Antioxidant potential in <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> callus in response to polyethylene glycol,paclobutrazol and gibberellin treatments

The study on Stevia callus has the potential to advance the knowledge of antioxidant mechanisms involved in unorganized cells response to drought stress. The effects of polyethylene glycol (PEG; 0 and 4% w/v) in combination with paclobutrazol (PBZ; 0 and 2 mg l^?1) and gibberellin (GA; 0 and 2 mg l^?1) were studied on Stevia rebaudiana callus. PEG treatment led to an oxidative stress, as indicated by increased H₂O₂ content whose accumulation was prevented with PBZ and GA treatments. All treatments of PEG, PBZ and GA increased the total antioxidant capacity, with the highest antioxidant power in PBZ and GA treatments without PEG. The activity of superoxide dismutase, catalase and ascorbate peroxidase significantly increased in PEG treatment alone or in combination with PBZ and GA. All treatments of PEG, PBZ and GA significantly increased proteins, amino acids and proline contents, with the highest increase in presence of PBZ in medium culture. In contrary to proline, the activity of pyrroline-5-carboxylate synthetase and proline dehydrogenase did not change in response to any of the treatments. Collectively, our results demonstrated that PBZ and GA increased reactive oxygen species scavenging and osmolytes in PEG-treated calli more than PEG treatment alone to alleviate negative effects of PEG on Stevia calli. These findings will enable us to design effective genetic engineering strategies in callus culture to generate some somaclonal variation that may be useful in enhancing drought resistance in Stevia.     

Endogenous Salicylic Acid Levels and Signaling Positively Regulate <Emphasis Type="Italic">Arabidopsis</Emphasis> Response to Polyethylene Glycol-Simulated Drought Stress

Salicylic acid (SA) functions in the plant response to drought stress were assessed using SA-altering Arabidopsis mutants, including snc1 (with constitutively high levels of SA) and its nahG-transformed plants (named as snc1/nahG, with a comparable SA level to the wild type), sid2 and transgenic line nahG (both with SA deficiency), and npr1-1 (with SA signaling blockage). The drought stress was simulated by polyethylene glycol (PEG)-6000 treatment. Compared with wild-type (wt) plants, the snc1 plants displayed obvious easing of PEG-induced growth inhibition, leaf water loss, and photosynthesis-related impairment, whereas in nahG, sid2, and npr1-1 mutants the effect was more severe. PEG stress reduced stomatal conductance, to a higher extent in the snc1 line, whereas it was lower in nahG, sid2, and npr1-1 lines as compared with the wt. The snc1 plants accumulated higher levels of H₂O₂ than the other genotypes tested. PEG stress increased activities of superoxide dismutase and peroxidase, but decreased activities of catalase in all lines tested, to a greater extent in snc1 and less in sid2, nahG, and npr1-1 relative to wt. Proline was significantly increased, especially in snc1 line at 6 % and higher PEG stress. Noticeably, the performance of snc1 under PEG stress was dependent on SA levels, as the expression of nahG in snc1 plants did not only significantly reduce SA levels, but largely reversed the above-mentioned parameters, as well as eliminated the drought tolerance. Based on these data, it was concluded that endogenous SA levels and signaling provided a protective role in the Arabidopsis response to PEG-simulated drought.     

Drought-inducible expression of <Emphasis Type="Italic">Hv</Emphasis>-miR827 enhances drought tolerance in transgenic barley

