Photosynthetic gas exchange characteristics in three different almond species during drought stress and subsequent recovery

Three different drought stress levels (water potential of the nutrient solution, Ψ_s = −0.6, −1.2 and −1.8 MPa, respectively), and a control treatment (Ψ_s = −0.1 MPa), were applied during 2 weeks to three almond species, followed by 3 weeks of recovery. The selected test species were Prunus dulcis (Miller) D.Webb (bitter almond) and two wild almond species, P. lycioides (Spach) C.K. Schneider and P. scoparia (Spach) C.K. Schneider. All three are species native to Iran, and can be used as rootstock, but only P. dulcis is actually used for commercial almond production.In the absence of drought stress, maximum net assimilation rate (A_max) is highest for P. scoparia and lowest for P. dulcis. For all species A_max was above 16 μmol CO₂ m⁻² s⁻¹. A similar relationship between A_max and dark respiration rate (R_d), was observed for all species. This relationship suggests that optimisation of the carbon budget is independent of species.The three investigated species seem to have a different reaction to a similar stress, indicating different drought stress coping strategies. P. scoparia lost all its leaves during the experiment, while P. lycioides only kept some leaves, however, the remaining leaves were almost totally wilted and did not allow for any photosynthesis measurement. P. scoparia did not recover during the experiment, as no new leaves were developed once Ψ_s was restored to pre-drought stress levels. However, this species has green stems, indicating that stem photosynthesis might play an important role in the plants’ overall carbon balance. This species is an opportunistic one (sensu [Higgins, S.S., Larsen, F.E., Bendel, R.B., Radamaker, G.K., Bassman, J.H., Bidlake, W.R., Alwir, A., 1992. Comparative gas-exchange characteristics of potted, glasshouse-grown almond, apple, fig, grape, olive, peach and Asian pear. Sci. Hortic. 52 (4), 313–329]), where assimilation is seriously limited by non-stomatal processes as evidenced by measurements of intercellular CO₂ concentration, eventually resulting in total leaf loss. All P. lycioides leaves almost completely wilted during the experiment, but this species recovered rather quickly. Leaves, newly formed at the end of the experiment, obtained maximal assimilation rates under control Ψ_s levels, equivalent to those measured in the control treatment. Finally, P. dulcis did keep at least part of its leaves during drought stress. However, assimilation rates after 2 weeks of drought treatment and 3 weeks of recovery were only about half of those measured in the control treatment. Of the three investigated species, non-stomatal limitation of assimilation seems to be the least important in P. dulcis.Intrinsic water use efficiency, defined as the ratio of assimilation rate over stomatal conductance, increased for P. dulcis with increasing drought stress, while a different pattern was observed for P. lycioides and P. scoparia, indicating non-stomatal processes prevail over stomatal limitations of the assimilation process. It was concluded that P. dulcis is the species most tolerant to drought. P. scoparia tries to avoid drought, whereas P. lycioides has an intermediate behaviour. Besides P. dulcis, also P. lycioides seems to have some potential for use as rootstock for commercial almond production.     

Ultraviolet-B-induced oxidative stress and responses of the ascorbate-glutathione cycle in a marine macroalga Ulva fasciata

