Overexpression of <Emphasis Type="Italic">Thellungiella halophila</Emphasis> H<Superscript>+</Superscript>-PPase (<Emphasis Type="Italic">TsVP</Emphasis>) in cotton enhances drought stress resistance of plants

An H⁺-PPase gene, TsVP from Thellungiella halophila, was transferred into two cotton (Gossypium hirsutum) varieties (Lumianyan19 and Lumianyan 21) and southern and northern blotting analysis showed the foreign gene was integrated into the cotton genome and expressed. The measurement of isolated vacuolar membrane vesicles demonstrated that the transgenic plants had higher V–H⁺-PPase activity compared with wild-type plants (WT). Overexpressing TsVP in cotton improved shoot and root growth, and transgenic plants were much more resistant to osmotic/drought stress than the WT. Under drought stress conditions, transgenic plants had higher chlorophyll content, improved photosynthesis, higher relative water content of leaves and less cell membrane damage than WT. We ascribe these properties to improved root development and the lower solute potential resulting from higher solute content such as soluble sugars and free amino acids in the transgenic plants. In this study, the average seed cotton yields of transgenic plants from Lumianyan 19 and Lumianyan 21 were significantly increased compared with those of WT after exposing to drought stress for 21 days at flowering stage. The average seed cotton yields were 51 and 40% higher than in their WT counterparts, respectively. This study benefits efforts to improve cotton yields in arid and semiarid regions.     

Overexpression of <Emphasis Type="Italic">OsVP1</Emphasis> and <Emphasis Type="Italic">OsNHX1</Emphasis> Increases Tolerance to Drought and Salinity in Rice

Drought and salinity are major abiotic stresses affecting rice production. To improve plant tolerance to salinity and drought, we overexpressed rice Na⁺/H⁺ exchangers (OsNHX1) and H⁺-pyrophosphatase in tonoplasts (OsVP1) in a japonica elite rice cultivar, Zhonghua 11. Compared with our wild-type control, transgenic plants overexpressing both genes incurred less damage when exposed to long-term treatment with 100 mM NaCl or water deprivation. Under high-saline conditions, the transformants accumulated less Na⁺ and malondialdehyde in the leaves, thereby allowing the plants to maintain a low level of leaf water potential and reduce stress-induced damage. Those transgenics also had higher photosynthetic activity during the stress period. Under those conditions, they also showed an increase in root biomass, which enabled more water uptake. These results suggest that OsVP1 and OsNHX1 improve the tolerance of rice crops against drought and salt by employing multiple strategies in addition to osmotic regulation.     

Cloning and Functional Characterization of a Novel Glutathione <Emphasis Type="Italic">S</Emphasis>-Transferase Gene from <Emphasis Type="Italic">Limonium bicolor</Emphasis>

Plant glutathione S-transferases (GSTs) are involved in protecting plants against both diverse biotic and abiotic stresses. In the present study, a novel GST gene (LbGST1) was cloned from Limonium bicolor (Bunge) Kuntze (Plumbaginaceae). To characterize its function in salt tolerance, tobacco lines transformed with LbGST1 were generated. Compared with wild-type (WT) tobacco, transgenic plants overexpressing LbGST1 exhibited both GST and glutathione peroxidase activities. Moreover, superoxide dismutase, peroxidase (POD), and catalase activities in transgenic plants were significantly higher than those in WT plants, particularly when grown under conditions of salt stress. Similarly, levels of proline in transgenic plants were also higher than those in WT plants grown under NaCl stress conditions. Whereas, Malondialdehyde contents in transgenic plants were lower than those in WT plants under NaCl conditions. Furthermore, Na⁺ content in transgenic plants was lower than that in WT plants under these stress conditions. Subcellular localization analysis revealed that the LbGST1 protein was localized in the nucleus. These results suggested that overexpression of LbGST1 gene can affect many physiological processes associated with plant salt tolerance. Therefore, we hypothesize that LbGST1 gene can mediate many physiological pathways that enhance stress resistance in plants.     

