Micha Volokita

Response of the Cultivated Tomato and Its Wild Salt-tolerant Relative Lycopersicon Pennellii to Salt-dependent Oxidative Stress: Increased Activities of Antioxidant Enzymes in Root Plastids

Root plastids of the cultivated tomato Lycopersicon esculentum (Lem) exhibited salt-induced oxidative stress as indicated by the increased H 2 O 2 and lipid peroxidation levels which were accompanied with increased contents of the oxidized forms of ascorbate and glutathione. In contrast, H 2 O 2 level decreased, lipid peroxidation level slightly decreased and the levels of the reduced forms of ascorbate and glutathione increased in plastids of L. pennellii (Lpa) species in response to salinity. This better protection of Lpa root plastids from salt-induced oxidative stress was correlated with increased activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (POD), monodehydroascorbate reductase (MDHAR), glutathione peroxidase (GPX), glutathione- S -transferase (GST) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). In the plastids of both species, activities of SOD, APX, and POD could be resolved into several isozymes. In Lem plastids two Cu/ZnSOD isozymes were found whereas in Lpa an additional FeSOD type could also be detected. In response to salinity, activities of selected SOD, APX, and POD isozymes were increased in Lpa, while in Lem plastids the activities of most of SOD and POD isozymes decreased. Taken together, it is suggested that plastids play an important role in the adaptation of Lpa roots to salinity.

Ascorbate peroxidase gene family in tomato: its identification and characterization

The antioxidative response, where ascorbate peroxidase (APX) is a key enzyme, is an integral part of the plant tolerance response to environmental stresses. As a first step towards the study of the physiological role and the regulation of the members of the Apx gene family, the orthologs of the stress-sensitive cultivated tomato Solanum lycopersicum cv. M82 (Slm) and of the wild salt-tolerant species S. pennellii acc. Atico (Spa) were identified by utilizing the tomato EST database, and characterized. A redundant list of 16 virtual Apx transcripts and four singleton ESTs was shown to correspond to seven genuine Apx genes. The complete tomato Apx gene family is comprised of genes encoding three cytosolic, two peroxisomal, and two chloroplastic APXs. These genes attained differential regulatory patterns in various Slm organs. More detailed study of Apx1 and Apx2 genes, that are the products of a recent gene duplication event, shows that they have already attained differential regulation within and between Slm and Spa under control and stress conditions. It is also suggested that due to lineage-specific gene duplication and lose events, intricate phylogenetic relationships exist among the members of the Apx gene families.

Combining Comparative Sequence and Genomic Data to Ascertain Phylogenetic Relationships and Explore the Evolution of the Large GDSL-Lipase Family in Land Plants

The GDSL-lipase gene family is a very large subfamily within the supergene family of SGNH esterases, defined by the distinct GDSL amino acid motif and several highly conserved domains. Plants retain a large number of GDSL-lipases indicating that they have acquired important functions. Yet, in planta functions have been demonstrated for only a few GDSL-lipases from diverse species. Considering that orthologs often retain equivalent functions, we determined the phylogenetic relationships between GDSL-lipases from genome-sequenced species representing bryophytes, gymnosperms, monocots, and eudicots. An unrooted phylogenetic tree was constructed from the amino acid sequences of 604 GDSL-lipases from seven species. The topology of the tree depicts two major and one minor subfamily. This division is also supported by the unique gene structure of each subfamily. Because GDSL-lipase genes of all species are present in each of the three subfamilies, we conclude that the last common ancestor of the land plants already possessed at least one ancestral GDSL-lipase gene of each subfamily. Combined gene structure and synteny analyses revealed events of segmental duplications, gene transposition, and gene degeneration in the evolution of the GDSL-lipase gene family. Furthermore, these analyses showed that independent events of intron gain and loss also contributed to the extant repertoire of the GDSL-lipase gene family. Our findings suggest that underlying many of the intron losses was a spliceosomal-mediated mechanism followed by gene conversion. Sorting the phylogenetic relationships among the members of the GDSL-lipase gene family, as depicted by the tree and supported by synteny analyses, provides a framework for extrapolation of demonstrated functional data to GDSL-lipases, whose function is yet unknown. Furthermore, function(s) associated with specific lineage(s)-enriched branches may reveal correlations between acquired and/or lost functions and speciation.

