal structures of several primary sulfur assimilatory enzymes, complemented by kinetics, have revealed mechanisms of enzymatic regulation (i.e., via redox state and protein-protein interaction) in these biosynthetic pathways, in addition to the chemical mechanisms of catalysis. This review summarizes the state of our structural knowledge of primary and secondary sulfur assimilatory enzymes from plants.     

Blue light-induced chloroplast reorientations in Lemna trisulca L. (duckweed) are controlled by two separable cellular mechanisms as suggested by different sensitivity to wortmannin

Chloroplast reorientations within mesophyll cells are among the most rapid physiological responses of higher plants to blue light. At light intensities below the saturation point of photosynthesis, chloroplasts move to the cell walls perpendicular to the direction of light and maximize light absorption (low-fluence rate response [LFR]). At light intensities above the saturation point of photosynthesis, chloroplasts redistribute to cell walls parallel to the direction of light (high-fluence rate response [HFR]). The actin-based mechanism is responsible for the light-induced chloroplast movements. We have found that an inhibitor of phosphoinositide-3-kinases, wortmannin, potently and irreversibly inhibited LFR and HFR chloroplast responses to blue light in Lemna trisulca L. mesophyll cells. Microscopic observations and photometric measurement indicated that 100 nM wortmannin specifically inhibited LFR in Lemna, whereas HFR displayed no sensitivity to the inhibitor at this concentration. A complete inhibition of the HFR could be obtained by 1 microM wortmannin. These data indicate that LFR is more sensitive to wortmannin than HFR and suggest that these two responses may be under the control of different cellular mechanisms. Our results suggest that phosphoinositide kinases and other phosphoinositide cycle enzymes may play a role in the transduction of the light signal to the actin cytoskeleton in Lemna as factors specifying the direction of chloroplast movements. A hypothetical model assuming three signaling pathways regulating light-induced chloroplast reorientations in mesophyll cells is proposed.     

Autophosphorylation, electrophoretic mobility and immunoreaction of oat phototropin 1 under UV and blue Light

Phototropins are UV-A/blue light photoreceptors containing two flavin mononucleotide (FMN)-binding domains, light, oxygen and voltage (LOV)1 and LOV2, of which LOV2 is more sensitive toward light and more important for the physiological response compared with LOV1. Some physiological responses are plant phototropism, chloroplast migration and stomatal opening. Oat phototropin 1 together with light-dependent autophosphorylation shows a reduced electrophoretic mobility and reduced immunoreaction against a heterologous antiserum; both effects were suggested to be caused by phosphorylation at the same sites (M. Salomon, E. Knieb, T. von Zeppelin and W. Rudiger [2003] Biochemistry 42, 4217-4225). In this study, we show that both effects can be separated from each other: at low temperature, reduced immunoreaction preceded the mobility shift, and irradiation with UV-C light led to the mobility shift without the loss of immunoreactivity. We demonstrated that UV-C light at 280 nm, which does not match any absorption maximum of FMN, leads to autophosphorylation of phototropin. It is hypothesized that UV-C light causes differential activation of the LOV domains via energy transfer from aromatic amino acids.     

Phototropins and associated signaling: providing the power of movement in higher plants

Recent developments in phototropin biology have provided exciting new findings on the roles of these photoreceptor proteins in plants. Much of the recent work has focused on phototropin photochemistry and the structural alterations in both the chromophoric and peptide components of the molecule associated with light perception. In this review, specific aspects of phototropin action in higher plants will be discussed in the context of these new findings. Although, as their name suggests, phototropins play a key role in phototropic responses in plants, increasing evidence shows they have many other functions in plants. In this review, the roles of phototropins in additional plant "movement" responses will be addressed; in particular their roles in stomatal aperture control and chloroplast movements. In discussing these various movement responses special attention will be given to identified and hypothesized downstream signaling partners or events that enable the phototropins to selectively participate in any one or more of these responses in a given light condition.     

Elicitor-Induced Cellular and Molecular Events Are Responsible for Productivity Enhancement in Hairy Root Cultures: An Insight Study

A wide range of external stress stimuli triggers a plant cell to undergo a complex network of reactions that ultimately lead to the synthesis and accumulation of secondary metabolites. These secondary metabolites help the plant to survive under stress challenge. The potential of biotic and abiotic elicitors for the induction and enhancement of secondary metabolite production in various culture systems including hairy root (HR) cultures is well-known. The elicitor-induced defense responses involves signal perception of elicitor by a cell surface receptor followed by its transduction involving some major cellular and molecular events including activation of major secondary message signaling pathways. This result in induction of gene expressions escorting to the synthesis of various proteins mainly associated with plant defense responses and secondary metabolite synthesis and accumulation. The review discusses the elicitor-induced various cellular and molecular events and correlates them with enhanced secondary metabolite synthesis in HR systems. Further, this review also concludes that combining elicitation with in-silico approaches enhances the usefulness of this practice in better understanding and identifying the rate-limiting steps of biosynthetic pathways existing in HRs which in turn can contribute towards better productivity by utilizing metabolic engineering aspects.     

The role of MAPK signal transduction pathways in the response to oxidative stress in the fungal pathogen Candida albicans: implications in virulence

In recent years, Mitogen-Activated Protein Kinase (MAPK) pathways have emerged as major regulators of cellular physiology. In the fungal pathogen Candida albicans, three different MAPK pathways have been characterized in the last years. The HOG pathway is mainly a stress response pathway that is activated in response to osmotic and oxidative stress and also participates regulating other pathways. The SVG pathway (or mediated by the Cek1 MAPK) is involved in cell wall formation under vegetative and filamentous growth, while the Mkc1-mediated pathway is involved in cell wall integrity. Oxidative stress is one of the types of stress that every fungal cell has to face during colonization of the host, where the cell encounters both hypoxia niches (i.e. gut) and high concentrations of reactive oxygen species (upon challenge with immune cells). Two pathways have been shown to be activated in response to oxidative stress: the HOG pathway and the MKC1-mediated pathway while the third, the Cek1 pathway is deactivated. The timing, kinetics, stimuli and functional responses generated upon oxidative stress differ among them; however, they have essential functional consequences that severely influence pathogenesis. MAPK pathways are, therefore, valuable targets to be explored in antifungal research.     

Optimizing the energy status of skin cells during solar radiation.

Ionizing- and ultraviolet-radiation cause cell damage or death by directly altering DNA and protein structures and by production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). These processes disrupt cellular energy metabolism at multiple levels. The formation of DNA strand breaks activates signaling pathways that consume NAD, which can lead to the depletion of cellular ATP. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteasome that degrades damaged nuclear proteins including histones. Identifying approaches to optimize these responses while maintaining the energy status of cells is likely to be very important in minimizing the deleterious effects of solar radiation on skin.