Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles,water stress and abscisic acid

Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.     

Heat shock protein 70 regulates the abscisic acid-induced antioxidant response of maize to combined drought and heat stress

We investigated the interaction between heat shock protein 70 (HSP70) and abscisic acid (ABA)-induced antioxidant response of maize to the combination of drought and heat stress. First, the increased activities of enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT), induced by drought were less than those by heat or combined drought and heat stress, except some individual cases (e.g. CAT in leaves, GR in roots). Second, both HSP70 synthesis and H₂O₂ production increased prominently under drought, heat or their combination stress; the increase in leaves induced by drought and heat combination was the highest, followed by heat and by drought, while the increase in roots had not visible difference. Third, either in leaves or roots, pretreatment with ABA inhibitor, HSP70 inhibitor and H₂O₂ scavenger, significantly arrested the stress-induced increase of antioxidant enzyme activities, and ABA inhibitor and H₂O₂ scavenger obviously suppressed HSP70 synthesis, while HSP70 inhibitor slightly heightened H₂O₂ accumulation. Finally, 100 μM ABA significantly enhanced the activities of antioxidant enzymes, HSP70 expression and H₂O₂ production under stresses in comparison with ABA-deficient mutant vp5 maize plants without pretreatment. Thus, ABA-induced H₂O₂ production enhances the HSP70 synthesis and up-regulates the activities of antioxidant enzymes, resulting in the suppression of cellular reactive oxygen species (ROS) levels. Our results suggest that HSP70 may play a crucial role in ABA-induced antioxidant defense of maize to drought and heat combination.     

Leaf δ18O of remaining trees is affected by thinning intensity in a semiarid pine forest

Silvicultural thinning usually improves the water status of remaining trees in water‐limited forests. We evaluated the usefulness of a dual stable isotope approach (δ¹³C, δ¹⁸O) for comparing the physiological performance of remaining trees between forest stands subjected to two different thinning intensities (moderate versus heavy) in a 60‐year‐old Pinus halepensis Mill. plantation in semiarid southeastern Spain. We measured bulk leaf δ¹³C and δ¹⁸O, foliar elemental concentrations, stem water content, stem water δ¹⁸O (δ¹⁸O_{stem water}), tree ring widths and leaf gas exchange rates to assess the influence of forest stand density on tree performance. Remaining trees in low‐density stands (heavily thinned) showed lower leaf δ¹⁸O, and higher stomatal conductance (g_s), photosynthetic rate and radial growth than those in moderate‐density stands (moderately thinned). By contrast, leaf δ¹³C, intrinsic water‐use efficiency, foliar elemental concentrations and δ¹⁸O_{stem water} were unaffected by stand density. Lower foliar δ¹⁸O in heavily thinned stands reflected higher g_s of remaining trees due to decreased inter‐tree competition for water, whereas higher photosynthetic rate was largely attributable to reduced stomatal limitation to CO₂ uptake. The dual isotope approach provided insight into the early (12 months) effects of stand density manipulation on the physiological performance of remaining trees.     

Cloning of a 9-<Emphasis Type="Italic">cis</Emphasis>-epoxycarotenoid dioxygenase gene (<Emphasis Type="Italic">SgNCED1</Emphasis>) from <Emphasis Type="Italic">Stylosanthes guianensis </Emphasis>and its expression in response to abiotic stresses

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. Oxidative cleavage of cis-epoxycarotenoids catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is the main regulatory step in the biosynthesis of ABA in higher plants. A NCED gene, SgNCED1, was cloned from the dehydrated leaves of Stylosanthes guianensis. The 2,241-bp full-length SgNCED1 had a 1,809-bp ORF, which encodes a peptide of 602 amino acids. The deduced amino acid sequence of SgNCED1 protein shared high identity with other NCEDs. At the N-terminus of the SgNCED1 located a chloroplast transit peptide sequence. DNA blot analysis revealed that SgNCED1 was a single copy gene in the genome of S. guianensis. The relationship between expression of SgNCED1 and endogenous ABA level was investigated. The expression of SgNCED1 was induced in both leaves and roots of S. guianensis under drought stress. Dehydration and salt stress induced the expression of SgNCED1 strongly and rapidly. The ABA accumulation was coincidently induced with the SgNCED1 mRNA under drought, dehydration and salt stress. The expression of SgNCED1 and ABA accumulation were also induced under chilling condition.     

