Morphological, physiochemical and antioxidant responses of Maclura pomifera to drought stress.

Drought is one of the most important environmental factor limiting the growth of woody and non woody plants. In the present paper, we aimed to explore the performance of Maclura pomifera under a prolonged drought period followed by re-watering. M. pomifera plants were exposed to four different watering regimes (100%, 75%, 50% and 30% of the field capacity (FC)) for three weeks and then rewatered. The exposure to drought affected physiological, morphological and biochemical traits of M. pomifera. Leaf area, relative water content and water potential of leaf decreased in parallel with increased water deficit. Malondialdehyde content increased along with the drought stress experiment. Soluble carbohydrates (sucrose, glucose and fructose) accumulated during drought stress, but decreased after 22 days of water deficit in severe stressed plants (30% FC). Proline and mannitol, two compatible osmolytes, were higher in drought stresses plants than in control plants. Additionally the activity of antioxidant enzymes (SOD, APX, DHAR and GR) resulted affected by drought stress. In the recovery period, the physiological parameters as well as the proline content recovered at control levels, whereas soluble sugars, mannitol and total activity of antioxidant enzymes remained slight higher than in control plants, presumably to allow plants a complete recovery after stress. Our results suggest that M. pomifera has a good adaptive response to drought stress, probably corresponded to decreasing oxidative injury by induction of the antioxidant system and accumulation of stable and protective osmolytes such as proline and mannitol at higher rates.

Cross-talk between physiological and biochemical adjustments by Punica granatum cv. Dente di cavallo mitigates the effects of salinity and ozone stress.

Plants are exposed to a broad range of environmental stresses, such as salinity and ozone (O3), and survive due to their ability to adjust their metabolism. The aim of this study was to evaluate the physiological and biochemical adjustments adopted by pomegranate (Punica granatum L. cv. Dente di cavallo) under realistic field conditions. One-year-old saplings were exposed to O3 [two levels denoted as ambient (AO) and elevated (EO) O3 concentrations] and salinity [S (salt, 50 mM NaCl)] for three consecutive months. No salt (NS) plants received distilled water. Despite the accumulation of Na+ and Cl- in the aboveground biomass, no evidence of visible injury due to salt (e.g. tip yellow-brown lesions) was found. The maintenance of leaf water status (i.e. unchanged values of electrolytic leakage and relative water content), the significant increase of abscisic acid, proline and starch content (+98, +65 and +59% compared to AO_NS) and stomatal closure (-24%) are suggested to act as adaptive mechanisms against salt stress in AO_S plants. By contrast, EO_NS plants were unable to protect cells against the negative impact of O3, as confirmed by the reduction of the CO2 assimilation rate (-21%), accumulation of reactive oxygen species (+10 and +225% of superoxide anion and hydrogen peroxide) and malondialdehyde by-product (about 2-fold higher than AO_NS). Plants tried to preserve themselves from further oxidative damage by adopting some biochemical adjustments [i.e. increase in proline content (+41%) and induction of catalase activity (8-fold higher than in AO_NS)]. The interaction of the two stressors induced responses considerably different to those observed when each stressor was applied independently. An analysis of the antioxidant pool revealed that the biochemical adjustments adopted by P. granatum under EO_S conditions (e.g. reduction of total ascorbate; increased activities of superoxide dismutase and catalase) were not sufficient to ameliorate the O3-induced oxidative stress.

Root colonization by Pseudomonas chlororaphis primes tomato (Lycopersicum esculentum) plants for enhanced tolerance to water stress.

Previous research demonstrated that Pseudomonas chlororaphis subsp. aureofaciens strain M71, a plant growth promoting bacterium (PGPB), exerts beneficial effects on plant metabolism and primes tolerance mechanisms against biotic stresses in tomatoes. We designed an experiment to assess whether root colonization with P. chlororaphis is also able to improve tolerance to water stress in tomatoes. Our results show that inoculation with P. chlororaphis stimulates the antioxidant activity of well-watered tomatoes while maintaining a steady-state level of reactive oxygen species (ROS), increases the expression of genes encoding for the biosynthesis of leaf terpenes, stimulates the production of both the phytohormones ABA and IAA, in turn affecting plant shape (number of leaves) and height (length of internodes), without altering photosynthesis. Upon exposure to mild water stress conditions, an improved antioxidant activity in tomatoes 'primed' by P. chlororaphis inoculation limited the accumulation of reactive oxygen species (ROS) in leaves and thus enhanced tolerance, also through increase of the (osmolyte) proline content. Moreover, P. chlororaphis inoculation further enhanced the ABA level in leaves of water-stressed tomatoes allowing a more efficient modulation of stomatal closure that resulted in an improved water use efficiency (WUE) and biomass accumulation.

