Adaptive responses of <Emphasis Type="Italic">Populus przewalskii</Emphasis> to drought stress and SNP application

In this study we used the cuttings of Populus przewalskii Maximowicz as experimental material and sodium nitroprusside (SNP) as nitric oxide (NO) donor to determine the physiological and biochemical responses to drought stress and the effect of NO on drought tolerance in woody plants. The results indicated that drought stress not only significantly decreased biomass production, but also significantly increased hydrogen peroxide content and caused oxidative stress to lipids and proteins assessed by the increase in malondialdehyde and total carbonyl contents, respectively. The cuttings of P. przewalskii accumulated many amino acids for osmotic adjustment to lower water potential, and activated the antioxidant enzymes such as superoxide dismutase, guaiacol peroxidase and ascorbate peroxidase to maintain the balance of generation and quenching of reactive oxygen species. Moreover, exogenous SNP application significantly heightened the growth performance of P. przewalskii cuttings under drought treatment by promotion of proline accumulation and activation of antioxidant enzyme activities, while under well-watered treatment the effect of SNP application was very little.     

The <Emphasis Type="Italic">ABI2</Emphasis>-dependent abscisic acid signalling controls HrpN-induced drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.Hong-Ping Dong and Haiqin Yu contributed equally to this study and are regarded as joint first authors.     

NaCl induced morpho-biochemical and anatomical changes in mulberry (<Emphasis Type="Italic">Morus</Emphasis> spp.)

Mulberry is an economically important tree, used for feeding the silkworm Bombyx mori L. Effect of different levels of NaCl on growth and development of mulberry has been studied using five mulberry genotypes selected on the basis of their performance under in vitro salinity. The study while endorsing the efficacy of in vitro screening of axillary buds of mulberry for salt tolerance, showed genotypic variability in its response to salinity. Salinity reduced growth and development of all genotypes. However, the putative tolerant genotypes showed better performance than the putative susceptible genotypes. Under low salinity (<0.5% NaCl) salt tolerant genotypes showed an increase in chlorophyll and protein concentrations, while in susceptible genotypes both were reduced by 3–58% at 0.5% NaCl and 50–64% at 1.00% NaCl. Leaf thickness increased by 16% at 1.00% NaCl in C776 and reduced by 1.0% in Mandalaya. The increase in chlorophyll concentration and leaf thickness under high salinity can be considered as preliminary selection parameters for salt tolerance in mulberry. The study confirmed the efficacy of in vitro method for screening of large number of genotypes for salt tolerance in mulberry.     

Functional roles of the pepper antimicrobial protein gene, <Emphasis Type="Italic">CaAMP1</Emphasis>, in abscisic acid signaling,and salt and drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Biotic signaling molecules including abscisic acid (ABA) are involved in signal transduction pathways that mediate the defense response of plants to environmental stresses. The antimicrobial protein gene CaAMP1, previously isolated from pepper (Capsicum annuum), was strongly induced in pepper leaves exposed to ABA, NaCl, drought, or low temperature. Because transformation is very difficult in pepper, we overexpressed CaAMP1 in Arabidopsis. CaAMP1-overexpressing (OX) transgenic plants exhibited reduced sensitivity to ABA during the seed germination and seedling stages. Overexpression of CaAMP1 conferred enhanced tolerance to high salinity and drought, accompanied by altered expression of the AtRD29A gene, which is correlated with ABA levels and environmental stresses. The transgenic plants were also highly tolerant to osmotic stress caused by high concentrations of mannitol. Together, these results suggest that overexpression of the CaAMP1 transgene modulates salt and drought tolerance in Arabidopsis through ABA-mediated cell signaling. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession number AY548741.     

Cryopreservation of white mulberry (<Emphasis Type="Italic">Morus alba</Emphasis> L.) by encapsulation-dehydration and vitrification

Shoot apices of in vitro-grown plantlets of white mulberry, Morus alba L. cv Florio, were cryopreserved using either encapsulation-dehydration or vitrification. For encapsulation-dehydration, alginate beads containing apices were dehydrated for 1, 3, 5 or 7 days in a liquid medium containing various sucrose concentrations (0.5, 0.75, 1.0 or 1.25 M). Bead desiccation was performed using silica gel for either 0, 4, 6, 8, 9 or 14 h. For vitrification, apices were directly immersed for either 5, 15, 30 or 60 min in a vitrification solution (PVS2). Following encapsulation-dehydration, treatment of alginate beads with 0.75 M sucrose was more effective in promoting re-growth of explants after immersion in liquid nitrogen than in the presence of 0.5 M sucrose for either 1 or 3 days. Re-growth of explants was also observed following vitrification and this reached 47% with increasing duration of the PVS2 treatment from 5 to 30 min. Overall, the highest frequency of explant re-growth was obtained when explants were subjected to encapsulation-dehydration in the presence of 0.75 M along with a 3 day sucrose dehydration pre-treatment and followed by desiccation for 9 h in silica gel.     

