Functional analysis of endo-1,4-ß-glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant-pathogen interactions.

Plant cell wall modification is a critical component in stress responses. Endo-1,4-ß-glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence-signalling network. A study of a set of Arabidopsis EG T-DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant-pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant-pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.

Kinetics of NH (4) (+) uptake by the arbuscular mycorrhizal fungus Rhizophagus irregularis.

The kinetics and energetics of (15)NH (4) (+) uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis were investigated. (15)NH (4) (+) uptake increased with increasing substrate concentration over the concentration range of 0.002 to 25 mM. Eadie-Hofstee plots showed that ammonium (NH (4) (+) ) uptake over this range was biphasic. At concentrations below 100 µM, NH (4) (+) uptake fits a Michaelis-Menten curve, typical of the activity of a saturable high-affinity transport system (HATS). At concentrations above 1 mM, NH (4) (+) influx showed a linear response typical of a nonsaturable low-affinity transport system (LATS). Both transport systems were dependent on external pH. The HATS and, to a lesser extent, the LATS were inhibited by the ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP) and the ATP-synthesis inhibitor 2,4-dinitrophenol. These data indicate that the two NH (4) (+) transport systems of R. irregularis are dependent on metabolic energy and on the electrochemical H(+) gradient. The HATS- and the LATS-mediated (15)NH (4) (+) influxes were also regulated by acetate. This first report of the existence of active high- and low-affinity NH4(+) transport systems in the extraradical mycelium of an arbuscular mycorrhizal fungus and provides novel information on the mechanisms underlying mycosymbiont uptake of nitrogen from the soil environment.

Effect of a novel chemical mixture on senescence processes and plant--fungus interaction in Solanaceae plants.

The effects of exogenous application of a chemical mixture consisting of adipic acid monoethyl ester, furfurylamine, and 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranose (FGA) on various metabolic pathways and the plant-fungus interaction have been studied in Solanaceae plants. Tomato and pepper plants were sprayed with the FGA mixture, and different biochemical parameters such as gas exchange, chlorophyll concentration, protein, cell wall sugar and phenolics contents, and peroxidase and phenylalanine ammonia lyase (PAL) activities were measured. FGA-treated plants showed, in general, an increase in cell wall sugar content and decreases in the chlorophyll degrading rate and the peroxidase activity. These results suggest that FGA (a possible synthetic regulator) could act as a retardant--antisenescence agent in Solanaceae plants. The FGA mixture increased the PAL activity and promoted an overall rise in the concentration of flavonoids and phenolic compounds. Therefore, FGA induced the synthesis of compounds that could give protection to plants against pathogens or insects. To further verify this putative protection, several fungi were inoculated in intact plants. Exogenous FGA applications on intact plants delayed fungus-provoked lesion development. In addition, data also showed that applications of 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranose inhibited fungal growth in vitro. These results confirm that FGA can activate protective mechanisms in plants upon contact with invaders such as fungi.

Characterization and regulation of ammonium transport systems in Citrus plants.

We have investigated both the kinetics and regulation of 15NH4+ influx in roots of 3-month-old hydroponically grown Citrus (Citrus sinensis L. Osbeck x Poncirus trifoliata Blanco) seedlings. The 15NH4+ influx is saturable below an external ammonium concentration of 1 mM, indicating the action of a high-affinity transport system (HATS). The HATS is under feedback repression by the N status of the plant, being down-regulated in plants adequately supplied with N during growth, and up-regulated by N-starvation. When assayed between 1 and 50 mM [15NH4+]0, the 15NH4+ influx showed a linear response typical of a low-affinity transport system (LATS). The activity of the LATS increased in plants supplied with NH4+ as compared with plants grown on an N-free medium. Transfer of the plants to N-free solution resulted in a marked decrease in the LATS-mediated 15NH4+ influx. Accordingly, resupply of NH4+ after N-starvation triggered a dramatic stimulation of the activity of the LATS. These data provide evidence that in Citrus plants, the LATS or at least one of its components is inducible by NH4+. Even when up-regulated, both the HATS and the LATS displayed a limited capacity, as compared with that usually found in herbaceous species. The use of various metabolic uncouplers or inhibitors indicated that 15NH4+ influx mediated by the HATS is strongly dependent on energy metabolism and H+ transmembrane electrochemical gradient. By contrast, the LATS is not affected by protonophores or inhibitors of the H(+)-ATPase, suggesting that its activity is mostly driven by the NH4+/NH3 transmembrane gradient. In agreement with these hypotheses, the HATS-mediated 15NH4+ influx was strongly inhibited when the solution pH was raised from 4 to 7, whereas influx mediated by the LATS was slightly stimulated.

