Shimon Gepstein

Delayed leaf senescence induces extreme drought tolerance in a flowering plant

Drought, the most prominent threat to agricultural production worldwide, accelerates leaf senescence, leading to a decrease in canopy size, loss in photosynthesis and reduced yields. On the basis of the assumption that senescence is a type of cell death program that could be inappropriately activated during drought, we hypothesized that it may be possible to enhance drought tolerance by delaying drought-induced leaf senescence. We generated transgenic plants expressing an isopentenyltransferase gene driven by a stress- and maturation-induced promoter. Remarkably, the suppression of drought-induced leaf senescence resulted in outstanding drought tolerance as shown by, among other responses, vigorous growth after a long drought period that killed the control plants. The transgenic plants maintained high water contents and retained photosynthetic activity (albeit at a reduced level) during the drought. Moreover, the transgenic plants displayed minimal yield loss when watered with only 30% of the amount of water used under control conditions. The production of drought-tolerant crops able to grow under restricted water regimes without diminution of yield would minimize drought-related losses and ensure food production in water-limited lands.

Use of a Scanning Densitometer or an ELISA Plate Reader for Measurement of Nanogram Amounts of Protein in Crude Extracts from Biological Tissues

Protein contents of crude extracts from plant and animal tissues can be rapidly assayed using a Coomassie blue dye-binding procedure combined with scanning densitometry. Total protein is extracted from 100 mg of fresh-frozen or dried-ground tissue using 1 ml of extraction buffer. One-microliter aliquots of standard solutions or crude extracts are spotted in rows on a suitably sized sheet of Whatman 3MM chromatography paper. The dried samples are stained with Coomassie brilliant blue R-250 (0.2%, w/v, in acidified 50% MeOH) for 20 min and rinsed twice with acidified 20% MeOH. After drying, protein concentrations are read as reflectance using a scanning densitometer and peak heights or peak areas recorded using a digital integrator. In an alternative procedure, each spot is cut from the sample sheet and the dye-protein complex eluted in 1% sodium dodecyl sulfate (SDS) using an ultrasonic cleaner. Absorbance is subsequently read in a microwell sample holder at 590 nm with an enzyme-linked immunosorbent assay plate reader. Both procedures offer distinct advantages over previously reported methods. They are significantly faster when large numbers of samples are processed, they avoid interference by chlorophyll, dithiothreitol, SDS, 2-mercaptoethanol, Nonidet P-40, and phenylmethylsulfonyl fluoride (and other protease inhibitors) and they yield marked improvements in sensitivity, providing measurements of protein concentration below 100 and 200 ng.microliter-1, respectively.

Progressive Inhibition by Water Deficit of Cell Wall Extensibility and Growth along the Elongation Zone of Maize Roots Is Related to Increased Lignin Metabolism and Progressive Stelar Accumulation of Wall Phenolics

Water deficit caused by addition of polyethylene glycol 6000 at −0.5 MPa water potential to well-aerated nutrient solution for 48 h inhibited the elongation of maize (Zea mays) seedling primary roots. Segmental growth rates in the root elongation zone were maintained 0 to 3 mm behind the tip, but in comparison with well-watered control roots, progressive growth inhibition was initiated by water deficit as expanding cells crossed the region 3 to 9 mm behind the tip. The mechanical extensibility of the cell walls was also progressively inhibited. We investigated the possible involvement in root growth inhibition by water deficit of alterations in metabolism and accumulation of wall-linked phenolic substances. Water deficit increased expression in the root elongation zone of transcripts of two genes involved in lignin biosynthesis, cinnamoyl-CoA reductase 1 and 2, after only 1 h, i.e. before decreases in wall extensibility. Further increases in transcript expression and increased lignin staining were detected after 48 h. Progressive stress-induced increases in wall-linked phenolics at 3 to 6 and 6 to 9 mm behind the root tip were detected by comparing Fourier transform infrared spectra and UV-fluorescence images of isolated cell walls from water deficit and control roots. Increased UV fluorescence and lignin staining colocated to vascular tissues in the stele. Longitudinal bisection of the elongation zone resulted in inward curvature, suggesting that inner, stelar tissues were also rate limiting for root growth. We suggest that spatially localized changes in wall-phenolic metabolism are involved in the progressive inhibition of wall extensibility and root growth and may facilitate root acclimation to drying environments.

Branched-chain and aromatic amino acid catabolism into aroma volatiles in Cucumis melo L. fruit.

The unique aroma of melons (Cucumis melo L., Cucurbitaceae) is composed of many volatile compounds biosynthetically derived from fatty acids, carotenoids, amino acids, and terpenes. Although amino acids are known precursors of aroma compounds in the plant kingdom, the initial steps in the catabolism of amino acids into aroma volatiles have received little attention. Incubation of melon fruit cubes with amino acids and α-keto acids led to the enhanced formation of aroma compounds bearing the side chain of the exogenous amino or keto acid supplied. Moreover, L-[13C6]phenylalanine was also incorporated into aromatic volatile compounds. Amino acid transaminase activities extracted from the flesh of mature melon fruits converted L-isoleucine, L-leucine, L-valine, L-methionine, or L-phenylalanine into their respective α-keto acids, utilizing α-ketoglutarate as the amine acceptor. Two novel genes were isolated and characterized (CmArAT1 and CmBCAT1) encoding 45.6 kDa and 42.7 kDa proteins, respectively, that displayed aromatic and branched-chain amino acid transaminase activities, respectively, when expressed in Escherichia coli. The expression of CmBCAT1 and CmArAT1 was low in vegetative tissues, but increased in flesh and rind tissues during fruit ripening. In addition, ripe fruits of climacteric aromatic cultivars generally showed high expression of CmBCAT1 and CmArAT1 in contrast to non-climacteric non-aromatic fruits. The results presented here indicate that in melon fruit tissues, the catabolism of amino acids into aroma volatiles can initiate through a transamination mechanism, rather than decarboxylation or direct aldehyde synthesis, as has been demonstrated in other plants.

