Drought and Abscisic Acid Effects on Aquaporin Content Translate into Changes in Hydraulic Conductivity and Leaf Growth Rate: A Trans-Scale Approach

The effects of abscisic acid (ABA) on aquaporin content, root hydraulic conductivity (Lp_r), whole plant hydraulic conductance, and leaf growth are controversial. We addressed these effects via a combination of experiments at different scales of plant organization and tested their consistency via a model. We analyzed under moderate water deficit a series of transformed maize (Zea mays) lines, one sense and three antisense, affected in NCED (for 9-cis-epoxycarotenoid dioxygenase) gene expression and that differed in the concentration of ABA in the xylem sap. In roots, the mRNA expression of most aquaporin PIP (for plasma membrane intrinsic protein) genes was increased in sense plants and decreased in antisense plants. The same pattern was observed for the protein contents of four PIPs. This resulted in more than 6-fold differences between lines in Lp_r under both hydrostatic and osmotic gradients of water potential. This effect was probably due to differences in aquaporin activity, because it was nearly abolished by a hydrogen peroxide treatment, which blocks the water channel activity of aquaporins. The hydraulic conductance of intact whole plants was affected in the same way when measured either in steady-state conditions or via the rate of recovery of leaf water potential after rewatering. The recoveries of leaf water potential and elongation upon rehydration differed between lines and were accounted for by the experimentally measured Lp_r in a model of water transfer. Overall, these results suggest that ABA has long-lasting effects on plant hydraulic properties via aquaporin activity, which contributes to the maintenance of a favorable plant water status.     

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.     

华北落叶松夜间树干液流特征及生长季补水格局

在宁夏六盘山北侧半干旱区的叠叠沟小流域,采用热扩散探针法在2011年生长季监测了华北落叶松(Larix principisrupprechtii)人工林的树干液流速率,分析了夜间树干液流和补水量的变化特征及与气象、土壤水分等环境因子的关系.结果表明:树干液流速率日变化表现为典型的单峰宽峰曲线,且整个生长季均存在微弱的夜间液流,一般表现为逐渐减小,特别是在晴天,且晴天的变幅显著大于雨天.除生长季中期雨天夜间液流平均速率显著高于晴天,生长季初期及末期雨天与晴天的差异并不显著.生长季内,夜间树干补水总量为11.03 mm,占总蒸腾量的7.22％;5月份的树干补水量最大(4.19mm),其他月份的树干补水量明显减小,在0.9-1.7mm的范围波动.但不同月份间的补水贡献率存在明显差异,表现为生长季末期(9、10月)＞初期(5月)＞中期(6-8月).相关分析表明,日补水量与各气象因子关系不大,仅与降水量显著正相关(P＜0.05),与土壤含水率、日间蒸腾量、日蒸腾总量极显著正相关(P＜0.01).夜间补水的月蒸腾贡献率与月均土壤含水率、月均气温、月均日间蒸腾量、月总蒸腾量等显著相关(P＜0.05);而夜间补水的日蒸腾贡献率与日最高气温、日均气温、日间蒸腾量、日均饱和水汽压差、日总蒸腾量、日均太阳辐射强度、日最低气温、日均空气相对湿度、日降水量、土壤含水率等极显著相关(P＜0.01),经逐步回归分析建立了日补水量蒸腾贡献率与环境因子的多元线性模型.     

Aquaporin genes GintAQPF1 and GintAQPF2 from Glomus intraradices contribute to plant drought tolerance

Arbuscular mycorrhizal (AM) symbiosis, established between AM fungi (AMF) and roots of higher plants, occurs in most terrestrial ecosystems. It has been well demonstrated that AM symbiosis can improve plant performance under various environmental stresses, including drought stress. However, the molecular basis for the direct involvement of AMF in plant drought tolerance has not yet been established. Most recently, we cloned two functional aquaporin genes, GintAQPF1 and GintAQPF2, from AM fungus Glomus intraradices. By heterologous gene expression in yeast, aquaporin localization, activities and water permeability were examined. Gene expressions during symbiosis in expose to drought stress were also analyzed. Our data strongly supported potential water transport via AMF to host plants. As a complement, here we adopted the monoxenic culture system for AMF, in which carrot roots transformed by Ri-T DNA were cultured with Glomus intraradices in two-compartment Petri dishes, to verify the aquaporin gene functions in assisting AMF survival under polyethylene glycol (PEG) treatment. Our results showed that 25% PEG significantly upregulated the expression of two aquaporin genes, which was in line with the gene functions examined in yeast. We therefore concluded that the aquaporins function similarly in AMF as in yeast subjected to osmotic stress. The study provided further evidence to the direct involvement of AMF in improving plant water relations under drought stresses.     

