Thiourea mediated regulation in the expression profile of aquaporins and its impact on water homeostasis under salinity stress in Brassica juncea roots

Bioregulatory molecules such as thiourea (TU) play an important role in imparting stress tolerance to crops. However, the molecular mechanism involved in the TU-mediated tolerance has not been elucidated. Towards this endeavour, the expression profile of various PIPs (plasma membrane intrinsic proteins) was studied under salt stress (NaCl; 700 mM) with/without thiourea (TU; 6.5 mM) at different time periods in roots of Brassica juncea. Various aquaporin isoforms demonstrated an upregulation upon salinity stress imposition, whereas they were downregulated upon TU supplementation. TU treatment also led to a decrease in the accumulation of reactive oxygen species and delimited the need for an enhanced accumulation of osmolytes. The vacuolar pH was also maintained in NaCl + TU treatment as demonstrated by in vivo ³¹P NMR of roots. In conclusion, TU supplementation to salt stressed seedlings was found to maintain the water homeostasis of roots through coordinated regulation of different PIP isoforms.     

Peroxidase mediated hydrogen peroxide production in barley roots grown under stress conditions

All applied metals (Co, Al, Cu, Cd) and NaCl inhibited barley root growth. No root growth inhibition was caused by drought exposure, in contrast to cold treatment. 0.01 mM H₂O₂ stimulated root growth and GA application did not affect root growth at all. Other activators and inhibitors of H₂O₂ production (SHAM, DTT, 10 mM H₂O₂, 2,4-D) inhibited root growth. Loss of cell viability was most significant after Al treatment, followed by Cd and Cu, but no cell death was induced by Co. Drought led to slight increase in Evans blue uptake, whereas neither NaCl nor cold influenced this parameter. DTT treatment caused slight increase in Evans blue uptake and significant increases were detected after 2,4-D and 10 mM H₂O₂ treatment, but were not induced by others stressors. Metal exposure increased guaiacol-POD activity, which was correlated with oxidation of NADH and production of H₂O₂. Exposure to drought caused a minor change in NADH oxidation, but neither H₂O₂ production nor guaiacol-POD activity was increased. Cold and NaCl application decreased all monitored activities. Increase in NADH oxidation and guaiacol-POD activity was caused by 10 mM H₂O₂ and 0.01 mM 2,4-D treatment, which also caused enhancement of H₂O₂ production. Slight inhibition of all activities was caused by 0.01 mM H₂O₂, GA, DTT; more pronounced inhibition was detected after SHAM treatment. The role of H₂O₂ production mediated by POD activity in relation to root growth and cell viability under exposure to some abiotic stress factors is discussed.     

Identification of aquaporins and deciphering their role under salinity stress in pomegranate (Punica granatum)

Journal of Plant Biochemistry and Biotechnology - Aquaporins (AQPs), pore-forming proteins, are known to involve in the transport of water and many other small solutes, and play a diverse role in...     

Water-deficit tolerance in citrus is mediated by the down regulation of PIP gene expression in the roots

Water deficit (WD) is a growing problem in agriculture. In citrus crops, genetically-determined rootstock characteristics are important factors influencing plant responses to WD. Aquaporins are involved in regulating the water supply to the plant by mediating water flow through the cell membranes. Recent studies support a direct role for aquaporins in plant water relations and demonstrate their involvement in WD tolerance. This study investigates the relationship between photosynthetic and water-balance parameters with aquaporin expression levels and hydraulic conductance of roots (Kr) in conditions of moderate WD in citrus rootstocks. The plant materials used were the rootstocks Poncirus trifoliata (L.) Raf. (PT), Cleopatra mandarin (Citrus reshni Hort ex Tan.) (CM) and 030115 (a hybrid of the two former rootstocks), all grafted with the citrus variety ??Valencia Late?? (C. sinensis (L.) Osb). Plants were irrigated with two differents irrigation doses (normal irrigation and moderate WD) during 70 days and leaf water potential (??s), net CO₂ assimilation (A_CO2), transpiration, stomatal conductance (gs) and substomatal CO₂ concentration (Ci) were measured periodically under both irrigation conditions. Kr and PIP1 and PIP2 gene expression levels in fine roots of control plants and plants subjected to WD on day 43 of the experiment were determined. Under WD conditions, the hybrid 030115 drastically reduced aquaporin expression and Kr, accompanied by a loss of plant vigour but without reducing the net CO₂ assimilation (A_CO2). PT maintained the same aquaporin expression level and similar Kr under WD as under normal irrigation conditions, but suffered a sharp reduction in A_CO2. CM, which has lower Kr and aquaporin expression than PT under both normal irrigation conditions and WD, responded better to water stress conditions than PT. Low aquaporin levels, or down-regulated aquaporin expression, accompanied by decreased plant vigour led to decreased plasma membrane permeability, thereby facilitating water retention in the cells under water stress conditions. This may induce water stress tolerance in citrus rootstocks.     