The regulation of the antioxidant defence system by ultraviolet-B (UV-B) was determined in a marine macroalga Ulva fasciata Delile exposed to low (0.5, 1 W m(-2)), medium (2.5, 5 W m(-2)), and high (10, 20 W m(-2)) UV-B irradiance. UV-B > or =2.5 W m(-2) increased H2O2 contents that are positively correlated with lipid peroxidation and total peroxide contents. Inhibition of the UV-B-induced H2O2 increase by a specific O2.- scavenger, 1,2-dihydroxy-benzene-3,5-disulphonic acid, shows that O2.- is the primary source of H2O2. Superoxide dismutase activity was increased by UV-B with a peak at 2.5 W m(-2), which did not match the H2O2 pattern. Alleviation of UV-B-induced oxidative damage by a H2O2 scavenger, dimethylthiourea, and a free radical scavenger, sodium benzoate, which inhibited UV-B-induced H2O2 accumulation, suggests that oxidative damage caused by UV-B > or = 2.5 W m(-2) is ascribed to accumulated H2O2. However, a decrease in growth rate and TTC reduction ability only at high UV-B doses indicates that the defence and repairing systems operate at low and medium UV-B doses. H2O2 not only can be excreted but can also be detoxified via the ascorbate-glutathione cycle. Increases in catalase, peroxidase, ascorbate peroxidase, and glutathione reductase activities and ascorbate (AsA) and glutathione pools, as well as AsA regeneration ability, function to keep the balance of cellular H2O2 under low UV-B doses. Dehydroascorbate reductase and monodehydroascorbate reductase are responsible for AsA regeneration under low and medium UV-B radiation, respectively. The appearance of oxidative damage in medium and high UV-B flux is attributable to a lower induction of the ascorbate-glutathione cycle as an antioxidant defence system. Overall, the availability of antioxidants and the induction of antioxidant enzyme activities for detoxifying reactive oxygen species (ROS) are regulated in U. fasciata against UV-B-induced oxidative stress, and experiments using ROS scavengers demonstrate that the antioxidant defence system is modulated by O2.- or H2O2.     

Function of the ascorbate-glutathione cycle in aged sunflower seeds

The function of the ascorbate-glutathione (AsA/GSH) cycle was analyzed in seeds of sunflower ( Helianthus annuus L. cv. Peredovik) subjected to accelerated ageing at 43°C and 75% relative humidity for 1 to 11 days. The study was performed using dry seeds and seeds hydrated by imbibition in distilled water for 4 h at 25 °C. Lipid peroxidation was also determined by measuring the malondialdehyde (MDA) level. As the ageing period increased, a progressive loss of seed viability became increasingly evident. Even though high levels of MDA were delected, the MDA level did not change during accelerated ageing, suggesting that lipid peroxidation might occur to some extent. The study of the ascorbate/glutathione (AsA/GSH) cycle revealed that the GSH system is the major detoxifying mechanism in both dry and imbibed sunflower seeds. The GSH system is mainly located in the embryo, and its protective role is mediated by reactions that consume the GSH pool and, thereby, minimize the increase of the oxidized form (GSSG). Seed imbibition activates cellular metabolism and allows some antioxidant enzymes like glutathione reductase (EC 1,6,4,2) to act upon toxic agents. These reactions provide a reducing status, so that repair of damage becomes possible. However, prolonged ageing conditions (11 days) result in an irreversible damage, as evidenced by the appearance of dead seeds when the germination period ended. Multiple regression analysis revealed the effectiveness of the GSH system in aged seeds, especially upon imbibition and until the AsA/GSH cycle became completely functional.     

外源NO对铜胁迫下番茄幼苗根系抗坏血酸-谷胱甘肽循环的影响

采用营养液培养方法,研究外源NO对铜胁迫下番茄(Lycopersicon esculentum Mill.)幼苗根系抗坏血酸(AsA)-谷胱甘肽(GSH)循环中抗氧化物质和抗氧化酶系的影响.结果表明:外施适量NO(硝普钠)可提高铜胁迫下番茄幼苗根系AsA、GSH含量和AsA/DHA(氧化型抗坏血酸)、GSH/GSSG(氧化型谷胱甘肽),降低DHA和GSSG含量.添加100 μmol·L-1 BSO(谷胱甘肽合成酶抑制剂)处理下,外源NO可提高铜胁迫下番茄幼苗根系的AsA含量、AsA/DHA及抗坏血酸酶(AAO)、单脱氢抗坏血酸还原酶(MDHAR)和脱氢抗坏血酸还原酶(DHAR)比活性,降低DHA、GSH、GSSG含量及抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)比活性;添加250 μmol·L-1 BSO处理下,外源NO提高了铜胁迫下番茄幼苗根系的AsA、GSH、GSSG含量、AsA/DHA及APX和GR比活性,降低了DHA含量及AAO、DHAR和MDHAR比活性.说明外源NO影响了铜胁迫下番茄根系的AsA-GSH代谢循环,并通过调节AsA/DHA、GSH/GSSG的变化来减轻氧化胁迫,从而缓解铜胁迫对番茄根系的伤害.     