Adaptation of the Common Ice Plant to High Copper and Zinc Concentrations and Their Potential Using for Phytoremediation

A facultative halophite Mesembryanthemum crystallinum L. (the common ice plant) was shown to grow successively at the high concentrations of Cu and Zn. Although 25 μM CuSO₄ or 800 μM ZnSO₄ retarded markedly plant growth, they did not interfere with the completion of plant development and the formation of viable seeds. In such plants, leaves accumulated more than 200 μg of Cu and 1700 μg of Zn per 1 g of dry weight. A damaging effect of heavy metals (HMs) was manifested in a reduced content of water in leaves and proline accumulation in them. As copper is a metal with transient valence, copper salts are more toxic than zinc salts, which was manifested in a stronger inhibition of the chlorophyll synthesis. Both HMs induced oxidative stress, as evident from increased activities of guaiacol peroxidase and lipoxygenase. Moderate Cu and Zn concentrations did not damage cell membranes in leaves, as evident from the absence of their action on electrolyte leakage either under optimum conditions or after heat treatment. A capability of a substantial HM accumulation by the common ice plant and their considerable transport to shoots (up to 50 μg of Cu and 560 μg of Zn per plant) make it possible to consider the common ice plant as a promising phytoremediator. __________ Translated from Fiziologiya Rastenii, Vol. 52, No. 6, 2005, pp. 848–858. Original Russian Text Copyright ? 2005 by Kholodova, Volkov, Kuznetsov.     

Stomatal and hydraulic conductance in growing sugarcane: stomatal adjustment to water transport capacity*

Abstract Stomatal conductance per unit leaf area in well-irrigated field- and greenhouse-grown sugarcane increased with leaf area up to 0.2 m² plant ¹, then declined so that maximum transpiration per plant tended to saturate rather than increase linearly with further increase in leaf area. Conductance to liquid water transport exhibited parallel changes with plant size. This coordiantion of vapour phase and liquid phase conductances resulted in a balance between water loss and water transport capacity, maintaining leaf water status remarkably constant over a wide range of plant size and growing conditions. The changes in stomatal conductance were not related to plant or leaf age. Partial defoliation caused rapid increases in stomatal conductance, to re-establish the original relationship with remaining leaf area. Similarly, pruning of roots caused rapid reductions in stomatal conductance, which maintained or improved leaf water status. These results suggest that sugarcane stomata adjusted to the ratio of total hydraulic conductance to total transpiring leaf area. This could be mediated by root metabolites in the transpiration stream, whose delivery per unit leaf area would be a function of the relative magnitudes of root system size, transpiration rate and leaf area.     

Quantitative analysis of transpiration stream dynamics in an intact cucumber stem by a heat flux control method

Water flux of transpiration stream in an intact stem of the 10 leaf stage cucumber plant (Cucumis sativus L. cv. Chojitsu-Ochiai) was measured by a novel system of heat flux control method with a resolution of 1 × 10⁻³ grams per second and a time constant of 1 minute; two heat flux control sensors were attached to the seventh internode and the stem base. The transpiration stream responded clearly to leaf transpiration and root water absorption when the plant was exposed to light, and the water flux at the stem base corresponded to the transpiration rate per plant in steady state. Root water absorption lagged about 10 minutes behind leaf transpiration. Dynamics of water fluxes were affected by the lag of water absorption in roots, and temporary water loss caused by rapid increase in leaf transpiration was buffered by about 5% of the water content in the stem.     