Potential Use of Cutinase in Enzymatic Scouring of Cotton Fiber Cuticle

The present study characterized the ability of a bacterial cutinase to improve the wettability of raw cotton fabrics by specific hydrolysis of the cutin structure of the cuticle. The effect of cutinase was studied alone and in coreaction with pectin lyase. The changes in both the fabric and the reaction fluid were measured and compared to enzymatic hydrolysis with polygalacturonase, and to chemical hydrolysis with boiling NaOH. Water absorbancy, specific staining, fabric weight loss, and evaporative light-scattering reversephase high-performance liquid chromatography analysis of chloroform extract of the reaction fluid were measured to assess the enzymatic hydrolysis of the cuticle waxy layer. The pattern and extent of hydrolysis of the major cuticle constituents depended on the enzyme type and titers employed and paralleled the degree of wettability obtained. The combination of cutinase and pectin lyase resulted in a synergistic effect. The use of detergents improved enzymatic scouring. The major products released to the reaction medium by the cutinase treatment were identified by gas chromatography/mass spectrometry analysis as C:16 and C:18 saturated fatty acid chains.

Leaf senescence - not just a 'wear and tear' phenomenon

A recent, genome-wide study shows that the transcriptional program underlying leaf senescence is active and complex, reflecting the activation of more than 2,000 genes in Arabidopsis, with gene products involved in a broad spectrum of regulatory, biochemical and cellular events.

Proteomics of the response of Arabidopsis thaliana to infection with Alternaria brassicicola.

We have studied the proteome of the model plant Arabidopsis thaliana infected with a necrotrophic fungal pathogen, Alternaria brassicicola. The Arabidopsis-A. brassicicola host-pathogen pair is being developed as a model genetic system for incompatible plant-fungal interactions, in which the spread of disease is limited by plant defense responses. After confirming that a defense response was induced at the transcriptional level, we identified proteins whose abundance on 2-DE gels increased or decreased in infected leaves. At least 11 protein spots showed reproducible differences in abundance, increasing or decreasing during the progress of the infection. The pathogenesis-related protein PR4, a glycosyl hydrolase, and the antifungal protein osmotin are strongly up-regulated. Two members of the Arabidopsis glutathione S-transferase (GST) family increased in abundance in infected leaves. The spots in which these GST proteins were identified contain additional members of the GST family. Representation of GST family members in several protein spots migrating at similar molecular weight suggests post-translational modifications. The signature of GST regulation may be specific for the type of plant-pathogen interaction. The proteomic view of the defense response to A. brassicicola can be compared with other types of plant-pathogen interactions, and to leaf senescence, identifying unique regulatory patterns.

Differential expression profiles of growth-related genes in the elongation zone of maize primary roots.

Growth in the apical elongation zone of plant roots is central to the development of functional root systems. Rates of root segmental elongation change from accelerating to decelerating as cell development proceeds from newly formed to fully elongated status. One of the primary variables regulating these changes in elongation rates is the extensibility of the elongating cell walls. To help decipher the complex molecular mechanisms involved in spatially variable root growth, we performed a gene identification study along primary root tips of maize (Zea mays) seedlings using suppression subtractive hybridization (SSH) and candidate gene approaches. Using SSH we isolated 150 non-redundant cDNA clones representing root growth-related genes (RGGs) that were preferentially expressed in the elongation zone. Differential expression patterns were revealed by Northern blot analysis for 41 of the identified genes and several candidate genes. Many of the genes have not been previously reported to be involved in root growth processes in maize. Genes were classified into groups based on the predicted function of the encoded proteins: cell wall metabolism, cytoskeleton, general metabolism, signaling and unknown. In-situ hybridization performed for two selected genes, confirmed the spatial distribution of expression shown by Northern blots and revealed subtle differences in tissue localization. Interestingly, spatial profiles of expression for some cell wall related genes appeared to correlate with the profile of accelerating root elongation and changed appropriately under growth-inhibitory water deficit.

Reactive Oxygen Species and Induction of Lignin Peroxidase in Phanerochaete chrysosporium

ABSTRACT We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O2) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O2 gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O2 (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O2 concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O2 is at least partially mediated by the intracellular ROS.

Cytokinin, acting through ethylene, restores gravitropism to Arabidopsis seedlings grown under red light.

Cytokinin replaces light in several aspects of the photomorphogenesis of dicot seedlings. Arabidopsis thaliana seedlings grown under red light have been shown to become disoriented, losing the negative hypocotyl gravitropism that has been observed in seedlings grown in darkness or white light. We report here that cytokinin at micromolar concentrations restores gravitropism to seedlings grown under red light. Cytokinin cancels the effect of red light on the gravity-sensing system and at the same time replaces light in the inhibition of hypocotyl elongation. Furthermore, application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid acts similarly to cytokinin. Cytokinin cannot restore gravitropism under red light to an ethylene-insensitive mutant that is defective at the EIN2 locus. Stimulation of ethylene production, therefore, can explain the action of cytokinin in restoring negative gravitropism to the hypocotyls of Arabidopsis seedlings grown under continuous red light.