Seasonal changes in antioxidant activity,total phenolic and anthocyanin constituent of the stems of two <Emphasis Type="Italic">Morus</Emphasis> species (<Emphasis Type="Italic">Morus alba</Emphasis> L. and <Emphasis Type="Italic">Morus nigra</Emphasis> L.)

Seasonal changes of antioxidant activity, total phenolic and anthocyanin constituent of Morus nigra L. and Morus alba L. stems were investigated. Stems tissues of these species exhibited different antioxidant activity. Antioxidant action as well as anthocyanin constituents of these species reached the highest value in October whereas the lowest in February. Also, total phenolic constituents showed the similar pattern. An erratum to this article is available at .     

Differences in abscisic acid concentration in roots and leaves of two young Olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) cultivars in response to water deficit

Differences in abscisic acid (ABA) accumulation between two olive cultivars were studied by enzyme-linked immunosorbent assay in roots and leaves, leaf water potential (Ψ_l), stomatal conductance (g _s) as well as photosynthetic rate (A) were also determined in well-watered (WW) and water-stressed (WS) plants of two olive cultivars ‘Chemlali’ and ‘Chetoui’. ‘Chemlali’ was able to maintain higher leaf CO₂ assimilation rate and leaf stomatal conductance throughout the drought cycle when compared with ‘Chetoui’. Furthermore, leaf water potential of ‘Chemlali’ decreased in lower extent than in Chetoui in response to water deficit. Interestingly, significant differences in water-stress-induced ABA accumulation were observed between the two olive cultivars and reflect the degree of stress experienced. Chemlali, a drought tolerant cultivar, accumulated lower levels of ABA in their leaves to regulate stomatal control in response to water stress compared to the drought sensitive olive cultivar ‘Chetoui’ which accumulated ABA in large amount.     

Effects of salt stress in relation to osmotic adjustment on sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) callus cultures

In vitro responses of embryogenic sugarcane (Saccharum officinarum L.; cv. CoC-671) calli stressed with different levels of NaCl (0.0, 42.8, 85.6, 128.3, 171.1, 213.9 or 256.7 mM) were studied. The results showed that a significant decrease in callus growth and cell viability occurred with ≥85.6 mM NaCl. Higher amounts of free proline and glycine betaine were accumulated in NaCl-stressed calli. Although the leached and retained Na⁺ contents increased, the retained K⁺ content decreased with increasing levels of NaCl. Such a mechanism implies that sugarcane can be considered as a Na⁺-excluder. The accumulation of salt ions and osmolytes could play an important role in osmotic adjustment in sugarcane cells under salt stress.     

Non-enzymatic antioxidative defence in drought-stressed mulberry (<Emphasis Type="Italic">Morus indica</Emphasis> L.) genotypes

The present study investigated drought-induced responses of non-enzymatic antioxidants in four diverse mulberry genotypes (Morus indica L. S-36, M-5, MR-2 and V-1). Inside the glasshouse, potted plants were subjected to four water regimes for 75 days: (a) control: pots maintained at 100% pot water holding capacity (PC) (b) low water stress: 75% PC (c) medium water stress: 50% PC and (d) high water stress: 25% PC. Photosynthetic leaf gas exchange and non-enzymatic antioxidants including α-tocopherol, ascorbic acid (AA), glutathione, proline and total carotenoids were measured in leaves at regular intervals. Amongst all, V-1 was relatively drought tolerant and showed exceeded accumulation of α-tocopherol and AA-glutathione pool in association with higher carotenoids and proline contents. Susceptible S-36, M-5 and MR-2 could not induce any significant up-regulation in AA-glutathione pool leading to endogenous loss of α-tocopherol and more lipid peroxidation. Reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂) and superoxide (O₂ ^{· ?}) showed apparent accumulation in water-stressed leaves and significantly contributed to lipid peroxidation in susceptible genotypes when compared to V-1. Our study demonstrated that proline, AA and glutathione were the major non-enzymatic antioxidants in mulberry with α-tocopherol and carotenoids as good additional indicators for drought stress tolerance. These non-enzymatic antioxidants can cumulatively render effective protection against oxidative damage and can be considered as reliable markers for screening drought-tolerant mulberry genotypes.     