Photosynthetic activity of poikilochlorophyllous desiccation tolerant plant <Emphasis Type="Italic">Reaumuria soongorica</Emphasis> during dehydration and re-hydration

Diurnal patterns of gas exchange and chlorophyll (Chl) fluorescence parameters of photosystem 2 (PS2) as well as Chl content were analyzed in Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub during dehydration and rehydration. The net photosynthetic rate (P _N), maximum photochemical efficiency of PS2 (variable to maximum fluorescence ratio, F_v/F_m), quantum efficiency of non-cyclic electron transport of PS2, and Chl content decreased, but non-photochemical quenching of fluorescence and carotenoid content increased in stems with the increasing of drought stress. 6 d after re-hydration, new leaves budded from stems. In the re-watered plants, the chloroplast function was restored and Chl a fluorescence returned to a similar level as in the control plants. This improved hydraulic adjustment in plant triggered a positive effect on ion flow in the tissues and increased shoot electrical admittance. Thus R. soongorica plants are able to sustain drought stress through leaf abscission and keep part of Chl content in stems.     

Photosynthetic characteristics of <Emphasis Type="Italic">Hordeum,Triticum, Rumex</Emphasis>, and <Emphasis Type="Italic">Trifolium</Emphasis> species at contrasting altitudes

Photosynthetic characteristics were compared between plants of low altitude (LA) grown at LA (Palampur; 1 300 m) and at high altitude, HA (Kibber; 4 200 m), and plants naturally occurring at different altitudes (Palampur, 1 300 m; Palchan, 2 250 m; and Marhi, 3 250 m). Net photosynthetic rate (P _N) was not significantly different between altitudes. However, the slopes of the curve relating P _N to intercellular CO₂ concentration (C _i) were higher in plants at Palchan, Marhi, and Kibber compared to those at Palampur, indicating that plants had higher efficiency of carbon uptake (the initial slope of P _N/C _i curve is an indication) at HA. They had also higher stomatal conductance (g _s), transpiration rate, and lower water use efficiency at HA. g _s was insensitive to photosynthetic photon flux density (PPFD) for plants naturally occurring at Palampur, Palchan, and Marhi, whereas plants from LA grown at Palampur and Kibber responded linearly to increasing PPFD. Insensitivity of g _s to PPFD could be one of the adaptive features allowing wider altitudinal distribution of the plants.This research is supported by the Department of Biotechnology (DBT), Government of India vide grant number BT/PR/502/AGR/08/39/966-VI.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the medicinal plant <Emphasis Type="Italic">Centaurea montana</Emphasis>

An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter. A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including PCR, Southern blotting and RT-PCR, were performed on T₀ and T₁ plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants. Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay.     

Hydrogen peroxide-induced salt tolerance in the <Emphasis Type="Italic">Arabidopsis</Emphasis> salicylate-deficient transformants <Emphasis Type="Italic">NahG</Emphasis>

The effect of hydrogen peroxide treatment on the salt tolerance of wild-type Arabidopsis thaliana L. plants (Col-0) and plants transformed with the bacterial salicylate hydroxylase gene (NahG) was studied. The base tolerance to salt stress caused by 200 mM of NaCl in solution culture was higher in plants with the NahG genotype in comparison with the wild-type plants. Growth inhibition was observed for wild-type plants under the action of exogenous hydrogen peroxide, which was not observed for the NahG transformants; salt tolerance increased in the both types of plants after treatment, which was assessed based on the growth indicators and the ability to preserve the chlorophyll pool following NaCl treatment. The content of endogenous Н2О2 in the leaves of wild-type plants increased significantly following exogenous hydrogen peroxide treatment and salt stress, while it practically did not change in the leaves of the NahG genotype. The SOD activity increased in both genotypes after treatment with exogenous hydrogen peroxide, and remained at an elevated level after salt stress in comparison with the nontreated plants. Furthermore, the catalase activity increased in leaves of the salicylate-deficient genotype but not in the Col-0 genotype. The guaiacol peroxidase activity increased in plants of both genotypes under the action of hydrogen peroxide and salt stress, with the NahG plants demonstrating a higher degree of increase. The Н₂О₂ treatment facilitated the increase of the proline content in leaves of the plants of both genotypes under conditions of salt stress. It was concluded that there were hydrogen peroxide signal transduction pathways in Arabidopsis plants that were salicylic acid independent and that the antioxidant system functioned more effectively in salicylate-deficient Arabidopsis plants.     

