Cloning and characterisation of a pepper aquaporin,CaAQP, which reduces chilling stress in transgenic tobacco plants

Ubiquitous cell membrane proteins called aquaporins are members of major intrinsic proteins (MIPs), which control the specific transport of water molecules across cell membranes. A pepper aquaporin gene (CaAQP), which exhibits the structural features of tonoplast intrinsic proteins of the MIP subfamily, was isolated from the leaves of chilling-treated seedlings of pepper (Capsicum annuum L.) cv. P70. Assays indicated high levels of expression in young seeds, green fruits and flower buds and low levels of expression in the stems, leaves and roots of pepper. The expression patterns were strongly and rapidly induced by HgCl₂, low temperature, abscisic acid, fluridone and osmotic stresses. The responsiveness of pepper seedlings pretreated with abscisic acid at low temperatures demonstrated up-regulation of CaAQP by chilling, which is potentially involved in ABA signalling. Our results indicated that overexpression of CaAQP decreased chilling stress in transgenic plants, likely by increasing the stomatal aperture under stress, increasing the rate of membrane damage during the recovery stage, thereby affecting the intercellular CO₂ concentration with lower stomatal conductance and transpiration rates. VIGS of CaAQP in pepper plants caused significant growth retardation. These results suggested that CaAQP played a crucial role in the plant response to abiotic stresses.     

Growth,cell volume,and fine structure of Porphyra umbilicalis in relation to osmotic tolerance

Porphyra umbilicalis, a marine red alga occurring in the intertidal zone of the cold North Sea, tolerates a wide range of osmotic conditions from 0.2 x to 6 x artificial seawater medium ASP₁₂. In cells osmotically adapted for two weeks, photosynthesis and respiration are progressively inhibited in media more concentrated than 2 x. In both hypo- and hyperosmotic stress ranges, the most striking fine structural change is the development of vacuoles. In comparison to 1 x medium, where vacuoles are virtually lacking, the vacuolar part of the protoplasm increases 6-fold in 0.2 x and 10-fold in 3.5 x medium, respectively. However, at extreme hyperosmotic stress (6 x medium) the vacuolar part is extremely small. The largest cell volumes are found in 0.2 x and 3.5 x media, the smallest one in 6 x medium. In the osmotically regulated range (0.2–3.5 x medium), the regulated parameter is the volume of the protoplasm without the vacuolar system. It is suggested that at hyperosmotic stress the vacuoles may serve as osmotically active compartment, probably by accumulation of inorganic ions. The intracellular content of Floridean starch granules decreases with increasing osmotic pressure, possibly indicating the significance of soluble organic constituents as osmotically active solutes.Member of the Arbeitsgemeinschaft für Elektronenmikroskople un der Ticrärztlichen Hochschule Hannover     

Effect of osmotic stress on thylakoid fine structure inPorphyra umbilicalis

Summary The fine structural organization of thylakoid membranes in intact cells ofPorphyra umbilicalis, an intertidal red alga, was studied using the freeze-fracture method with special emphasis on changes induced by hypo- and hyperosmotic stresses. In osmotically adapted plants the density of intramembraneous particles on the PF-face increases considerably in the osmotic range from 5-fold diluted to 6-fold concentrated artifical seawater medium ASP₁₂, while that on the EF-face remains constant. The size of the particles on both fracture faces decreases strongly from extreme hypoosmotic to extreme hyperosmotic stress. These findings are discussed with relation to their biological significance.The author is member of the Arbeitsgemeinschaft für Elektronenmikroskopie an der Tierärztlichen Hochschule Hannover.     