Antioxidative Systems and Stress Tolerance: Insight from Wild and Cultivated Tomato Species

The role of antioxidative systems of the salt-sensitive cultivated tomato, L. esculentum (Lem) and its wild salt-tolerant relative L. pennellii (Lpa) in salt tolerance was studied. For this, leaf and root cell organelles (chloroplasts/plastids, mitochondria, and peroxisomes) isolated from control and salt-treated Lem and Lpa plants were characterized and compared. In general, the inherent organellar antioxidative systems of the two tomato species were highly similar. The activities of SOD and the ascorbate–glutathione cycle isozymes, in the various cell organelles, were separated into soluble and membrane-bound fractions. Latency and solubilization assays were used to map the in situ suborganellar localization of the ascorbate–glutathione cycle isozymes. These activities were localized to both faces of the organellar membranes and in the lumens of various suborganellar compartments and were modeled for peroxisomes, chloroplasts, and mitochondria. Differences between the inherent antioxidative systems of Lem and Lpa were found. These included different ratios of the soluble to membrane-bound activities and different SOD-type inventories. In Lem organelles, stress-induced downregulation of antioxidative isozymes, and oxidants, was correlated with increased oxidative damage, in contrast, in Lpa organelles a stress-induced upregulation of the antioxidative isozymes, and oxidants, was correlated with alleviation of oxidative stress. Similarly, cross-tolerance to imposed oxidative stress by SHAM and 3-AT was found only in Lpa plants grown in salinity and was dependent, at least in part, upon the capacity for de novo GSH synthesis. The failure of Lem to upregulate its antioxidative systems is discussed.

RESPONSE OF TOMATO PLANTS TO SALINE WATER AS AFFECTED BY CARBON DIOXIDE SUPPLEMENTATION. II. PHYSIOLOGICAL RESPONSES

SummaryPhotosynthesis of tomato plants (Lycopersicon esculentum (L.) Mill. cv. F144) was studied under conditions of CO2 supplementation and salinity. The purpose of the study was to elucidate the mechanisms underlying the effects of salinity on the acclimation of tomato plants to CO2 supplementation. Plants were grown under either low (355.mmol mol–1) or elevated (1200.6.50 mmol mol–1) CO2 and were irrigated with low concentrations of mixed salts. The highest salinity level (E.C. 7 dS m–1) was that used to produce quality tomatoes in the Negev highlands, in Israel. During early development (three weeks after planting), the net photosynthetic rate of the leaves was much higher under elevated CO2, and other than a slight decrease in quantum yield efficiency as measured by fluorescence (DF/F 9 m ), no signs of acclimation to high levels of CO2 were apparent. Clear acclimation to high CO2 concentration was evide t ten weeks after planting when the net photosynthetic rate, photosynthetic capacity, and carboxy...

Tethered ribozyme ligation enables detection of molecular proximity in homogeneous solutions

In contemporary drug discovery, bulk selection represents an important alternative to time consuming and expensive high-throughput screening. The selection methods, however, generally rely on affinity separation, a step that limits overall selection process efficiency. To overcome common drawbacks of conventional methods, we exploited the unique catalytic properties of an artificial enzyme, ribozyme ligase, to develop a selection methodology in which the entire detection process takes place in a homogeneous solution, thus eliminating the need for affinity separation. A molecular target is associated with the ribozyme, and library compounds are attached to a barcoded oligonucleotide that is a substrate for the ribozyme ligase. Spatial proximity resulting from specific target-compound interactions increases the probability of ribozyme ligation to the oligo-substrate, thus differentiating the interacting species from the bulk mixture. The covalent link formed between the ribozyme and target-interacting compounds diminishes the mass-action effect on the efficiency with which low-affinity and rare active species are detected. In addition, the magnitude of the detection signal associated with the interaction event renders the methodology an efficient platform for identifying inhibitors of intermolecular interactions. The proposed solution-based tethered ribozyme-ligation proximity detection method may facilitate the discovery of target-interacting compounds using both library selection and high-throughput screening approaches.