Microplate quantification of enzymes of the plant ascorbate-glutathione cycle

Here, we describe microplate assays for determining the specific activities of four enzymes that constitute the ascorbate-glutathione cycle: APX, MDHAR, DHAR, and GR. In plants, these enzymes play a major role in detoxifying reactive oxygen species produced in cells under environmental stress. This work presents the development of plate reader assays to allow rapid analysis of the ascorbate-glutathione cycle activity using tomato fruits subjected to salt stress as a model. With this method, it is possible to analyze easily in one day the activities of the four enzymes for 30 experimental samples, all in triplicate and with blanks.     

Calcium and L-histidine effects on ascorbate-glutathione cycle components under nickel-induced oxidative stress in tomato plants

The effects of NiSO₄, calcium, and L-histidine (His) on the components of ascorbate-glutathione cycle, antioxidant enzymes and lipid peroxidation in a tomato cultivar Early Urbana Y was investigated. The activities of enzymes including catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), lipoxygenase (LOX), and phenylalanine ammonia lyase (PAL) were measured. In addition, the content of H₂O₂, ascorbate (ASC), dehydroascorbate (DHA), reduced glutathione (GSH), chlorophyll (Chl) a+b, carotenoids, proteins, malondialdehyde (MDA), membrane aldehydes, and electrolyte leakage (EL) were determined. Results suggest that the excess of Ni increased the content of H₂O₂, MDA, membrane aldehydes and proteins in roots as well as GPX, LOX, APX activities, and EL in leaves, whereas Ca and His ameliorated these effects. Moreover, decreasing leaf GSH and DHA content and GR activity were observed under the Ni stress, but these parameters were raised by Ca plus His treatment. However, no improvement in leaf protein, ASC, root GSH content, and activities of PAL and CAT were observed by using Ca or His under Ni stress.     

Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism.

To understand the interaction between Zn, an essential micronutrient and Cd, a non-essential element, Cd-10 microM and Zn supplemented (10, 50, 100, and 200 microM) Cd 10 microM treated Ceratophyllum demersum L. (Coontail), a free floating freshwater macrophyte was chosen for the study. Cadmium at 10 microM concentration decreased thiol content, enhanced oxidation of ascorbate (AsA) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively, a clear indication of oxidative stress. Zinc supplementation to Cd (10 microM) treated plants effectively restored thiols, inhibited oxidation of AsA and GSH maintaining the redox molecules in reduced form. Cd-10 microM slightly induced ascorbate peroxidase (APX, E.C. 1.11.1.11) but inhibited monodehydroascorbate reductase (MDHAR, E.C. 1.6.5.4), dehydroascorbate reductase (DHAR, E.C. 1.8.5.1) and glutathione reductase (GR, E.C. 1.6.4.2), enzymes of ascorbate-glutathione cycle (AGC). Zn supplementation restored and enhanced the functional activity of all the AGC enzymes (APX, MDHAR, DHAR and GR). Gamma-glutamylcysteine synthetase (gamma-GCS, E.C. 6.3.2.2) was not affected by Cd as well as Zn, but Zn supplements increased glutathione-S-transferase (GST, E.C. 2.5.1.18) activity to a greater extent than Cd and simultaneously restored glutathione peroxidase (GSH-PX, E.C. 1.11.1.9) activity impaired by Cd toxicity. Zn-alone treatments did not change above investigated parameters. These results clearly indicate the protective role of Zn in modulating the redox status of the plant system through the antioxidant pathway AGC and GSH metabolic enzymes for combating Cd induced oxidative stress.     