Combined effect of boron and salinity on water transport: The role of aquaporins

Boron toxicity is an important disorder that can limit plant growth on soils of arid and semi arid environments throughout the world. Although there are several reports about the combined effect of salinity and boron toxicity on plant growth and yield, there is no consensus about the experimental results. A general antagonistic relationship between boron excess and salinity has been observed, however the mechanisms for this interaction is not clear and several options can be discussed. In addition, there is no information, concerning the interaction between boron toxicity and salinity with respect to water transport and aquaporins function in the plants. We recently documented in the highly boron- and salt-tolerant the ecotype of Zea mays L. amylacea from Lluta valley in Northern Chile that under salt stress, the activity of specific membrane components can be influenced directly by boron, regulating the water uptake and water transport through the functions of certain aquaporin isoforms.Key words: aquaporins, boron, salinity, water relations, Zea maysHigh concentrations of boron are often associated to saline soils in semi arid and/or arid climates and frequently crops are exposed to both stresses simultaneously.¹ As there is no a unique plant response to combination of salinity and boron toxicity, several mechanisms has been proposed to explain the experimental results. Some reports showed no additive effects of boron and salinity on shoot weight of different cultivars suggesting independent of the interaction.^2–5 However, additive effects^6–9 have been also proposed and the interaction of boron and salinity declined the rate of germination and limited growth in maize and sorghum plants.¹⁰ No explanation is currently available for these contradictory observations. Recently, the Abbot method has been applied to characterize the combined effect of boron and salinity at toxic levels in pepper plants, observing mainly an antagonistic relationship regarding growth and yield.¹¹ Antagonism between salinity and boron may be the result of decreased toxicity of boron in the presence of NaCl, reduced toxicity of NaCl in the presence of boron, or both together. Letey et al.,¹² have reported that increased soil salinity may also reduce boron movement to the broccoli plants and hence result in a reduction of boron toxicity symptoms. Reduction of boron accumulation in leaves in the presence of salinity has been also reported for melon,⁵ tomato⁸ jack pine¹³ and grapesvines¹⁴ and could be the result of the reduced rates of transpiration in plants where boron is transported via xylem as consequence of the osmotic effect of the salt. On the other hand, it has been observed that concentration of Na⁺ in leaves decreased with increasing addition of boron to the soil, probably due to the inhibition in root growth and reduction in root density caused by the boron treatment.¹⁵ Grieve and Poss⁷ found in wheat plants that the Cl⁻ content in the leaves was reduced when boron was increased. Similar results were reported in pepper plants suggesting that boron could reduce Cl⁻ toxicity.¹¹ Also, in our recent report although a nutrient imbalance resulted from the effect of salinity or boron alone, a general optimisation was observed when both treatments were applied together.¹⁶Under saline conditions, an optimal water balance is important in order to maintain the plant homeostasis and aquaporins may be one of the mechanisms involved under environmental and developmental changes.^17–19 However, there is no information concerning plant water uptake and transport in response to combined excess boron and salinity.It has been reported that, at high external B concentrations, considerable B transport occurs through the plasma membrane aquaporins, and a specific membrane intrinsic protein (MIP) has been described.²⁰ Thus boron uptake across the plasma membrane, by permeation through the lipid membrane and aquaporins, may be greatly influenced by the plant tolerance to salinity, through the associated changes in root hydraulic conductivity. Wimmer et al.,²¹ showed that salinity could interact with boron toxicity by a combined effect on boron and water uptake. In addition, we reported that the reduction of aquaporin functionality in NaCl-exposed plants could induce the reduction of plant boron concentration, producing a beneficial effect.²²Recently, we showed in a tolerant ecotype of maize a different pattern for PIP1 and PIP2 protein content under the application of excess of boron in combination with salinity, suggesting a differential aquaporin response in this cultivar and pointed out the complexity of the interaction.¹⁶ These results were in consonance with the previous observation that different aquaporin isoforms may represent a response to environmental changes.^18,19,²³ Thus, we concluded that the activity of specific membrane components can be influenced by boron under salt stress regulating the functions of certain aquaporin isoforms as possible components of the salinity tolerance mechanism. However, although a fine water transport control through the aquaporins could be necessary in order to reduce the accumulation of toxic boron levels in the tissues, the contribution of each isoform to water transport through the plasma membrane under boron-salinity combination must be elucidated.     