Characterization of the low affinity transport system for NO(3)(-) uptake by Citrus roots.

Three-month old citrange Troyer (hybrid of Citrus sinensis x Poncirus trifoliata) seedlings were grown hydroponically and, after a period of NO(3)(-) starvation, plants were transferred to solutions enriched with K(15)NO(3) (96% atoms 15N excess) to measure 15NO(3)(-) uptake rates as a function of external 15NO(3)(-) concentrations. Two different NO(3)(-) uptake systems were found. Between 1 and 50 mM 15NO(3)(-) in the uptake solution medium, the uptake rate increased linearly due to the low affinity transport system (LATS). Nitrate reductase activity showed the same response to external [NO(3)(-)], and also appears to be regulated by the rate of nitrate uptake. Nitrate pre-treatments had a represive effect on NO(3)(-) uptake rate measured at 5 or 30 mM external [15NO(3)(-)]. The extent of the inhibition depended on the [NO(3)(-)] during the pre-treatment and in the uptake solution. These results suggest that the LATS of Citrus seedlings is under feedback control by the N status of the plant. Accordingly, addition of amino acids (Glu, Asp, Asn, Gln) to the uptake solution resulted in a decrease in 15NO(3)(-) uptake rate. However, the inactivation of nitrate reductase activity after treatment of the seedlings with either 100 or 500 µM WO(4)(2-) did not affect the activity of the LATS. Metabolic uncouplers, 2,4-DNP and KCN, reduced the uptake rate by 43.3% and 41.4% respectively at 5mM external [15NO(3)(-)]. However, these compounds had little effect when 15NO(3)(-) uptake was assayed at 30 mM external concentration. The ATPase inhibitors DCCD and DES reduced 15NO(3)(-) uptake by 68.8%-35.6%, at both external [15NO(3)(-)]. Nitrate uptake by the LATS declined with the increase of the solution pH beyond pH 4. The data presented are discussed in the context of the kinetics, energy dependence and regulation of NO(3)(-) uptake.

Selection of a NaCl-tolerant Citrus plant.

The objective of the work was to select Citrus plants more tolerant to elevated NaCl concentrations in the irrigation water. For this purpose, unfertilized Troyer citrange ovules treated with a chemical mutagenic agent (ethyl methane sulphonate) were cultured in vitro. Whole plants were regenerated from embryos developed in the nucellar tissue of the ovule. The screening for salt tolerance was achieved by irrigating these plants with a nutrient solution containing 45 mM NaCl.Plants obtained from vegetative propagation of the selected plant show faster growth, less leaf damage and lower concentrations of Cl(-) and Na(+) in leaves than the original clone with increasing NaCl levels in the nutrient solution. In addition, the selected plant accumulated more Na(+) in shoots and roots and this was accompanied by a significant reduction in K(+)concentration. It can be concluded that the selected plant restricts the accumulation of Na(+) in leaves by concentrating Na(+) in shoots and roots.

Effect of the O chromosome of Drosophila subobscura on abdominal bristle number.

The role of the O chromosome of D. subobscura on the control of abdominal bristle number was analyzed. The study was performed using four strains derived from four lines selected for high (H1 and H2) and low (L1 and L2) bristle number. In all the lines, the factors for abdominal bristle number carried on the O chromosome showed additive effects and diminished the number of abdominal bristles. The negative effect of the O chromosomes from low selection lines was found to be stronger than the negative effect of the O chromosomes from high selection lines.