Habitat‐specific differences in plasticity of foliar δ13C in temperate steppe grasses

A decrease in foliar δ¹³C with increasing precipitation is a common tendency in steppe plants. However, the rate of decrease has been reported to differ between different species or populations. We here hypothesized that plant populations in the same habitat of temperate steppes may not differ in foliar δ¹³C response patterns to precipitation, but could differ in the levels of plasticity of foliar δ¹³C across different habitats. In order to test this hypothesis, we conducted controlled watering experiments in northeast China at five sites along a west–east transect at latitude 44°N, which show substantial interannual fluctuations and intra‐annual changes in precipitation among them. In 2001, watering treatment (six levels, three replicates) was assigned to 18 plots at each site. The responses of foliar δ¹³C to precipitation (i.e., the sum of watering and rainfall) were determined in populations of several grass species that were common across all sites. Although similar linear regression slopes were observed for populations of different species growing at the same site, significantly different slopes were obtained for populations of the same species growing at different sites. Further, the slope of the line progressively decreased from Site I to Site V for all species in this study. These results suggest habitat‐specific differences in plasticity of foliar δ¹³C in temperate steppe grasses. This indicates that species' δ¹³C response to precipitation is conservative at the same site due to their long‐term acclimation, but the mechanism responsible behind this needs further investigations.     

Stomatal responses to vapour pressure deficit are regulated by high speed gene expression in angiosperms

Plants dynamically regulate water use by the movement of stomata on the surface of leaves. Stomatal responses to changes in vapour pressure deficit (VPD) are the principal regulator of daytime transpiration and water use efficiency in land plants. In angiosperms, stomatal responses to VPD appear to be regulated by the phytohormone abscisic acid (ABA), yet the origin of this ABA is controversial. After a 20 min exposure of plants, from three diverse angiosperm species, to a doubling in VPD, stomata closed, foliar ABA levels increased and the expression of the gene encoding the key, rate‐limiting carotenoid cleavage enzyme (9‐cis‐epoxycarotenoid dioxygenase, NCED) in the ABA biosynthetic pathway was significantly up‐regulated. The NCED gene was the only gene in the ABA biosynthetic pathway to be up‐regulated over the short time scale corresponding to the response of stomata. The closure of stomata and rapid increase in foliar ABA levels could not be explained by the release of ABA from internal stores in the leaf or the hydrolysis of the conjugate ABA‐glucose ester. These results implicate an extremely rapid de novo biosynthesis of ABA, mediated by a single gene, as the means by which angiosperm stomata respond to natural changes in VPD.     

PeCHYR1, a ubiquitin E3 ligase from Populus euphratica,enhances drought tolerance via ABA‐induced stomatal closure by ROS production in Populus

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H₂O₂ production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild‐type) controls. Moreover, up‐regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA‐induced stomatal closure caused by hydrogen peroxide (H₂O₂) production in transgenic poplar plants.     

PdEPF1 regulates water‐use efficiency and drought tolerance by modulating stomatal density in poplar

Water deficiency is a critical environmental condition that is seriously reducing global plant production. Improved water‐use efficiency (WUE) and drought tolerance are effective strategies to address this problem. In this study, PdEPF1, a member of the EPIDERMAL PATTERNING FACTOR (EPF) family, was isolated from the fast‐growing poplar clone NE‐19 [Populus nigra × (Populus deltoides × Populus nigra)]. Significantly, higher PdEPF1 levels were detected after induction by dehydration and abscisic acid. To explore the biological functions of PdEPF1, transgenic triploid white poplars (Populus tomentosa ‘YiXianCiZhu B385’) overexpressing PdEPF1 were constructed. PdEPF1 overexpression resulted in increased water deficit tolerance and greater WUE. We confirmed that the transgenic lines with greater instantaneous WUE had approximately 30% lower transpiration but equivalent CO₂ assimilation. Lower transpiration was associated with a 28% reduction in abaxial stomatal density. PdEPF1 overexpression not only strongly enhanced WUE, but also greatly improved drought tolerance, as measured by the leaf relative water content and water potential, under limited water conditions. In addition, the growth of these oxPdEPF1 plants was less adversely affected by reduced water availability than plants with a higher stomatal density, indicating that plants with a low stomatal density may be well suited to grow in water‐scarce environments. Taken together, our data suggest that PdEPF1 improves WUE and confers drought tolerance in poplar; thus, it could be used to breed drought‐tolerant plants with increased production under conditions of water deficiency.     