Analyses of abiotic stress and brassinosteroid-related some genes in barley roots grown under salinity stress and HBR treatments: Expression profiles and phylogeny

We aimed to investigate abiotic stress (WAK, HvPIP1.1, HvPIP1.2, HvPIP1.3, HvPIP1.5, CYCD3, DREB2) and brassinosteroid-related gene (DWARF4) expressions in barley (Hordeum vulgare L. cv. “Hilal”) roots grown under different salt (150 and 250 mM), HBR (0.5 and 1 μM), and salt + HBR applications during 48 and 72 h at dark with their controls. Phylogenetic trees were also constructed to observe relationships among genes found in other plants. The expression of HvPIP1.2 and WAK reduced after salt treatment while HvPIP1.3, DREB2 and DWARF4 expressions increased. HvPIP1.1, HvPIP1.2, HvPIP1.3, HvPIP1.5 and DWARF4 expressions were upregulated under only HBR applications. Salt + HBR treatments increased HvPIP1.1, DREB2 and DWARF4 but decreased HvPIP1.2. Phylogenetic analyses indicated that Oryza sativa L. shared similar sequences with HvPIP1.5. CYCD3 could diverge relatively earlier from cyclin genes during evolution as it segregates in a distinct clade. Sorghum bicolor showed sequence homology with DREB2. Oryza australiensis L. and DWARF4 were found in the same clade. To our knowledge, this is the first detailed report related to salt stress and HBR applications in terms of the expression of different genes in barley, providing a valuable information for molecular breeding improvement of stress-related traits.     

Tissue and cell-specific localization of rice aquaporins and their water transport activities

Water transport in plants is greatly dependent on the expression and activity of water transport channels, called aquaporins. Here, we have clarified the tissue- and cell-specific localization of aquaporins in rice plants by immunoblotting and immunocytochemistry using seven isoform-specific aquaporin antibodies. We also examined water transport activities of typical aquaporin family members using a yeast expression system in combination with a stopped-flow spectrophotometry assay. OsPIP1 members, OsPIP2;1, OsTIP1;1 and OsTIP2;2 were expressed in both leaf blades and roots, while OsPIP2;3, OsPIP2;5 and OsTIP2;1 were expressed only in roots. In roots, large amounts of aquaporins accumulated in the region adjacent to the root tip (around 1.5-4 mm from the root tip). In this region, cell-specific localization of the various aquaporin members was observed. OsPIP1 members and OsTIP2;2 accumulated predominantly in the endodermis and the central cylinder, respectively. OsTIP1;1 showed specific localization in the rhizodermis and exodermis. OsPIP2;1, OsPIP2;3 and OsPIP2;5 accumulated in all root cells, but they showed higher levels of accumulation in endodermis than other cells. In the region at 35 mm from the root tip, where aerenchyma develops, aquaporins accumulated at low levels. In leaf blades, OsPIP1 members and OsPIP2;1 were localized mainly in mesophyll cells. OsPIP2;1, OsPIP2;3, OsPIP2;5 and OsTIP2;2 expressed in yeast showed high water transport activities. These results suggest that rice aquaporins with various water transport activities may play distinct roles in facilitating water flux and maintaining the water potential in different tissues and cells.     