An <Emphasis Type="Italic">Arabidopsis</Emphasis> senescence-associated protein SAG29 regulates cell viability under high salinity

The plasma membrane is an important cellular organ that perceives incoming developmental and environmental signals and integrates these signals into cellular regulatory mechanisms. It also acts as a barrier against unfavorable extracellular factors to maintain cell viability. Despite its importance for cell viability, molecular components determining cell viability and underlying mechanisms are largely unknown. Here, we show that a plasma membrane-localized MtN3 protein SAG29 regulates cell viability under high salinity in Arabidopsis. The SAG29 gene is expressed primarily in senescing plant tissues. It is induced by osmotic stresses via an abscisic acid-dependent pathway. Whereas the SAG29-overexpressing transgenic plants (35S:SAG29) exhibited an accelerated senescence and were hypersensitive to salt stress, the SAG29-deficient mutants were less sensitive to high salinity. Consistent with this, the 35S:SAG29 transgenic plants showed reduced cell viability in the roots under normal growth condition. In contrast, cell viability in the SAG29-deficient mutant roots was indistinguishable from that in the roots of control plants. Notably, the mutant roots exhibited enhanced cell viability under high salinity. Our observations indicate that the senescence-associated SAG29 protein is associated with cell viability under high salinity and other osmotic stress conditions. We propose that the SAG29 protein may serve as a molecular link that integrates environmental stress responses into senescing process.     

Salicylic acid and salicylic acid glucoside in xylem sap of <Emphasis Type="Italic">Brassica napus</Emphasis> infected with <Emphasis Type="Italic">Verticillium longisporum</Emphasis>

Salicylic acid (SA) and its glucoside (SAG) were detected in xylem sap of Brassica napus by HPLC–MS. Concentrations of SA and SAG in xylem sap from the root and hypocotyl of the plant, and in extracts of shoots above the hypocotyl, increased after infection with the vascular pathogen Verticillium longisporum. Both concentrations were correlated with disease severity assessed as the reduction in shoot length. Furthermore, SAG levels in shoot extracts were correlated with the amount of V. longisporum DNA in the hypocotyls. Although the concentration of SAG (but not SA) in xylem sap of infected plants gradually declined from 14 to 35 days post infection, SAG levels remained significantly higher than in uninfected plants during the whole experiment. Jasmonic acid (JA) and abscisic acid (ABA) levels in xylem sap were not affected by infection with V. longisporum. SA and SAG extend the list of phytohormones potentially transported from root to shoot with the transpiration stream. The physiological relevance of this transport and its contribution to the distribution of SA in plants remain to be elucidated.     

Responses of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> to high irradiance

The effect of high irradiance (HI) during desiccation and subsequent rehydration of the homoiochlorophyllous desiccation-tolerant shade plant Haberlea rhodopensis was investigated. Plants were irradiated with a high quantum fluence rate (HI; 350 μmol m⁻² s⁻¹ compared to ca. 30 μmol m⁻² s⁻¹ at the natural rock habitat below trees) and subjected either to fast desiccation (tufts dehydrated with naturally occurring thin soil layers) or slow desiccation (tufts planted in pots in peat-soil dehydrated by withholding irrigation). Leaf water content was 5 % of the control after 4 d of fast and 19 d of slow desiccation. Haberlea was very sensitive to HI under all conditions. After 19 d at HI, even in well-watered plants there was a strong reduction of rates of net photosynthesis and transpiration, contents of chlorophyll (Chl) and carotenoids, as well as photosystem 2 activity (detected by the Chl fluorescence ratio R_Fd). Simultaneously, the blue/red and green/red fluorescence ratios increased considerably suggesting increased synthesis of polyphenolic compounds. Desiccation of plants in HI induced irreversible changes in the photosynthetic apparatus and leaves did not recover after rehydration regardless of fast or slow desiccation. Only young leaves survived desiccation.     

Photosynthetic adjustment after rehydration in <Emphasis Type="Italic">Annona emarginata</Emphasis>

Reports indicate that Annona emarginata is tolerant to drought and is also used as an alternative rootstock for atemoya under drought conditions. The photosynthetic process can be adjusted after rehydration, resulting on total or partial recovery. The aim of this study was to determine if A. emarginata shows adjusts in gas exchange and the chlorophyll a fluorescence pattern after rehydration. During water deficits, the gas exchange and water content in the leaf decreased. However, after 5 days of rehydration, the water content in the leaf recovered and rehydrated plants presented the water use efficiency better than irrigated plants. Further remaining gas exchange parameters were lower in relation to irrigated plants. In chlorophyll a fluorescence, the rehydrated plants showed higher dissipation of light energy as heat, maintaining high activity of photoprotection. After rehydration, A. emarginata shows a positive correlation between transpiration and CO₂ assimilation rate, which optimize the water use efficiency. Thus, A. emarginata presents adjustments in gas exchange and photochemical process, resulting on a possible long-term photosynthetic acclimation to water deficiency.     

