Lydia Faize

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.

Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings

Hydrogen peroxide (H(2)O(2)) increased the germination percentage of pea seeds, as well as the growth of seedlings in a concentration-dependent manner. The effect of H(2)O(2) on seedling growth was removed by incubation with 10 microm ABA. The H(2)O(2)-pretreatment produced an increase in ascorbate peroxidase (APX), peroxidase (POX) and ascorbate oxidase (AAO). The increases in these ascorbate-oxidizing enzymes correlated with the increase in the growth of the pea seedlings as well as with the decrease in the redox state of ascorbate. Moreover, the increase in APX activity was due to increases in the transcript levels of cytosolic and stromal APX (cytAPX, stAPX). The proteomic analysis showed that H(2)O(2) induced proteins related to plant signalling and development, cell elongation and division, and cell cycle control. A strong correlation between the effect of H(2)O(2) on plant growth and the decreases in ABA and zeatin riboside (ZR) was observed. The results suggest an interaction among the redox state and plant hormones, orchestrated by H(2)O(2), in the induction of proteins related to plant signalling and development during the early growth of pea seedlings.

Acibenzolar-s-methyl-induced resistance to Japanese pear scab is associated with potentiation of multiple defense responses.

ABSTRACT This study reports the mode of action of acibenzolar-S-methyl (ASM) against Japanese pear scab, caused by Venturia nashicola. Pretreatment of potted Japanese pear trees with ASM reduced scab symptoms and potentiated several lines of plant defense response. This included transcripts encoding polygalacturonase-inhibiting protein (PGIP) that were highly and transiently promoted after scab inoculation of plants pretreated with ASM, suggesting a possible role for defenses involved in direct interaction with the pathogen. The activity of the key enzyme of phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), was enhanced in scab-inoculated leaves pretreated with ASM only 7 days after inoculation, suggesting that it may play a minor role in induced resistance. In this work, salicylic acid (SA) accumulation was enhanced in ASM-treated leaves for the first time, according to an equivalent time course to that of PAL activity. However, a delayed induction of SA accumulation in ASM-treated leaves compared with kinetics of induction of several pathogenesis- related (PR) proteins or their encoding genes suggested that resistance triggered by ASM may be SA-independent. Among these PR proteins, PR-1, chitinase and PR-10 were promoted early by ASM after scab inoculation. Peroxidase, as well as enzymes involved in the oxidative burst such as superoxide dismutase, catalase, and ascorbate peroxidase were weakly activated with ASM treatment alone or pathogen inoculation alone and highly enhanced in ASM pretreated plants upon challenge inoculation, suggesting the occurrence of priming phenomenon during the interaction of Japanese pear-ASM-V. nashicola. An early potentiation of the activity of these enzymes after scab inoculation of leaves pretreated with ASM suggested that active oxygen species may be involved as a signal for the activation of PR proteins or genes.

Characterization of a leucine-rich repeat receptor-like protein kinase (LRPK) gene from Japanese pear and its possible involvement in scab resistance

A gene encoding a leucine-rich repeat receptor-like protein kinase (LRPK) was identified from Japanese pear (Pyrus pyrifolia var. culta) using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The Japanese pear LRPKp protein was composed of a leucine-rich extracellular ligand-binding domain, a transmembrane-spanning segment, and a cytoplasmic kinase domain; it had the highest sequence homology with apple LRPKm and Arabidopsis receptor-like protein kinase (RLK). Southern blot analysis revealed that the LRPKp belongs to a multigenic family, and a polymorphism in restriction fragment length was observed among Asian and European pear cultivars. The LRPKp transcripts were present at a high level in leaves and at a low level in flowers, while no expression was detected in fruits. The expression pattern of LRPKp was also studied by real-time RT-PCR in the scab susceptible cv. Kousui and resistant cvs. Kinchaku and Flemish Beauty after challenge inoculation with Venturia nashicola, the causal agent of Japanese pear scab. Transcripts of LRPKp were transiently enhanced in both the susceptible Kousui and to a great extent in the non-host-resistant Flemish Beauty. However, in resistant Kinchaku, expression of this gene was only weakly enhanced. Moreover, treatment with a resistance inducer, acibenzolar-S-methyl (ASM), which significantly reduces scab severity on potted trees of Kousui, was also able to induce and to potentiate the expression of this gene after inoculation. This suggests that the LRPKp gene is possibly involved in ASM-induced resistance.