SlERF52 regulates SlTIP1;1 expression to accelerate tomato pedicel abscission

Abscission of plant organs is induced by developmental signals and diverse environmental stimuli and involves multiple regulatory networks, including biotic or abiotic stress-impaired auxin flux in the abscission zone (AZ). Depletion of auxin activates AZ ethylene (ETH) production and triggers acceleration of abscission, a process that requires hydrogen peroxide (H₂O₂). However, the interaction between these networks and the underlying mechanisms that control abscission are poorly understood. Here, we found that expression of tonoplast intrinsic proteins, which belong to the aquaporin (AQP) family in the AZ was important for tomato (Solanum lycopersicum) pedicel abscission. Liquid chromatography–tandem mass spectrometry and in situ hybridization revealed that SlTIP1;1 was most abundant and specifically present in the tomato pedicel AZ. SlTIP1;1 localized in the plasma membrane and tonoplast. Knockout of SlTIP1;1 resulted in delayed abscission, whereas overexpression of SlTIP1;1 accelerated abscission. Further analysis indicated that SlTIP1;1 mediated abscission via gating of cytoplasmic H₂O₂ concentrations and osmotic water permeability (P_f). Elevated cytoplasmic levels of H₂O₂ caused a suppressed auxin signal in the early abscission stage and enhanced ETH production during abscission. Furthermore, we found that increasing P_f was required to enhance the turgor pressure to supply the break force for AZ cell separation. Moreover, we observed that SlERF52 bound directly to the SlTIP1;1 promoter to regulate its expression, demonstrating a positive loop in which cytoplasmic H₂O₂ activates ETH production, which activates SlERF52. This, in turn, induces SlTIP1;1, which leads to elevated cytoplasmic H₂O₂ and water influx.

A SlERF52-SlTIP1;1 regulatory module accelerates pedicel abscission by increasing cytoplasmic hydrogen peroxide contents and osmotic water permeability.     

Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using β-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K_m for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.     

脱硫废弃物对碱胁迫下油葵幼叶细胞钙分布及Ca2+-ATPase活性的影响

利用脱硫废弃物改良盐碱地对于确保国家粮食安全和生态安全,发展循环经济具有重要意义。为了探索脱硫废弃物提高植物抗盐碱机理,采用盆栽试验法, 研究了施入不同量脱硫废弃物和CaSO₄对碱胁迫下油葵叶片细胞钙分布、总钙含量以及质膜和液泡膜Ca²⁺-ATPase活性的影响。结果表明:在碱胁迫下(CK),Ca²⁺与焦锑酸钾结合成黑色颗粒成团零星分布于叶绿体和液泡中,叶绿体超微结构受到不同程度的破坏。施入脱硫废弃物和CaSO₄,叶绿体结构完整,细胞间隙、细胞壁和液泡中的钙颗粒逐渐增多,同时,质膜和液泡膜Ca²⁺-ATPase活性随脱硫废弃物和纯品硫酸钙施量的增加而增加,其中液泡膜Ca²⁺-ATPase活性无论是对照(CK)还是处理的活性均高于质膜Ca²⁺-ATPase活性。叶片细胞内总钙含量也随脱硫废弃物和CaSO₄施用量的增加呈升高趋势。说明脱硫废弃物和CaSO₄通过增加Ca²⁺-ATPase活性,有利于钙通过质膜和液泡膜进入细胞内,维持膜结构的稳定性,缓解碱对油葵的胁迫。     

Increased Polyamines Conjugated to Tonoplast Vesicles Correlate with Maintenance of the H<Superscript>+</Superscript>-ATPase and H<Superscript>+</Superscript>-PPase Activities and Enhanced Osmotic Stress Tolerance in Wheat

The effects of osmotic stress on H⁺-ATPase and H⁺-PPase activities and the levels of covalently conjugated polyamines (CC-PAs) and noncovalently conjugated polyamines (NCC-PAs) were investigated using tonoplast vesicles isolated from the roots of wheat (Triticum aestivum L.) seedlings differing in drought-tolerance. The results showed that after polyethylene glycol (PEG) 6,000 (–0.55MPa) treatment for 7 days, seedling leaf relative water content (LRWC), relative dry weight increase rate (RDWIR) and root H⁺-ATPase and H⁺-PPase activities from the drought-sensitive cultivar Yangmai No. 9 decreased more markedly than those from the drought-tolerant cultivar Yumai No. 18. At the same time, the increase of the NCC-spermidine (NCC-Spd) and CC-putrescine (CC-Put) levels in root tonoplast vesicles from Yumai No. 18 was more obvious than that from Yangmai No. 9. Exogenous Spd treatment alleviated osmotic stress injury to Yangmai No. 9 seedlings, coupled with marked increases of tonoplast NCC-Spd levels and H⁺-ATPase and H⁺-PPase activities. Treatments with methylglyoxyl bis (guanyl hydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), and phenanthrolin, an inhibitor of transglutaminase (TGase), significantly inhibited the osmotically induced increases of NCC-Spd and CC-Put levels, respectively, in root tonoplast vesicles from Yumai No. 18 seedlings. Both MGBG and phenanthrolin treatments markedly promoted osmotically induced decreases of tonoplast H⁺-ATPase and H⁺-PPase activities and osmotic stress tolerance of seedlings of this cultivar. These results suggest that the NCC-Spd and CC-Put present in tonoplast vesicles isolated from wheat seedling roots might enhance the adaptation of seedlings to osmotic stress via maintenance of tonoplast H⁺-ATPase and H⁺-PPase activities.     