Injured and uninjured leaf photosynthetic responses after mechanical injury on <Emphasis Type="Italic">Nerium oleander</Emphasis> leaves,and <Emphasis Type="Italic">Danaus plexippus</Emphasis> herbivory on <Emphasis Type="Italic">Asclepias curassavica</Emphasis> leaves

Insect herbivory has variable effects on plant physiology; so greater understanding is needed about how injury alters photosynthesis on individual injured and uninjured leaves. Gas exchange and light-adapted leaf chlorophyll fluorescence measurements were collected from uninjured and mechanical partial leaf defoliation in two experiments with Nerium oleander (Apocynaceae) leaves, and one experiment with Danaus plexippus herbivory on Asclepias curassavica (Asclepiadaceae) leaves. Gas exchange impairment (lower photosynthetic rate (P _n ), stomatal conductance (g _s)) indicates water stress in a leaf, suggests stomatal limitations causing injury P _n impairment. The same pattern of gas exchange impairment also occurred on uninjured leaves opposite from injured leaves in both N. oleander experiments. This is an interesting result because photosynthetic impairment is rarely reported on injured leaves near injured leaves. No photosynthetic changes occurred in uninjured A. curassavica leaves opposite from D. plexippus-fed leaves. Partially defoliated leaves that had P _n and g _s reductions lacked any significant changes in intercellular leaf [CO₂], C _i. These results neither support, nor are sufficient to reject, stomatal limitations to photosynthesis. Manually imposed midrib vein severance in N. oleander experiment #1 significantly increased leaf C _i, indicating mesophyll limitations to photosynthesis. Maximal light-adapted leaf photochemical efficiency () and also non-photochemical quenching (q _N) were reduced by mechanical or insect herbivory to both study species, suggesting leaf trouble handling excess light energy not used for photochemistry. Midrib injury on N. oleander leaves and D. plexippus herbivory on A. curassavica leaves also reduced effective quantum yield (ΦPSII) and photochemical quenching (q _P); so reduced plastoquinone pools could lead to additional PSII reaction center closure.     

Photosynthetic activity of homoiochlorophyllous desiccation tolerant plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> during dehydration and rehydration

The functional state of the photosynthetic apparatus of flowering homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and subsequent rehydration was investigated in order to characterize some of the mechanisms by which resurrection plants survive drought stress. The changes in the CO₂ assimilation rate, chlorophyll fluorescence parameters, thermoluminescence, fluorescence imaging and electrophoretic characteristics of the chloroplast proteins were measured in control, moderately dehydrated (50% water content), desiccated (5% water content) and rehydrated plants. During the first phase of desiccation the net CO₂ assimilation decline was influenced by stomatal closure. Further lowering of net CO₂ assimilation was caused by both the decrease in stomatal conductance and in the photochemical activity of photosystem II. Severe dehydration caused inhibition of quantum yield of PSII electron transport, disappearance of thermoluminescence B band and mainly charge recombination related to S₂Q_A⁻ takes place. The blue and green fluorescence emission in desiccated leaves strongly increased. It could be suggested that unchanged chlorophyll content and amounts of chlorophyll–proteins, reversible modifications in PSII electron transport and enhanced probability for non-radiative energy dissipation as well as increased polyphenolic synthesis during desiccation of Haberlea contribute to drought resistance and fast recovery after rehydration.     

Rose-scented geranium (<Emphasis Type="Italic">Pelargonium</Emphasis> sp.) generated by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A₄ and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A₄ strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A₄ and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A₄ and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.     

Tolerance to soil water stress by <Emphasis Type="Italic">Oryza sativa</Emphasis> cv. IR20 was improved by expression of <Emphasis Type="Italic">Wsi18</Emphasis> gene locus from <Emphasis Type="Italic">Oryza nivara</Emphasis>

Wild rice genotypes are rich in genetic diversity. This has potential to improve agronomic rice by allele mining for superior traits. Late embryogenesis abundant (LEA) proteins are often associated with desiccation tolerance and stress signalling. In the present study, a group 3 LEA gene, Wsi18 from the wild rice Oryza nivara was expressed under its own inducible promoter element in stress susceptible cultivated indica rice (cv. IR20). The resulting transgenic plants cultivated in a greenhouse showed enhanced tolerance to soil water deficit. Transgenic plants had higher grain yield, plant survival rate, and shoot relative water content compared to wild type (WT) IR20. Cell membrane stability index, proline and soluble sugar content were also greater in transgenic than WT plants under water stress. These results demonstrate the potential for improving SWS tolerance in agronomically important rice cultivar by incorporating Wsi18 gene from a wild rice O. nivara.