Exogenous proline alleviates the effects of H2O2-induced oxidative stress in wild almond species

The effect of proline on the antioxidant system in the leaves of eight species of wild almond (Prunus spp.) exposed to H₂O₂-mediated oxidative stress was studied. The levels of endogenous proline (Pro) and hydrogen peroxide, and the activities of total superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and guaiacol peroxidase (POD) were measured. The degradation of chlorophyll but not carotenoids occurred in leaves in the solution of 5 mM H₂O₂. An increase in membrane lipid peroxidation was observed in H₂O₂ treatment, as assessed by MDA level and percentage of membrane electrolyte leakage (EL). Significant increases in total SOD and CAT activities, as well as decreases in APX and POD activities, were detected in H₂O₂-treated leaves. The three SOD isoforms showed different behavior, as Mn-SOD activity was enhanced by H₂O₂, whereas Fe-SOD and Cu/Zn-SOD activities were inhibited. In addition, Pro accumulation up to 0.1 ??mol/g fr wt, accompanied by significant decreases in ascorbate and glutathione levels, was observed in H₂O₂-treated leaves. After two different treatments with 10 mM Pro + 5 mM H₂O₂, total SOD and CAT activities were similar to the levels in control plants, while POD and APX activities were higher if compared to the leaves exposed only to H₂O₂. Pro + H₂O₂ treatments also caused a strong reduction in the cellular H₂O₂ and MDA contents and EL. The results showed that Pro could have a key role in protecting against oxidative stress injury of wild almond species by decreasing membrane oxidative damage.     

Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

Salt stress induction of some key antioxidant enzymes and metabolites in eight Iranian wild almond species

The present work describes the changes in the activities of key antioxidant enzymes and the levels of some metabolites in relation to salt tolerance in eight wild almond species. All the species were exposed to four levels of NaCl (control, 40, 80 and 120 mM). Plant fresh biomass, α-, γ- and δ-tocopherol, total soluble proteins, malondialdehyde (MDA_eq), H₂O₂, total phenolics, and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were analyzed in leaves of salt-stressed and non-stressed plants of the eight almond species. In all the species, salt stress significantly enhanced the activities of SOD and POD, levels of total phenolics and γ- and δ-tocopherols. High levels of salt stress significantly depressed the levels of total soluble proteins, MDA and CAT activity, while salt stress did not significantly affect leaf H₂O₂ contents. Regression analysis showed that the relationship between salt levels and total soluble proteins, CAT, γ-tocopherol, MDA_eq, SOD and POD were statistically significant. Principal component analysis discriminated the almond species based on their degree of tolerance/sensitivity to saline conditions: Prunus reuteri and P. glauca were ranked as salt tolerant, P. lycioides and P. scoparia as moderately tolerant, and P. communis, P. eleagnifolia, P. arabica and P. orientalis as salt sensitive. The results could be used for selecting salt tolerant genotypes to be used as rootstocks for almond cultivation.     

Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide

Salinity stress causes ionic stress (mainly from high Na⁺ and Cl^- levels) and osmotic stress (as a result of inhibition of water uptake by roots and amplified water loss from plant tissue), resulting in cell death and inhibition of growth and ultimately adversely reducing crop productivity. In this report, changes in root nitric oxide content, shoot and root biomass, root H₂O₂ content, root lipid peroxidation, root cell death, root caspase-like enzymatic activity, root antioxidant enzymatic activity and root ascorbate and glutathione contents/redox states were investigated in maize (Zea mays L. cv Silverking) after long-term (21 d) salt stress (150 mM NaCl) with or without exogenously applied nitric oxide generated from the nitric oxide donor 2,2′-(Hydroxynitrosohydrazano)bis-ethane. In addition to reduced shoot and root biomass, salt stress increased the nitric oxide and H₂O₂ contents in the maize roots and resulted in elevated lipid peroxidation, caspase-like activity and cell death in the roots. Altered antioxidant enzymatic activities, along with changes in ascorbate and glutathione contents/redox status were observed in the roots in response to salt stress. The detrimental effects of salt stress in the roots were reversed by exogenously applied nitric oxide. These results demonstrate that exogenously applied nitric oxide confers salt stress tolerance in maize by reducing salt stress-induced oxidative stress and caspase-like activity through a process that limits accumulation of reactive oxygen species via enhanced antioxidant enzymatic activity.     