Morphological,physiological and biochemical responses to drought stress of Stone pine (<Emphasis Type="Italic">Pinus pinea</Emphasis> L.) seedlings

In this study, the effect of irrigation intervals (drought stress) on growth, predawn xylem water potential (Ψ _w), the osmotic potential at full turgor (Ψπ ₁₀₀), the osmotic potential at the turgor loss point (Ψπ _TLP), osmotic adjustment and osmotic solutes (soluble sugars and proline) of Pinus pinea L. seedlings were examined. An experiment was carried out under greenhouse conditions using four watering treatments (control, 7-, 14- and 21-day irrigation intervals) in the first growth season; from mid-July to early November. Results showed that irrigation interval had significant effect on growth characteristics, Ψ _w, water relation parameters, and osmotic solutes. The increasing irrigation interval significantly decreased the seedling height, root collar diameter, root, stem and needle dry weight, number of lateral branches, root percentage, root:shoot ratio and diameter:height ratio. Ψ _w and total soluble sugars decreased while proline content increased with the increase of drought stress. The Ψπ ₁₀₀ and Ψπ _TLP significantly decreased in drought-stressed seedlings compared to control (no stress) seedlings. The results suggest that the impact of drought stress increased with the increase of irrigation interval. Therefore, in the drought-stressed P. pinea seedlings were indicated osmotic adjustment by increasing the proline content and decreasing Ψπ ₁₀₀ and Ψπ _TLP during drought stress. Growth decreased under drought stress conditions in P. pinea seedlings.     

Enzymic and protein character of tonoplast from hippeastrum vacuoles

The membrane of anthocyanin containing Hippeatrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg²⁺-dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, α-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3-O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K₂HPO₄ or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels.     

Ammonium-induced proline and sucrose accumulation, and their significance in antioxidative activity and osmotic adjustment

Excess of ammonia generates oxidative and osmotic stress, and results in an accumulation of compatible solutes. The aim of this study was to investigate the physiological significance of excess ammonium-induced proline and sucrose accumulation on antioxidative activity and osmotic adjustment. The detached leaves of white clover (Trifolium repense L.) were fed with 0, 10, 50, 100, and 200 mM NH₄Cl, and the contribution of proline and sucrose to osmotic adjustment and their relationship with antioxidative enzymes activity were assessed. A gradual decline of relative water content and osmotic potential (Ψ_π) with increasing NH₄Cl feeding level was accompanied by an increase in ammonia concentration. Significant accumulation of proline and sucrose was observed when NH₄Cl was fed over 100 mM compared with control (0 mM NH₄Cl). The increase in enzyme activity was significant only at 200 mM for ascorbate peroxidase (APOD) and over 100 mM NH₄Cl for guaiacol peroxidase (GPOD) and catalase (CAT). The contribution of proline and sucrose to osmotic adjustment over 100 mM, where proline and sucrose accumulation was more important, maintained at control levels or significantly decreased. The content of proline and sucrose as affected by NH₄Cl feeding level was positively related with the activity of APOD, GPOD, and CAT. These results suggest that proline and sucrose accumulation induced by the excess of ammonium has a more influential role in antioxidative activity rather than osmotic adjustment.     

Reduced osmotic potential inhibition of photosynthesis : site-specific effects of osmotically induced stromal acidification

The effects of reduced reaction medium osmotic potential (0.67 molar sorbitol as compared to a control treatment with 0.33 molar sorbitol) on the enzymic steps of the photosynthetic carbon reduction cycle were investigated using isolated spinach (Spinacia oleracea L. var Longstanding Bloomsdale) chloroplasts. Reversal of reduced osmotic potential inhibition of photosynthetic rates by a stromal alkalating agent (NH₄Cl) was associated with specific steps of the cycle. Low osmotic potential induced stromal acidification was found to be facilitated by osmotically induced chloroplast shrinkage. However, the action of the alkalating agent was found not to be associated with reversal of osmotically induced morphological changes of the stromal compartment.