Drought is one of the major abiotic stresses reducing crop yield. Since the discovery of plant microRNAs (miRNAs), considerable progress has been made in clarifying their role in plant responses to abiotic stresses, including drought. miR827 was previously reported to confer drought tolerance in transgenic Arabidopsis. We examined barley (Hordeum vulgare L. ‘Golden Promise’) plants over-expressing miR827 for plant performance under drought. Transgenic plants constitutively expressing CaMV-35S::Ath-miR827 and drought-inducible Zm-Rab17::Hv-miR827 were phenotyped by non-destructive imaging for growth and whole plant water use efficiency (WUE_wp). We observed that the growth, WUE_wp, time to anthesis and grain weight of transgenic barley plants expressing CaMV-35S::Ath-miR827 were negatively affected in both well-watered and drought-treated growing conditions compared with the wild-type plants. In contrast, transgenic plants over-expressing Zm-Rab17::Hv-miR827 showed improved WUE_wp with no growth or reproductive timing change compared with the wild-type plants. The recovery of Zm-Rab17::Hv-miR827 over-expressing plants also improved following severe drought stress. Our results suggest that Hv-miR827 has the potential to improve the performance of barley under drought and that the choice of promoter to control the timing and specificity of miRNA expression is critical.     

Olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) plants transgenic for tobacco osmotin gene are less sensitive to in vitro-induced drought stress

Olive is one of the most important tree crops in the Mediterranean region, because of its ability to grow and produce acceptable yields under limited water availability. In this study, the drought tolerance of an olive cultivar Canino was compared to the performance of its derived transgenic line expressing osmotin gene from tobacco, obtained by Agrobacterium-mediated transformation of Canino cultivar. Shoot cultures of both wild-type (wt) and transgenic lines were exposed to drought stress over a 28-day period, and their differential responses to in vitro-drought stress were investigated. After exposure to PEG, most of the shoots from wt plants resulted in damage and exhibited decreased levels of chlorophyll, while those of transgenic line did not show injuries and showed a normal growth even when exposed to the highest PEG concentration (4%). After preliminary evaluation we characterized Canino AT17-1, by measuring several physiological parameters, including the activities of the antioxidant enzymes (POD and CAT), and the content of malondialdehyde (MDA). Both the activity of catalase and the proline content were higher in the leaves of the transgenic shoots compared to wt plants. Consequently, it was observed that the transgenic line accumulated less MDA indicating that the presence of the osmotin gene protected the cell membrane from damage by lipid peroxidation. Together, these results could suggest that the transgenic line Canino AT17-1 was more efficient in the activation of defense responses against oxidative stress with respect to the Canino wt. The further finding that the transgenic shoots also showed higher proline accumulation supported the hypothesis that the osmotin gene conferred to transgenic shoots increased tolerance to drought stress compared with the wt.     

Physiological,biochemical, antioxidant and growth characterizations of gibberellin and paclobutrazol-treated sweet leaf (<Emphasis Type="Italic">Stevia rebaudiana</Emphasis> B.) herb

A greenhouse experiment was designed to study the responses of Stevia rebaudiana herb to paclobutrazol (PBZ) and gibberellin (GA) treatments. GA and PBZ treatments caused no significant impact on photosynthesis pigments while they increased carbohydrates, amino acids and protein metabolites. Stevia showed a potent antioxidant activity through scavenging DPPH, NO^·; O ₂ ^·? and OH^· radicals which was highlighted in GA and PBZ treatments. The enzymatic and non-enzymatic antioxidant system of Stevia plant showed a significant increase in response to PBZ and GA treatments. PBZ treatment decreased plant growth while GA treatment had no significant effect on it. Collectively, both GA and PBZ treatments effectively increased metabolites and antioxidant property of Stevia herb.     

<Emphasis Type="Italic">Lsi1</Emphasis>-regulated Cd uptake and phytohormones accumulation in rice seedlings in presence of Si