Overexpression of <Emphasis Type="Italic">TMAC2</Emphasis>, a novel negative regulator of abscisic acid and salinity responses,has pleiotropic effects in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Phytohormone abscisic acid (ABA) regulates many aspects of plant development and growth. To explore the molecular mechanism of ABA, we identified the novel ABA-regulated genes in Arabidopsis thaliana by searching for genes possessing two or more ABREs (ABA-responsive elements). One of these genes, two or more ABREs-containing gene 2 (TMAC2) is highly induced by ABA and NaCl. Database searches revealed that TMAC2 encodes a protein with no domains of known function. Expression of TMAC2-GFP fusion protein in Arabidopsis mesophyll protoplasts indicated that TMAC2 is targeted to the nucleus. Although the gene has a basal level of expression in various Arabidopsis organs/tissues except for adult leaves, a high expression level was detected in roots. Constitutive overexpression of TMAC2 in plants resulted in the insensitivity to ABA and NaCl, suggesting that TMAC2 plays a negative role in ABA and salt stress responses. Furthermore, TMAC2-overexpressing plants exhibited the short roots, late flowering and starch-excess phenotypes. RT-PCR analysis showed that decreased expression of two floral- and one starch degradation-related genes, SOC1/AGL20 and SEP3/AGL9, and SEX1, respectively, may lead to altered phenotypes of TMAC2-overexpressing plants. Taken together, our data reveal that TMAC2 acts in the nucleus and is an important negative regulator of ABA and salt stress responses, and could play a critical role in controlling root elongation, floral initiation and starch degradation. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Differential responses of lipid peroxidation and antioxidants in <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis> subjected to drought stress

To evaluate oxidative stress and the plant antioxidant system of Alternanthera philoxeroides [Mart.] Griseb and Oryza sativa L. in the response to drought, root and leaf tissues of drought-treated A. philoxeroides and O. sativa were collected and relative water content, stomatal conductance, the concentrations of malondialdehyde, proline and the activities of superoxide dismutase, peroxidases, catalase and total antioxidative activity investigated. The results showed that drought treatment had almost no effect on relative water content in A. philoxeroides but reduced relative water content in O. sativa. A. philoxeroides maintained a greater stomatal conductance than O. sativa under drought stress. In A. philoxeroides levels of lipid peroxidation were lower than in O. sativa and did not change during the experiment. After exposure to drought, concentrations of proline and activities of superoxide dismutase, peroxidases and catalase in A. philoxeroides were between 10% and 30% higher than in O. sativa, whereas total antioxidative activity in A. philoxeroides was several-fold higher than in O. sativa.     

Biochemical changes during seed development in <Emphasis Type="Italic">Pinus taeda</Emphasis> L.

Amino acids, polyamines, 3-indoleacetic acid (IAA), abscisic acid (ABA), buffer-soluble protein and starch contents and dry matter accumulation were analyzed in megagametophytes containing developing embryos during seed development in Pinus taeda. The highest total amino acids and polyamine contents occurred at the cotyledonary stage, followed by a significant decrease in the mature seed. Free polyamines exhibited higher levels than conjugated ones, with putrescine being the predominant type until the cotyledonary stage, and spermidine at the mature seed stage. IAA content increased continually from the globular stage reaching the maximum at the cotyledonary stage, followed by a decrease in the mature seed. The highest ABA level occurred at the globular stage, followed by a continuous reduction until stabilization at the pre-cotyledonary stage. Buffer-soluble protein and starch contents, and dry matter increased progressively during development, reaching their maximum values at the mature stage.     

Abscisic Acid Response of Corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) Roots and Protoplasts to Lanthanum

Lanthanum ions antagonize calcium and are used as a Ca²⁺ channel blocker but their direct effects are unknown. We investigated lanthanum effects on endogenous abscisic acid (ABA) levels in protoplasts and intact primary roots of Zea mays L. Application of 1 mM La³⁺ reduced primary root elongation, caused swelling of root tips, and essentially doubled the ABA content in intact roots but decreased ABA in root protoplasts in a concentration-dependent manner. Osmotic stress increased ABA level in protoplasts more than in intact roots. Temporal ABA changes in response to La³⁺ treatment indicate that La³⁺ affects root growth at least partially via ABA pathway.     