Protective effect of three brown seaweed extracts against fungal and bacterial diseases of tomato

The effect of extracts of the brown algae Cystoseira myriophylloides, Laminaria digitata, and Fucus spiralis against the tomato pathogens Verticillium dahliae and Agrobacterium tumefaciens was evaluated in vitro and in the greenhouse. A significant inhibition of growth was observed only with methanolic seaweed extracts (MSE). Disease resistance was assessed in the greenhouse against Verticillium wilt using spray application of aqueous seaweed extracts (ASE) on the whole plant or using seed imbibition. Both methods significantly reduced disease severity whatever the algal species, though protection observed after seed treatments was higher than that observed after spray treatment. Spray application of ASE from C. myriophylloides and F. spiralis also resulted in significant reduction of Crown gall disease caused by the bacterial pathogen A. tumefaciens. ASE-treated plants had significantly higher levels of activity of defense enzymes polyphenol oxidase and peroxidase compared to the control. ASE did not inhibit mycelium growth of V. dahliae or development of A. tumefaciens in vitro; it is therefore suggested that induced resistance is probably the main mechanism of protection afforded by ASE.

Transformation of plum plants with a cytosolic ascorbate peroxidase transgene leads to enhanced water stress tolerance

BACKGROUND AND AIMS Water deficit is the most serious environmental factor limiting agricultural production. In this work, the tolerance to water stress (WS) of transgenic plum lines harbouring transgenes encoding cytosolic antioxidant enzymes was studied, with the aim of achieving the durable resistance of commercial plum trees. METHODS The acclimatization process was successful for two transgenic lines: line C3-1, co-expressing superoxide dismutase (two copies) and ascorbate peroxidase (one copy) transgenes simultaneously; and line J8-1, harbouring four copies of the cytosolic ascorbate peroxidase gene (cytapx). Plant water relations, chlorophyll fluorescence and the levels of antioxidant enzymes were analysed in both lines submitted to moderate (7 d) and severe (15 d) WS conditions. Additionally, in line J8-1, showing the best response in terms of stress tolerance, a proteomic analysis and determination of the relative gene expression of two stress-responsive genes were carried out. KEY RESULTS Line J8-1 exhibited an enhanced stress tolerance that correlated with better photosynthetic performance and a tighter control of water-use efficiency. Furthermore, this WS tolerance also correlated with a higher enzymatic antioxidant capacity than wild-type (WT) and line C3-1 plum plants. On the other hand, line C3-1 displayed an intermediate phenotype between WT plants and line J8-1 in terms of WS tolerance. Under severe WS, the tolerance displayed by J8-1 plants could be due to an enhanced capacity to cope with drought-induced oxidative stress. Moreover, proteomic analysis revealed differences between WT and J8-1 plants, mainly in terms of the abundance of proteins related to carbohydrate metabolism, photosynthesis, antioxidant defences and protein fate. CONCLUSIONS The transformation of plum plants with cytapx has a profound effect at the physiological, biochemical, proteomic and genetic levels, enhancing WS tolerance. Although further experiments under field conditions will be required, it is proposed that J8-1 plants would be an interesting Prunus rootstock for coping with climate change.

Media derived from brown seaweeds Cystoseira myriophylloides and Fucus spiralis for in vitro plant tissue culture