Regulation of specific genes during the cell cycle

Evidence for differential gene expresion during the cell cycle and approaches for studying cell-cycle-stage specific gene expression are summarized. Attention is focused on regulation of histone gene expression during the cell cycle of continuously dividing cells and after stimulation of nondividing cells to proliferate. The level(s) at which control of histone gene expression occurs and the possible involvement of chromosomal proteins in the regulation of histone gene expression are discussed. The preparation of cloned human histone sequences and their use in studying the structural and functional properties of human histone genes are presented.     

Responses of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits

Wild watermelon from the Botswana desert had an ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of leaf proteins, seven spots were newly induced after watering stopped. One with the molecular mass of 40 kilodaltons of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/Acy1/Cpg2/YscS protein family (including acetylornithine deacetylase, carboxypeptidase and aminoacylase-1). This suggests that the protein is involved in the release of free amino acid by hydrolyzing a peptidic bond. As the drought stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status and the content of citrulline reached about 50% in the total free amino acids. The accumulation of citrulline during the drought stress in wild watermelon is an unique phenomenon in C3-plants. These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance of the water status and (2) a metabolic change to accumulate citrulline.     

Regulation of metabolomics in Atriplex halimus growth under salt and drought stress

Forty-day-old Atriplex halimus seedlings were treated with either NaCl (50, 300 and 550?mM) for the subsequent 30?days or PEG for the following 3, 6 and 10?days. Shoot fresh and dry weights were significantly increased by 50?mM NaCl; nevertheless, the other concentrations had no effect. However, the growth was reduced by drought only after 10?days. Meanwhile, Na⁺ was accumulated in treated plants; the magnitude of accumulation was highest with high NaCl concentration or PEG for 10?days. The metabolite profiles showed discrimination particularly up-regulation of the amino acids proline, valine, isoleucine, and methionine. Moreover, the macro analysis revealed that NaCl- and PEG-treated plants shared 10?% of the metabolites in the positive mode, however, 87?% were unique to NaCl and 46?% were unique to PEG whereas in the negative mode, 8?% were in share while 90 or 53?% were restricted to NaCl or PEG, respectively. Additionally, sucrose in particular was significantly increased up to threefold and fivefold by 300 and 550?mM NaCl, respectively and up to 2.5-fold by drought for 10?days, nevertheless, the other sugar fractions remained largely unchanged. Also, proline was significantly increased by only the high NaCl concentrations and the long-term drought, nonetheless, the other treatments led, if any, to decreases. These results conclude that NaCl affects the metabolite profiles more than PEG and these metabolites might contribute to osmotic adjustments to act as osmoprotectants rather than osmolytes. These changes of metabolomics might function in many resistance and stress responses.     

Response of the ascorbate-glutathione cycle to re-aeration following hypoxia in lupine roots.

The response of the enzymes and metabolites of the ascorbate-glutathione pathway to oxidative stress caused by re-aeration following hypoxia was studied in roots of hydroponically grown lupine (Lupinus luteus L. cv. Juno) seedlings. Lupine roots were deprived of oxygen by subjecting them to hypoxia for 48 and 72 h and then re-aerated for up to 4 h. An increased content of total ascorbate was observed in lupine roots immediately after hypoxia, whereas total glutathione level decreased. However, a significant increase in the reduced forms of both metabolites was found directly after hypoxia. Re-admission of oxygen caused the decrease of the ratios of reduced to oxidized forms of ascorbate and glutathione, indicating oxidative stress. While monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activity remained unaltered during re-aeration the increase in activities of ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) was observed 30 min after transfer from hypoxic condition. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity approached the control level during a whole re-aeration period. Native gel electrophoresis combined with specific activity staining revealed seven isoforms of APX, five isoforms of GR and three different proteins with DHA reductase activity in roots extracts. However, immediately after hypoxic treatment APX-5 isoform and GR-1 isoform were not observed in roots. This experimental system was also used to investigate superoxide anion level in roots utilizing the superoxide anion-specific indicator dihydroethidium (DHE). Intense DHE-derived fluorescence was found in re-aerated root tips as compared to control roots, indicating that re-aeration induced superoxide anion production in hypoxically pretreated roots.