Phosphatidylethanolamine in wheat and barley leaves under water stress

Phosphatidylethanolamine could not be detected in the leaves of less drought-tolerant varieties of wheat (S-308) and barley (BG-25) when the plants wer     

A putative role for TIP and PIP aquaporins in dynamics of leaf hydraulic and stomatal conductances in grapevine under water stress and re‐watering

We examined the role of aquaporins (AQPs) in regulating leaf hydraulic conductance (K_leaf) in Vitis vinifera L. (cv Chardonnay) by studying effects of AQP inhibitors, and AQP gene expression during water stress (WS) and recovery (REC). K_leaf was measured after 3 h of petiole perfusion with different solutions and to introduce inhibitors. The addition of 0.1 mm HgCl₂ to 15 mm KCl reduced K_leaf compared with perfusion in 15 mM KNO₃ or KCl, and these solutions were used for leaves from control, WS and REC plants. Perfusion for 3 h did not significantly alter stomatal conductance (g_s) though expression of VvTIP1;1 was increased. WS decreased K_leaf by about 30% and was correlated with g_s. The expression of VvTIP2;1 and VvPIP2;1 correlated with K_leaf, and VvTIP2;1 was highly correlated with g_s. There was no association between the expression of particular AQPs during WS and REC and inhibition of K_leaf by HgCl₂; however, HgCl₂ treatment itself increased expression of VvPIP2;3 and decreased expression of VvPIP2;1 . Inhibition by HgCl₂ of K_leaf only at early stages of WS and then after REC suggested that apoplasmic pathways become more important during WS. This was confirmed using fluorescent dyes confined to apoplasm or preferentially accumulated in symplasm.     

Immunolocalization of PIP aquaporins in protoplasts from the suspension culture of sugar beet mesophyll under isoosmotic conditions and osmotic stress

Protoplasts from the suspension culture of sugar beet (Beta vulgaris L.) mesophyll were found to change their volume in response to short-term osmotic stress. When the sorbitol concentration in the external medium was increased 1.5-fold (from 0.4 to 0.6 M) or decreased from 0.4 to 0.25 M, the volume of protoplasts decreased and increased, respectively, by 55–60%. These changes started immediately after the shift in osmoticum concentration and completed within 1–3 min. In the presence of an endocytosis marker FM1-43, its fluorescence increased conspicuously after replacement of isotonic medium with the hypotonic solution but did not change after the substitution with hypertonic medium. At the same time, the hypertonic shrinkage of protoplasts was accompanied by accumulation of fluorescent material in the periplasmic space. The western blot analysis with the use of immune serum for conservative sequence of PIP-type aquaporins revealed their presence in the plasmalemma and intracellular membranes. This conclusion was confirmed by indirect immunofluorescence microscopy: the membrane-bound secondary antibodies labeled with a fluorescent probe Alexa-Fluor 488 were distributed comparatively uniformly on the boundary between the plasmalemma and the protoplast internal compartment. As evident from micrographs of protoplasts exposed to the hypotonic treatment, the fluorescence was smoothly distributed over the plasmalemma after protoplast swelling but its intensity was not so bright. The protoplast shrinkage during the hypertonic treatment resulted in heterogeneous alternate distribution of fluorescent and transparent plasmalemma regions, the fluorescence of stained regions being very intense. The results are interpreted as the evidence that the short-term osmotic stress activates exo-and endocytosis. The migrating regions of the plasmalemma were depleted of PIP-type aquaporins; hence, the induction of osmotic stress has no effect on the amount of this type aquaporins in the plasma membrane.     