In the present study, the effect of seaweed extract (SE) from Fucus spiralis (Fs), Cystoseira myriophylloides (Cm) and Laminaria digitata (Ld) on in vitro plant tissue culture was examined. Combination of 25 % of SE from Cm with 25 % of MS medium increased adventitious shoot regeneration from Nicotiana benthamiana leaf discs explants by 620 %, when compared to the conventional regeneration medium. Similarly SE from Fs and Ld enhanced regeneration by about 500 %. However, when increasing SE to 50 %, only Cm significantly enhanced shoot regeneration. The effect of SE was also evaluated on in vitro micropropagation of N. benthamiana, grape, plum and apricot by assessing shoot length, number of leaves and internodes. When used alone but at lower concentrations (2.5 and 12.5 %), SE from Fs and Cm resulted in at least the same efficacy as MS alone for micropropagation of N. benthamiana shoots. However, for micropropagation of grapevine, plum and apricot woody plants, a combination of 50 % of SE from Cm or Fs with 50 % of their conventional micropropagation media was necessary. Rooting was also enhanced in N. benthamiana and grapevine, and was correlated with their higher concentrations of indole acetic acid when compared to SE from Ld. This finding, in addition to mineral analysis data, suggests that SE of Fs and Cm contain necessary nutrients and growth regulators to allow their use as medium for in vitro plant culture.

Cytosolic ascorbate peroxidase and Cu, Zn-superoxide dismutase improve seed germination, plant growth, nutrient uptake and drought tolerance in tobacco

The effects of over-expression of two cytosolic antioxidant enzymes (Cu, Zn-SOD and/or APX) on plant nutrition, gas exchange, chlorophyll fluorescence, seed viability and germination in transgenic tobacco (Nicotiana tabacum cv. Xanthi) under deficit irrigation or salinity conditions were investigated. Three transgenic lines of tobacco were used in this study: line 17, harboring 2 copies of the cytosolic CuZn-SOD (cytsod) gene; line 51, with 2 copies of the cytosolic APX (cytapx) gene and line 39, harboring one copy of each gene. Over-expression of cytosolic antioxidants enzymes in tobacco plants resulted in a better growth performance that correlated with an improved photosynthetic capacity and nutrient uptake. Moreover, cytsod or cytapx genes promoted seed germination, and enhanced tolerance to mild water stress. In addition, this enhanced antioxidant capacity protected seeds from ageing during prolonged storage, and stimulated germination under salt stress conditions. These results suggest that cytosolic antioxidant transgenes are useful tools to improve drought tolerance, nutrient uptake and seed germination under stressful conditions.

Semisynthetic Triterpenes Derived from Euphorbia officinarum as Plant Growth Promoters and Inducers of Disease Resistance

This study evaluated the effect of application of the semisynthetic triterpenes 3β-acetoxy-norlup-20-one (F4) and 3-chloro-4α,14α-dimethyl-5α-cholest-8-ene (F6) triterpene derivatives from Euphorbia officinarum on the growth of tomato seedlings under normal conditions and when challenged with the pathogens Verticillium dahliae and Agrobacterium tumefaciens. Foliar spray of F4 and F6 significantly improved growth rate, fresh weight, dry weight, and leaf area. In addition, they enhanced several physiological parameters including photosynthetic pigments, proline content, and nitrate reductase activity. Moreover, they induced H2O2 accumulation and increased the activity of several antioxidant enzymes such as catalase, ascorbate peroxidase, and guaiacol peroxidase. They also enhanced disease resistance against V. dahliae and A. tumefaciens. These results suggest that the two semisynthetic triterpenes represent new plant growth regulators and inducers of plant disease resistance.

Towards the valorization of the invasive seaweeds Caulerpa cylindracea and Asparagopsis taxiformis in the Mediterranean Sea: applications for in vitro plant regeneration and crop protection

Caulerpa cylindracea and Asparagopsis taxiformis are both invasive seaweeds representing a major threat to indigenous species and the native ecosystems of certain regions of the Mediterranean Sea. This work examines the possibility of adding value to this abundant biomass in the fields of in vitro culture and plant protection. Results showed that using a 10% aqueous extract from C. cylindracea and A. taxiformis to in vitro culture media enhanced the regeneration percentage of apricot hypocotyl slices, as well as the diameter of explants and the number of regenerating buds per explant. The combination of extracts from these two seaweeds with an increasing concentration of conventional plant growth regulators further significantly enhanced most of these parameters. In addition, the data showed that soaking seeds of Nicotiana benthamiana in a 1.5% solution of aqueous extracts of C. cylindracea resulted in seedlings with reduced disease severity, when inoculated with the plum pox virus. These observations demonstrated that it would be possible to transform and perhaps manage the biomass of these invasive algae, which could pave the way to multiple valorizations.