Enhanced drought and salt tolerance by expression of AtGSK1 gene in poplar

A GSK3/shaggy-like kinase (AtGSK1) has been implicated in the regulation of drought and salt tolerance. We transferred AtGSK1 from Arabidopsis thaliana to a hybrid poplar (Populus alba × P. tremula var. grandulosa) to determine the effect of the transgene expression in the transgenic trees. The results from northern blot and RT-PCR analyses showed that the expression level varied among the transgenic lines. During their culture on tissue culture media, the transgenic poplars formed vigorous growing roots even in the presence of 125 mM NaCl and callus in the presence of 150 mM NaCl. When the transgenic poplars were growing in pots and provided with NaCl solution, they stayed much healthier than did nontransgenic poplars, showing higher rates of photosynthetic rates, stomatal conductance, and evaporation rates under the stress. Whereas the total level of leaf Na⁺ level increased dramatically in transgenic poplars under severe saline conditions (150 mM NaCl), that of leaf K⁺ decreased in the same plants under the same conditions. Total root Na⁺ level increased in nontransgenic poplars under severe saline conditions. In contrast, total root K⁺ level decreased in the same plants under the same conditions. The chloride content and relative electrical conductivity of the transgenic poplars after salt stress treatment were lower than those of nontransgenic poplars. The transgenic poplars were also tolerant to up to 20 % PEG remaining significantly healthy when compared with nontransgenic poplars with necrosis and chlorosis symptoms. Another dramatic feature of the transgenic poplars was wilting tolerance for prolonged drought treatment up to 2 weeks. The results provide evidence that the expression of AtGSK1 gene conferred drought and salt tolerance in the transgenic poplars.     

Genotypic variation in 'early warning signals' from roots in drying soil: intrinsic differences in ABA synthesising capacity rather than root density determines total ABA 'message' in cowpea (Vigna unguiculata L.)

In a comparison of six cowpea cultivars, we determined the variation in abscisic acid (ABA) production as an ‘early warning signal’ produced in response to drought stress. By imposing drought only to the upper 20 cm rooting zone, we compared the rates of ABA synthesis relative to (i) total root mass and (ii) inherent variation per unit root mass. We were able to relate the intensity of the stress response to these two factors, and determine which is quantitatively more important as the primary signal indicating responsiveness to drought stress. Plants were grown in 1.2 m long columns and a soil drying treatment imposed in such a way that that upper roots were in dry soil and deep roots in soil at field capacity. Relative water contents (RWC) of stressed plants were similar and not significantly different from those of well watered controls. However, roots accumulated ABA in the dehydrated zone, where root water content ranged from 10–12 g g^?1 DW. The soil moisture contents and root ‐water contents in the dry zone were similar for each of the different varieties. However, the ABA contents were significantly different in drought‐stressed (upper) roots and ranged from 7.82 nmol g^?1 DW in cv. APC 689 to 16.02 nmol g^?1 DW in cv. APC 370, such that for varieties with similar overall root weights (e.g. APC 580 and APC 540) the different ABA contents were related to the capacity for ABA synthesis. The relationship between stomatal conductance and total root ABA was assessed, with a negative relation (r= 0.90, n= 24, P= 0.05) suggesting that the intrinsic capacity of cowpea varieties for ABA synthesis could play an important role in regulating stomatal conductance in a drying soil and provide useful selection criteria for tolerance to drought stress.     

The distribution of poplar mosaic virus in hybrid poplars and virus detection by ELISA

Purified poplar mosaic virus (PMV) at a concentration of 8 ng/ml was readily detected by enzyme-linked immunosorbent assay (ELISA). Bioassay in Nicotiana megalosiphon was more sensitive (detecting 1–4 ng/ml) and latex flocculation less sensitive (c. 25 ng/ml) than ELISA assays. While the foliar sap of fresh, naturally-infected poplars (e.g. Populus. euramericana cv. Robusta) was not infective at dilutions greater than 2 . 10–², ELISA easily detected PMV antigen when sap was diluted 4 . 10–³ in buffer or when one part of infected tissue was triturated with 99 parts healthy leaf. Furthermore, although sap from poplar leaves stored at -20 °C for 6 months was not infective, PMV was still detectable in ELISA tests. PMV antigen in poplar leaves was not all pelleted after centrifugation for 2.5 h at 130 000 g yet parallel tests using unbuffered sap from systemically infected Nicotiana megalosiphon foliage showed that infectivity was restricted to the pellet. In poplar foliage, the concentration of PMV antigen was generally greatest where symptoms were most obvious; least antigen was detected in the overwintering leaves located at the bases of long shoots. In winter, when root and inner bark tissue in the trunk was an erratic source of PMV, the virus was readily detected in buds, the concentration being greatest in the bases, including the meristem, of terminal buds. Propagation from single node cuttings of P. euramericana cv. Regenerata allowed the selection of clones that consistently showed either ‘severe’ or ‘mild’ foliar symptoms. The associated virus isolates also infected another poplar clone causing symptoms characteristic of their source. ELISA consistently detected less PMV antigen in field-grown cv. Regenerata than in cv. Robusta foliage, but this was reversed when the associated virus isolates were propagated in Nicotiana glutinosa at 24 °C. During 6 yr, 21 out of 127 poplars at a site in Western England, became infected with PMV. By contrast, in Eastern England, none of 46 were infected. The aphids Pterocomma populea and Myzus persicae did not transmit PMV.