Phospholipid changes in wheat and barley leaves under water stress

Total phospholipid content of leaves of wheat and barley increased and phospholipid components changed under water stress. Notable among these were the absence of phosphatidyl serine in barley varieties, decrease in phosphatidyl glycerol content in a less drought-tolerant variety of wheat (S-308) and barley (BG-25), and appearance of phosphatidic acid in both crops. The phospholipid content and its components did not return to normal upon release of the stress by subsequent irrigations. Such observations are indicative of water stress effected alterations in membranes.     

Glycolipid changes in wheat and barley chloroplast under water stress

The effect of water stress was studied on the glycolipids of wheat and barley chloroplasts. Glycolipid content of chloroplast decreased in both the crops, when plants were stressed at tillering, ear emergence and grain filling stages. Thin-layer chromatography of glycolipids revealed MGDG and DGDG as the major chloroplast glycolipids. Both of these decreased in both the crops under water stress and the reduction was more in the more water-requiring cultivars of wheat (S-308) and barley (BG-25). Chloroplast glycolipids of less water-requiring cultivars of wheat (C-306) and barley (C-138) showed a good recovery when water stress was released by subsequent irrigations.     

Time‐course analysis of salicylic acid effects on ROS regulation and antioxidant defense in roots of hulled and hulless barley under combined stress of drought,heat and salinity

Greater crop losses can result from simultaneous exposure to a combination of drought, heat and salinity in the field. Salicylic acid (SA), a phenolic phytohormone, can affect a range of physiological and biochemical processes in plants and significantly impacts their resistance to these abiotic stresses. Despite numerous reports involving the positive effects of SA by applying each abiotic stress separately, the mechanism of SA‐mediated adaptation to combined stresses remains elusive. This study, via a time‐course analysis, investigated the role of SA on the roots of hulled and hulless (naked) barley (Hordeum vulgare L. ‘Tarm’ and ‘Özen’, respectively), which differed in salt tolerance, under the combined stress of drought, heat and salt. The combined stress caused marked reductions in root length and increases in proline content in both genotypes; however, Tarm exhibited better adaptation to the triple stress. Under the first 24 h of stress, superoxide dismutase (SOD; EC.1.15.1.1) and peroxidase (POX; EC.1.11.1.7) activity in the Tarm roots increased remarkably, while decreasing in the Özen roots. Furthermore, the Tarm roots showed higher catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) activity than the Özen during the combined stresses. The sensitivity of hulless barley roots may be related to decreasing SOD, POX, CAT and GR activity under stress. Over 72 h of stress, the SA pretreatment improved the APX and GR activity in Tarm and that of POX and CAT in Özen, demonstrating that exogenously applied SA regulates antioxidant defense enzymes in order to detoxify reactive oxygen species. The results of this study suggest that SA treatment may improve the triple‐stress combination tolerance in hulled and hulless barley cultivars by increasing the level of antioxidant enzyme activity and promoting the accumulation of proline. Thus, SA alleviated the damaging effects of the triple stress by improving the antioxidant system, although these effects differed depending on characteristic of the hull of the grain.     

Water transport in maize roots under the influence of mercuric chloride and water stress: a role of water channels

The influence of inhibitor of water channels, HgCl₂, on water diffusion in maize (Zea mays L.) seedling roots was investigated with the pulsed nuclear magnetic resonance (NMR) method. Blocking of water channels decreased the water permeability of cell membranes by 1.5 – 2 times. This effect of HgCl₂ was exhibited only in the roots of seedlings grown in a nutrient solution containing Ca²⁺ and was reversed with Hg-scavenging agent β-mercaptoethanol. Subsequent incubation of Ca²⁺-deficient roots in the nutrient solution with Ca²⁺ recovered the sensitivity to HgCl₂. The water stress decreased water diffusion rates similarly to HgCl₂ and the effects of water stress and HgCl₂ were not additive. The obtained data demonstrate the possibilities of the pulsed NMR method for study of the transmembrane water exchange in vivo in connection with water channel functioning.     