Silicon (Si) is known for its role in regulating the response of plants to imposed abiotic stresses. Since the stresses generally hinder production of a crop, such as rice, the exploration of the biochemistry and plant physiology relating to the function is of interest. Indeed, recently, there were reports on the function of Lsi1 in regulating the tolerance of rice to cadmium (Cd) stress. This study compared the kinetics of the Cd uptakes in Lemont wild type rice and its transgenic lines exposed to Cd with or without exogenous Si supply. At the same time, changes on the endogenous phytohormones and growth of the rice seedlings were monitored. Genetically, Lsi1 overexpression was found to downregulate K_m and V_max of Cd uptake kinetics in the plants under Cd stress, especially in the presence of Si. On the other hand, Lsi1 RNAi upregulated K_m and V_max regardless whether Si was present or not. It implied that Lsi1 could be capable of regulating Si as well as Cd transports. Under Cd stress, addition of Si reduced the Cd uptake of the rice lines in the order of Lsi1-overexpression line?>?Lemont?>?Lsi1-RNAi line. In addition, it also affected the chlorophyll biosynthesis and dry mass accumulation of the rice plants under Cd stress. Analyses on phytohormones including IAA, GA₃, JA, SA and ABA, as well as physiological functions, of the seedlings further verified the active involvement of Lsi1 in the complex defense system of the plants against Cd stress.     

Physiological and molecular responses to drought and salinity in soybean

Drought and salinity are severe environmental stresses and limit soybean growth. In this study, a comparative analysis of physiological and molecular responses of two soybean (Glycine max L.) genotypes to these stresses was carried out. Plants of drought-tolerant genotype RD (cv. FD92) and sensitive genotype SD (cv. Z1303) were exposed to 15 % (m/v) PEG 6000, which simulated drought stress, or 150 mM NaCl. The RD plants maintained larger leaf area and higher net photosynthetic rate, chlorophyll content, stomatal conductance, and relative water content compared with the SD plants. Leaf proline content increased under both stresses more in RD than in SD. The drought tolerance of RD plants was also correlated with greater antioxidant activity and lower content of hydrogen peroxide and malondialdehyde under stress conditions. Amounts of abscisic acid, jasmonic acid, and salicylic acid under stress increased to a greater extent in RD than in SD plants. At the molecular level, the effects of 20-d stress treatments were manifested by relatively higher expression of drought- or salt-related genes: GmP5CS, GmDREB1a, GmGOLS, GmBADH, and GmNCED1 in RD plants than in SD plants. These results form the basis for understanding the mechanisms of the drought- and salt-stress tolerance in soybean.     

Gender-related differences in adaptability to drought stress in the dioecious tree <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Ginkgo (Ginkgo biloba) as a precious relict plant is cultivated around the world, and it is also a typical dioecious tree. Drought is a major environmental stress that limits the growth and development of ginkgo. Although many studies have examined the impact of drought on ginkgo, few have investigated gender-related under drought treatment in the species. In our research, we examined comparative morphology, physiology and the ultrastructure of mesophyll cell in male and female ginkgoes to determine which gender shows superior adaptability to drought stress. Two-year-old cutting-propagated male and female ginkgoes suffered to drought treatment. The experiments showed that drought significantly limited growth and development, disrupted photosynthesis, and destoried the antioxidant protection system in both male and female ginkgoes. When the gender differences in the species were compared, females showed better growth, activities of SOD and POD, concentrations of chl t, chl a/b ratio and proline, P _n, C _i, g _s, qP and NPQ under drought, but lower concentrations of H₂O₂ and O₂ ^?, and relative electrolyte leakage. In the aspect of cell ultrastructure, female plants showed a slower rate of cell breakdown and chloroplast decomposition under drought stress than males. The results indicate that female plants of ginkgo show superior growth performance and self-protective mechanisms and higher photosynthetic capacity than male plants under drought stress. Thus, we conclude that female individuals of ginkgo possess better adaptability to drought stress than male individuals.     