Labeled metabolite analyses indicated that the osmotic stress treatment caused the substrate for fructose 1,6-bisphosphatase (FBPase) to build up in the absence of NH₄Cl, and the substrate for phosphoribulokinase to increase in the presence of NH₄Cl. These data were interpreted as indicating that the most severe effect of osmotic stress on photosynthesis is at the site of FBPase, and that this inhibition is mediated by osmotically induced stromal acidification. Phosphoribulokinase activity inhibition at the low osmotic potential treatment was apparently less severe and not mediated by stromal acidification. A third site of osmotic inhibition, which was reversed by NH₄Cl, and therefore was assumed to be mediated by stromal acidification, was at the step of ribulose 1,5-bisphosphate carboxylase.

Additions of NH₄Cl also enhanced the activity of the pH-insensitive phase of the photosynthetic carbon reduction cycle, 3-phosphoglyceric acid reduction, at the stress treatment. This effect was thought to be mediated by the removal of the block at FBPase. A model was proposed to outline the relative severity of osmotic stress effects at various sites of the photosynthetic carbon reduction cycle.

     

Dynamics of activity and structure of the tonoplast vacuolar-type H+-ATPase in plants with differing CAM expression and in a C3 plant under salt stress

Summary Differences in the activity and structure of the vacuolar H⁺-ATPase (V-ATPase, EC 3.6.1.3) were investigated in the C₃/CAM intermediate plantKalanchoë blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, andHordeum vulgare cv. Carina, grown with or without 150 mM NaCl. InK. blossfeldiana ATP-hydrolysis and H⁺-transport activity were higher with higher expression of CAM than in plants with very weak CAM. This was mainly due to a larger amount of V-ATPase. Statistical analysis of the diameter of intramembrane particles (IMPs) on freeze-fractures of tonoplast vesicles showed that IMPs were larger in tonoplast vesicle preparations ofK. blossfeldiana with strong CAM expression (9.1 nm) than in preparations ofK. blossfeldiana with low CAM expression (7.3 nm). As there is evidence that the majority of IMPs on freeze-fractures of tonoplast vesicles corresponds to the V₀ domain of V-ATPase, the higher activity of V-ATPase inK. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase inK. blossfeldiana with stronger CAM is discussed in relation to the requirement for a higher proton pumping capacity for nocturnal malate accumulation in the vacuole. The ATP-dependent H⁺-pumping activity inH. vulgare was higher under salt stress than in control plants, while the rates of ATP-hydrolysis and the size of IMPs were not affected by the salt treatment. The data presented here indicate that different mechanisms might increase the transport capacity of V-ATPase to meet the higher requirements of secondary active transport related to CAM expression and adaptation to salt stress.Abbrevations ATP adenosine triphosphate - CAM crassulacean acid metabolism - IMP intramembrane particles - V-ATPase vacuolar proton-translocating adenosine triphosphatase - V₀ domain membrane-integral domain of V-ATPase - V₁ domain membrane-peripheral domain of V-ATPase Dedicated to Prof. Dr. Eberhard Schnepf on the occasion of his retirement     

Early changes of water diffusional transfer in maize roots under the influence of water stress

The time dependent response of the hydrodynamic root system to PEG-induced water stress was studied in intact maize Zea mays L. seedlings at intervals varying from several seconds to 3 h by detecting diffusional water transfer with the use of pulsed NMR. In order to establish the contribution of water transfer through aquaporins in response to water stress, the transmembrane water transport in control roots and roots treated with aquaporin blocker was detected. Changes in diffusional water transfer under stress were shown to depend on the duration of osmotic treatment, and include the series of heterogeneous processes. A transient pulsed jump in diffusional water transfer detected several seconds after beginning the osmotic treatment is associated with the spread of the wave of hydraulic pressure along the root. It is proposed that early responses of the hydrodynamic system of maize roots to PEG-induced water stress lies in the unequal change in water permeability of the plasmalemma and tonoplast resulting from the changes in aquaporin activity and perhaps in the escalation of water transfer along the cell vacuome.     