三角叶滨藜根吸水特点与其抗盐性的关系

采用压力室和冰点渗透压计测定了三角叶滨藜在不同浓度NaCl的根系环境溶液中根木质部的压力势和伤流液的渗透势,并利用原子吸收分光光度计测定了植株和伤流液以及环境溶液中Na 含量。结果表明:随着根环境溶液NaCl浓度的增加,三角叶滨藜植株和木质部伤流液中Na 含量虽呈上升趋势,但根系的过滤系数和体内Na 相对累积量逐渐降低,说明三角叶滨藜根细胞对盐分有很强的过滤作用;木质部伤流液的渗透势随着环境溶液渗透势的降低而降低,但根木质部溶液的水势则逐渐高出根外环境溶液的渗透势;表明三角叶滨藜能够利用较低的木质部负压来抵抗根外溶液的低渗透势而反渗透吸水,并利用根细胞对盐分的过滤作用来避免从环境摄取过量的盐分。     

Alterations in glycolipids of wheat and barley leaves under water stress

Glycolipids of leaves from water-stressed and stress-recovered wheat and barley plants were studied. A decrease in the content of total glycolipid, mon     

Additive effects of Na+ and Cl- ions on barley growth under salinity stress

Soil salinity affects large areas of the world's cultivated land, causing significant reductions in crop yield. Despite the fact that most plants accumulate both sodium (Na(+)) and chloride (Cl(-)) ions in high concentrations in their shoot tissues when grown in saline soils, most research on salt tolerance in annual plants has focused on the toxic effects of Na(+) accumulation. It has previously been suggested that Cl(-) toxicity may also be an important cause of growth reduction in barley plants. Here, the extent to which specific ion toxicities of Na(+) and Cl(-) reduce the growth of barley grown in saline soils is shown under varying salinity treatments using four barley genotypes differing in their salt tolerance in solution and soil-based systems. High Na(+), Cl(-), and NaCl separately reduced the growth of barley, however, the reductions in growth and photosynthesis were greatest under NaCl stress and were mainly additive of the effects of Na(+) and Cl(-) stress. The results demonstrated that Na(+) and Cl(-) exclusion among barley genotypes are independent mechanisms and different genotypes expressed different combinations of the two mechanisms. High concentrations of Na(+) reduced K(+) and Ca(2+) uptake and reduced photosynthesis mainly by reducing stomatal conductance. By comparison, high Cl(-) concentration reduced photosynthetic capacity due to non-stomatal effects: there was chlorophyll degradation, and a reduction in the actual quantum yield of PSII electron transport which was associated with both photochemical quenching and the efficiency of excitation energy capture. The results also showed that there are fundamental differences in salinity responses between soil and solution culture, and that the importance of the different mechanisms of salt damage varies according to the system under which the plants were grown.     

Boron tolerance in barley is mediated by efflux of boron from the roots

Many plants are known to reduce the toxic effects of high soil boron (B) by reducing uptake of B, but no mechanism for limiting uptake has previously been identified. The B-tolerant cultivar of barley (Hordeum vulgare L.), Sahara, was shown to be able to maintain root B concentrations up to 50% lower than in the B-sensitive cultivar, Schooner. This translated into xylem concentrations that were approximately 64% lower and leaf concentrations 73% lower in the tolerant cultivar. In both cultivars, B accumulation was rapid and reached a steady-state concentration in roots within 3 h. In Schooner, this concentration was similar to the external medium, whereas in Sahara, the root concentration was maintained at a lower concentration. For this to occur, B must be actively extruded from the root in Sahara, and this is presumed to be the basis for B tolerance in barley. The extrusion mechanism was inhibited by sodium azide but not by treatment at low temperature. Several anion channel inhibitors were also effective in limiting extrusion, but it was not clear whether they acted directly or via metabolic inhibition. The ability of Sahara to maintain lower root B concentrations was constitutive and occurred across a wide range of B concentrations. This ability was lost at high pH, and both Schooner and Sahara then had similar root B concentrations. A predictive model that is consistent with the empirical results and explains the tolerance mechanism based on the presence of a borate anion efflux transporter in Sahara is presented.