Overexpression of <Emphasis Type="Italic">AlTMP2</Emphasis> gene from the halophyte grass <Emphasis Type="Italic">Aeluropus littoralis</Emphasis> in transgenic tobacco enhances tolerance to different abiotic stresses by improving membrane stability and deregulating some stress-related genes

Herein, we report isolation of the AlTMP2 gene from the halophytic C4 grass Aeluropus littoralis. The subcellular localization suggested that AlTMP2 is a plasma membrane protein. In A. littoralis exposed to salt and osmotic stresses, the AlTMP2 gene was induced early and at a high rate, but was upregulated relatively later in response to abscisic acid and cold treatments. Expression of AlTMP2 in tobacco conferred improved tolerance against salinity, osmotic, H₂O₂, heat, and freezing stresses at the germination and seedling stages. Under control conditions, no growth or yield penalty were mentioned in transgenic plants due to the constitutive expression of AlTMP2. Interestingly, under greenhouse conditions, the seed yield of transgenic plants was significantly higher than that of non-transgenic (NT) plants grown under salt or drought stress. Furthermore, AlTMP2 plants had less electrolyte leakage, higher membrane stability, and lower Na⁺ and higher K⁺ accumulation than NT plants. Finally, six stress-related genes were shown to be deregulated in AlTMP2 plants relative to NT plants under both control and stress conditions. Collectively, these results indicate that AlTMP2 confers abiotic stress tolerance by improving ion homeostasis and membrane integrity, and by deregulating certain stress-related genes.     

Photosynthetic,physiological and biochemical events associated with polyethylene glycol-mediated osmotic stress tolerance in taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> L. Schott)

Six genotypes of taro (Colocasia esculenta L. Schott) were evaluated under in vitro and in vivo polyethylene glycol (PEG–6000)-mediated osmotic stress conditions. A significant variation in growth response was observed among the taro genotypes under in vitro-induced stress conditions. In vivo results indicated a significant effect of osmotic stress on photosynthetic parameters, such as net photosynthetic rate, transpiration rate, stomatal conductance, stomatal resistance, internal CO₂ concentration, carboxylation efficiency, and transpiration efficiency on the tested genotypes at the tuberization stage. Lesser variations in photosynthesis and higher accumulation of proline, phenols, and antioxidative enzymes, namely, superoxide dismutase and guaiacol peroxidase, were associated with yield maintenance under osmotic stress conditions. The genotypes DP–89, IGCOL–4, and Ramhipur showed a higher degree of tolerance towards osmotic stress with a minimum variation in the studied parameters. These genotypes could be lines of interest for intensification of breeding strategies to develop drought-tolerant plants.     

Responses of in vitro-cultured <Emphasis Type="Italic">Allium hirtifolium</Emphasis> to exogenous sodium nitroprusside under PEG-imposed drought stress

Drought stress is a major threat to plant production in semi-arid and arid areas of the world. This research was laid out to asses the effects of sodium nitroprusside (SNP) as a nitric oxide donor on growth, physiological and biochemical changes of in vitro-cultured Allium hirtifolium under polyethylene glycol (PEG) induced drought stress. Basal plate explants of A. hirtifolium were cultured on MS medium containing different levels of PEG (0, 2, 4, 8 and 16 mM) and SNP (0, 10, 40 and 70 µM). After prolonged drought, growth responses, oxidative stress indicators, and phytochemical variations of regenerated plantlets with or without PEG and/or SNP treatments were recorded. Water limitation reduced regeneration potential of explants and consequently number of shoots per explant. Relative water content, total chlorophyll and carotenoid contents of regenerated A. hirtifolium plantlets decreased, but accumulation of malondialdehyde, H₂O₂ and proline and the activities of superoxide dismutase, ascorbate peroxidase, catalase and peroxidase enzymes increased with decreasing water availability. Total phenol and allicin contents were also increased in response to drought stress. Exogenous SNP in 10 and particularly in 40 µM was effective in enhancing regeneration rate and relative water content as well as protecting photosynthetic pigments under different levels of water availability. SNP also inhibited the hydrogen peroxide (H₂O₂) accumulation and lipid peroxidation in cell membranes via increasing the activities of superoxide dismutase and ascorbate peroxidase enzymes and accumulating proline and allicin. In general, these results suggest that exogenous SNP at 40 µM not only could somewhat protect A. hirtifolium from drought stress, but also can help to improve the propagation and allicin production of that plant under in vitro condition.