Concanamycin 4-B: A Potent Inhibitor of Vacuolar pH Regulation in Chara Cells

Concanamycin 4-B, a macrolide antibiotic with an 18-membered lactone ring, is known as a specific inhibitor of the vacuolar type of H⁺-ATPase, as is bafilomycin A₁. The drug was tested for its effect on regulation of the vacuolar pH (pH_v) of internodal cells of a fresh water characean alga, Chara corallina, under normal conditions and under salt stress. The pH_v was measured either on isolated vacuolar sap with a conventional pH electrode or directly by inserting a pH-sensitive glass microelectrode into the vacuole. Proton-pumping into tonoplast vesicles was almost completely inhibited by concanamycin 4-B at 1 nM. Concanamycin 4-B at 1 μM significantly increased pH_v while bafilomycin A₁ was ineffective when applied at 1 μM. Concanamycin 4-B did not affect pH_v when applied at 0.1 μM and increasing the concentration to 10 μM did not amplify the degree of alkalization. Concanamycin 4-B also inhibited pH_v regulation under NaCl stress. When Chara cells were treated with 100 mM NaCl, pH_v promptly increased and then recovered to the original level. The reacidification was completely inhibited by concanamycin 4-B (1 μM), suggesting that the reacidification was achieved by the H⁺-ATPase of the tonoplast.     

Visualisation of xylem sap flow direction in isolated fine lateral roots and estimation of the xylem sap osmotic potential

Xylem sap outflow from fine lateral roots (FLRs) isolated from hydroponically grown young maize (Zea mays L.) plants was visualized by local brightening of test solutions contrasted with purified Indian ink particles. Flow into the vessels was indicated by the adsorption of Evans Blue in their walls. The fraction of the FLRs able to exude xylem sap in a mineral medium with 30 mM mannitol decreased with increasing incubation time. This change was strongly retarded, when the FLRs were incubated in a medium containing glucose instead of mannitol. There was a broad range of variation of the osmotic potential of the test solutions (Ψ_so), wherein the fraction of the FLRs showing an initially reversed flow of the xylem sap varied between zero and unity. A median (M) of the osmotic potential of the xylem sap in FLRs (Ψ_sx) was estimated. It represents the value of Ψ_so that was lower than Ψ_sx in half of the roots of a sample before their transfer to the test solutions (Ψ_sxo). M was dependent on the osmotic potential of the medium used for growth or pre-incubation of the FLRs. Its value was not dependent on the molecular size of the osmolytes used to adjust Ψ_so, including dextran 8, which is excluded from cell walls. In all of the studied plants, M was lower than the osmotic potential of the xylem sap collected from the root before isolation of the FLRs. To explain this finding it is assumed that FLRs with Ψ_sxo > M had a higher hydraulic conductivity and a larger volume contributed to the exuded sap than those with Ψ_sx < M.     

The role of hydrogen peroxide in chitosan-induced resistance to osmotic stress in rice (Oryza sativa L.)

Chitosan is a biopolymer with multiple agricultural applications. The objective of this research was to identify the mechanism required for the chitosan response. Chitosan clearly induced resistance to osmotic stress (a surrogate for drought stress) in the ‘Leung Pratew 123’ (‘LPT123’) rice (Oryza sativa L. ‘Leung Pratew123’) by enhancing plant growth and maintenance of the photosynthetic pigments during osmotic stress, but not in the derived mutated line, LPT123-TC171. Hydrogen peroxide (H₂O₂) was increased after osmotic stress in both lines, but higher levels were found in the LPT123 cultivar. Chitosan application did not affect the H₂O₂ or glutathione content under the osmotic stress condition in the LPT123 cultivar, but decreased H₂O₂ accumulation in the LPT123-TC171 line. The 20-fold lower glutathione level in the LPT123 cultivar suggested a low glutathione-ascorbate cycle activity that would lead to the higher H₂O₂ levels. Whereas, the chitosan-mediated reduction in glutathione levels in the LPT123-TC171 line during osmotic stress suggested a higher glutathione-ascorbate cycle activity leading to low H₂O₂ levels. Additionally, a higher peroxidase and catalase activity following chitosan treatment of the LPT123-TC171 line supports the lower observed H₂O₂ level. The lipid peroxidation after osmotic stress was decreased by chitosan treatment in LPT123, but not in LPT123-TC171. The exogenous H₂O₂ application with chitosan treatment in LPT123-TC171 could enhance plant growth during osmotic stress. It is concluded that the limited H₂O₂ level, the signal molecule for chitosan responses in the LPT123-TC171 line, resulted in no beneficial effects of chitosan application for osmotic stress. Therefore, H₂O₂ is proposed to be one of the key components for plant growth stimulation during osmotic (drought) stress by chitosan.     