In vitro screening of mulberry (<Emphasis Type="Italic">Morus</Emphasis> spp.) for salinity tolerance

An efficient in vitro screening method has been developed for mulberry (Morus spp. ) to screen salinity-tolerant genotypes from a large population. Axillary buds from field-grown plants were cultured on MS medium containing five different concentrations (0.0%, 0.25%, 0.5%, 0.75% and 1.00%) of sodium chloride (NaCl) in order to study the shoot growth pattern. Rooting was also tested at four different concentrations of NaCl (0.0%, 0.1%, 0.2% and 0.3.%). NaCl has been found to inhibit the growth and development of mulberry shoots and roots in vitro. The survivability of the axillary buds of the genotypes tested was reduced from 83.7% for the controls to 6.1% in 1.0% NaCl. The average number of roots developed by the genotypes ranged from 11.9 (controls) to 0.2 (0.3% NaCl). Out of the 63 genotypes tested, only seven—Rotundiloba, English black, Kolitha-3, Berhampore-A, Kajli, BC₂59 and C776—developed roots in 0.3% NaCl. Root growth was also reduced drastically from 1.8 cm for the controls to 0.1 cm in 0.3% NaCl. To test the reproducibility of the results in soil, five tolerant and two susceptible genotypes, identified in this in vitro study, were selected and tested under ex vitro conditions. The significant correlation coefficients obtained between the performances of these genotypes under both types of cultural conditions revealed that in vitro screening of mulberry through axillary bud culture is an easy and efficient method to identify salt-adapted genotypes within a limited space and time period.Abbreviations BA 6-Benzylaminopurine - dS m^-1 Deci-Siemens per meter - EC Electric conductivity - NAA -Naphthaleneacetic acidCommunicated by G.C. Phillips     

Endophytic bacteria in sunflower (<Emphasis Type="Italic">Helianthus annuus </Emphasis>L.): isolation,characterization, and production of jasmonates and abscisic acid in culture medium

This study was designed to isolate and characterize endophytic bacteria from sunflower (Helianthus annuus) grown under irrigation and water stress (drought) conditions, to analyze growth of isolated bacteria under drought condition, and to evaluate the ability of bacteria isolated from plants cultivated under drought to produce jasmonates (JAs) and abscisic acid (ABA). Bacteria were isolated from soil samples collected when sunflower plants were at the end of the vegetative stage. A total of 29 endophytic strains were isolated from plants grown under irrigation or drought condition. Eight strains (termed SF1 through SF8) were selected based on nitrogen-fixing ability. All eight strains showed positive catalase and oxidase activities; five strains (SF2, SF3, SF4, SF5, SF7) solubilized phosphates; none of the strains produced siderophores. Strains SF2, SF3, SF4, and SF5, the ones with the highest phosphate solubilization ability, strongly inhibited growth of the pathogenic fungi Verticillum orense and Sclerotinia sclerotiorum but had less inhibitory effect on Alternaria sp. Among the eight strains, SF2 showed 99.9% sequence homology with Achromobacter xiloxidans or Alcaligenes sp., while the other seven showed 99.9% homology with Bacillus pumilus. Strains SF2, SF3, and SF4 grown in control medium produced jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), and ABA. These three strains did not differ in amount of JA or OPDA produced. ABA content was higher than that of JA, and production of both ABA and JA increased under drought condition. The characteristics of these isolated bacterial strains have technological implications for inoculant formulation and improved growth of sunflower crops.     

Differential plant growth and osmotic effects of two maize (<Emphasis Type="Italic">Zea mays</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) cultivars to exogenous glycinebetaine application under drought stress

We investigated the effects of exogenous glycinebetaine (GB) and drought stress (DS) on grain yield (GY) and production of dry matter (DM) and osmolytes in two maize (Zea mays L.) cultivars i.e. Shaandan 9 (S₉) and Shaandan 911 (S₉₁₁) during the entire growing period. Drought stress substantially reduced DM and GY but increased free proline, endogenous GB, soluble sugar and K⁺ concentrations in leaves of both cultivars. The DM production, GY, drought index (DI) and concentrations of these osmolytes were greater for S₉ than those for S₉₁₁ under DS. The significant differences in these parameters suggested that S₉ was more drought-tolerant as compared to S₉₁₁. Additionally, foliar application of GB increased the concentrations of all osmolytes measured, DM and GY of both cultivars under DS. These positive responses of exogenous GB spray were more pronounced in S₉₁₁ as compared to those in S₉. Further correlation analysis involving a number of parameters indicated that maize production was tighterly correlated with accumulation of the osmolytes measured during DS rather than well-watered controls. Accordingly, this study demonstrated the notion of an anti-drought role of exogenous GB by osmoregulation under DS, particularly in this drought sensitive cultivar. Thus, exogenous GB application might be firstly used with drought sensitive species/cultivars when exposed to DS.