Physiological and metabolomic analysis of Punica granatum (L.) under drought stress.

MAIN CONCLUSION: Punica granatum has a noticeable adaptation to drought stress. The levels of the green leaf volatile trans-2-hexenal increased in response to drought stress suggesting a possible role of this compound in drought stress response in pomegranate. Punica granatum (L.) is a highly valued fruit crop for its health-promoting effects and it is mainly cultivated in semi-arid areas. Thus, understanding the response mechanisms to drought stress is of great importance. In the present research, a metabolomics analysis was performed to evaluate the effects of drought stress on volatile organic compounds extracted from the leaves of pomegranate plants grown under water shortage conditions. The time course experiment (7 days of water deprivation and 24-h recovery) consisted of three treatments (control, drought stress, and rehydration of drought-stressed plants). Plant weights were recorded and control plants were irrigated daily at pot capacity to provide the lost water. Fraction of transpirable soil water has been evaluated as indicator of soil water availability in stressed plants. The levels of proline, hydrogen peroxide and lipid peroxidation as well as of the photosynthetic parameters such as photosynthesis rate (A), stomatal conductance (g s), photosynthetic efficiency of photosystem II, and photochemical quenching were monitored after the imposition of drought stress and recovery as markers of plant stress. Constitutive carbon volatile components were analyzed in the leaf of control and drought-stressed leaves using Head Space Solid Phase Micro Extraction sampling coupled with Gas Chromatography Mass Spectrometry. A total of 12 volatile compounds were found in pomegranate leaf profiles, mainly aldehydes, alcohols, and organic acids. Among them, the trans-2-hexenal showed a significant increase in water-stressed and recovered leaves respect to the well-watered ones. These data evidence a possible role of the oxylipin pathway in the response to water stress in pomegranate plants.

Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na+ and Cl- accumulation behaviour.

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes ('Cleopatra' and 'Willow leaf' mandarins), which differ for Na(+) and Cl(-) accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator 'Cleopatra' mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator 'Willow leaf' mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× 'Cleopatra' and 4× 'Willow leaf' mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× 'Cleopatra' mandarin and 4× 'Willow leaf' mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.

Itraconazole inhibits HMEC-1 angiogenesis.

Abnormal angiogenesis is implicated in a number of human diseases and endothelial growth inhibition represents a common approach in tumor therapy. Recently itraconazole, frequently used in humans as antifungal drug, which blocks the biosynthesis of cholesterol, has been found to be antiangiogenic in primary umbilical vein endothelial cells. However, the exact antiangiogenic mechanisms remain largely unknown. In this paper, we studied the effect of itraconazole in human dermal microvascular endothelial cells (HMEC-1), an immortalized cell line to study adult angiogenesis. A 50% reduction of microtubule formation was observed after itraconazole treatment which was partially rescued by cholesterol addition. We found that itraconazole inhibits angiogenesis markers such as VEGF, AAMP and e-NOS. mTOR and ERK1/2 phosphorylation as well as the expression of Gli1, one of the main controllers of the Shh pathway, were also inhibited by itraconazole. Cholesterol addition did not completely rescue inhibition of these pathways, suggesting that the itraconazole antiangiogenic activity could be due to multiple mechanisms. Our results may contribute to novel approaches to block angiogenesis with therapeutic application.

A stress responsive alternative splicing mechanism in Citrus clementina leaves.

Chitinases are often considered pathogenesis-related proteins since their activity can be induced by viral infections, fungal and bacterial cell wall components, and also by more general sources of stress such as wounding, salicylic acid, ethylene, auxins and cytokinins. In the present study, comparative proteomic analysis showed the defense-related acidic chitinase II to be specifically induced in Citrus clementina leaves infested by the two-spotted spider mite Tetranychus urticae or treated with MeJA. In parallel, changes in the mRNA profiles of two partially homologous chitinase forms were shown by RT-PCR. In particular, the appearance of an additional cDNA chitinase fragment in T. urticae-infested and MeJA-treated leaves was observed. This finding may indicate a specific regulatory mechanism of chitinase expression. We report evidence for alternative splicing in T. urticae-infested C. clementina, where a premature stop codon after the first 135 amino acids was introduced. We observed inducible chitinase activity after MeJA treatment, indicative of a rapid plant response to infestation. This work provides the first evidence of chitinase alternative splicing in C. clementina. In addition, the presence of the dual-band pattern for chitinase cDNA by RT-PCR may represent a suitable predictive marker for early diagnosis of plant biotic stress.