Oxygen transfer efficiency and environmental osmolarity response to neutralizing agents on l-lactic acid production efficiency by Lactobacillus paracasei

The effects of Ca(OH)₂, NH₄OH and NaOH as neutralizing agents on the efficiency of l-lactic acid production by Lactobacillus paracasei were investigated in this study. Fermentation performance with Ca(OH)₂ was superior to NH₄OH and NaOH because it had the highest oxygen transfer rate (OTR) and lowest environmental osmotic pressure. Much smaller bubbles were generated using a calcium lactate solution compared with those generated using ammonium lactate and sodium lactate solutions, indicating that Ca(OH)₂ had the highest OTR. Moreover, experiments demonstrated that ammonium lactate and sodium lactate caused more severe osmotic stress on cell growth than calcium lactate. In conclusion, the effects of neutralizing agents on l-lactic acid production efficiency could be ascribed to the contribution of lactates to OTR and environmental osmotic stress.     

Strategies of ROS regulation and antioxidant defense during transition from C3 to C4 photosynthesis in the genus Flaveria under PEG-induced osmotic stress

In the present study, we aimed to elucidate how strategies of reactive oxygen species (ROS) regulation and the antioxidant defense system changed during transition from C₃ to C₄ photosynthesis, by using the model genus Flaveria, which contains species belonging to different steps in C₄ evolution. For this reason, four Flaveria species that have different carboxylation mechanisms, Flaveria robusta (C₃), Flaveria anomala (C₃–C₄), Flaveria brownii (C₄-like) and Flaveria bidentis (C₄), were used. Physiological (growth, relative water content (RWC), osmotic potential), and photosynthetical parameters (stomatal conductance (g_s), assimilation rate (A), electron transport rate (ETR)), antioxidant defense enzymes (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductases(GR)) and their isoenzymes, non-enzymatic antioxidant contents (ascorbate, glutathione), NADPH oxidase (NOX) activity, hydrogen peroxide (H₂O₂) content and lipid peroxidation levels (TBARS) were measured comparatively under polyethylene glycol (PEG 6000) induced osmotic stress. Under non-stressed conditions, there was a correlation only between CAT (decreasing), APX and GR (both increasing) and the type of carboxylation pathways through C₃ to C₄ in Flaveria species. However, they responded differently to PEG-induced osmotic stress in regards to antioxidant defense. The greatest increase in H₂O₂ and TBARS content was observed in C₃F. robusta, while the least substantial increase was detected in C₄-like F. brownii and C₄F. bidentis, suggesting that oxidative stress is more effectively countered in C₄-like and C₄ species. This was achieved by a better induced enzymatic defense in F. bidentis (increased SOD, CAT, POX, and APX activity) and non-enzymatic antioxidants in F. brownii. As a response to PEG-induced oxidative stress, changes in activities of isoenzymes and also isoenzymatic patterns were observed in all Flaveria species, which might be related to ROS produced in different compartments of cells.     

Osmotic Adjustment in Cotton (Gossypium hirsutum L.) Leaves and Roots in Response to Water Stress

The relative magnitude of adjustment in osmotic potential (ψ_s) of water-stressed cotton (Gossypium hirsutum L.) leaves and roots was studied using plants raised in pots of sand and grown in a growth chamber. One and three water-stress preconditioning cycles were imposed by withholding water, and the subsequent adjustment in solute potential upon relief of the stress and complete rehydration was monitored with thermocouple psychrometers. Both leaves and roots exhibited a substantial adjustment in ψ_s in response to water stress with the former exhibiting the larger absolute adjustment. The osmotic adjustment of leaves was 0.41 megapascal compared to 0.19 megapascal in the roots. The roots, however, exhibited much larger percentage osmotic adjustments of 46 and 63% in the one and three stress cycles, respectively, compared to 22 and 40% in the leaves in similar stress cycles. The osmotically adjusted condition of leaves and roots decreased after relief of the single cycle stress to about half the initial value within 3 days, and to the well-watered control level within 6 days. In contrast, increasing the number of water-stress preconditioning cycles resulted in significant percentage osmotic adjustment still being present after 6 days in roots but not in the leaves. The decrease in ψ_s of leaves persisted longer in field-grown cotton plants compared to plants of the same age grown in the growth chamber. The advantage of decreased ψ_s in leaves and roots of water-stressed cotton plants was associated with the maintenance of turgor during periods of decreasing water potentials.