Quantitative neutron imaging of water distribution,venation network and sap flow in leaves

Main conclusion

Quantitative neutron imaging is a promising technique to investigate leaf water flow and transpiration in real time and has perspectives towards studies of plant response to environmental conditions and plant water stress. The leaf hydraulic architecture is a key determinant of plant sap transport and plant–atmosphere exchange processes. Non-destructive imaging with neutrons shows large potential for unveiling the complex internal features of the venation network and the transport therein. However, it was only used for two-dimensional imaging without addressing flow dynamics and was still unsuccessful in accurate quantification of the amount of water. Quantitative neutron imaging was used to investigate, for the first time, the water distribution in veins and lamina, the three-dimensional venation architecture and sap flow dynamics in leaves. The latter was visualised using D₂O as a contrast liquid. A high dynamic resolution was obtained by using cold neutrons and imaging relied on radiography (2D) as well as tomography (3D). The principle of the technique was shown for detached leaves, but can be applied to in vivo leaves as well. The venation network architecture and the water distribution in the veins and lamina unveiled clear differences between plant species. The leaf water content could be successfully quantified, though still included the contribution of the leaf dry matter. The flow measurements exposed the hierarchical structure of the water transport pathways, and an accurate quantification of the absolute amount of water uptake in the leaf was possible. Particular advantages of neutron imaging, as compared to X-ray imaging, were identified. Quantitative neutron imaging is a promising technique to investigate leaf water flow and transpiration in real time and has perspectives towards studies of plant response to environmental conditions and plant water stress.     

Zinc treatment of hydroponically grown barley plants causes a reduction in root and cell hydraulic conductivity and isoform‐dependent decrease in aquaporin gene expression

The cellular and molecular basis of a reduction in root water uptake in plants exposed to heavy metals such as zinc (Zn) is poorly studied. The aim of the present study on hydroponically grown barley (Hordeum vulgare) was to test whether any reduction in root hydraulic conductivity (Lp) in response to Zn treatment is accompanied by a reduction in cell Lp and gene expression level of aquaporin (AQP) isoforms. Plants were grown in the presence of 0.25 μM, (control), 0.1 and 1 mM Zn in the root medium and analysed when they were 16–18 days old. Root and cell Lp was determined through exudation and cell pressure probe analyses, respectively, and gene expression of five candidate AQPs was analysed [real time quantitative polymerase chain reaction (PCR)]. Zinc treatments caused significant reductions (25–83%) in transpiration rate, root and shoot fresh weight, surface area and stomatal conductance. Zinc concentrations in tissues increased more than 100‐fold. Root Lp decreased by 24% (0.1 mM Zn) and 58% (1 mM Zn), while cell Lp decreased by 45 and 71%, respectively. Gene expression of AQPs decreased by 14–80%; decreases were statistically significant for HvPIP1;3, HvPIP2;4 and HvPIP2;5. Turgor in root cortex cells was not reduced by Zn treatments. It is concluded that reductions in plant water flow in response to Zn treatment are facilitated through decreases in root (Lp) and shoot (stomata) hydraulics. The decrease in root Lp is facilitated through reductions in cell Lp and AQP gene expression and may also reflect increased suberization in the endodermis.     

Over-expression of PIP2;5 aquaporin alleviates gas exchange and growth inhibition in poplars exposed to mild osmotic stress with polyethylene glycol

The effects of mild osmotic stress conditions on aquaporin-mediated water transport are not well understood. In the present study, mild osmotic stress treatments with 20 and 50 g L^?1 polyethylene glycol 6000 (PEG) in Hoagland’s mineral solution were applied for 3 weeks under controlled environmental conditions to transgenic Populus tremula × Populus alba plants constitutively over-expressing a Populus PIP2;5 aquaporin and compared with the wild-type plants. The PEG treatments resulted in growth reductions and triggered changes in net photosynthesis, transpiration, stomatal conductance and root hydraulic conductivity in the wild-type plants. However, height growth, leaf area, gas exchange, and root hydraulic conductivity were less affected by the PEG treatments in PIP2;5-over-expressing poplar lines. These results suggest that water transport across the PIP2;5 aquaporin is an important process contributing to tolerance of mild osmotic stress in poplar. Greater membrane abundance of PIP2;5 was most likely the factor that was responsible for higher root hydraulic conductivity leading to improved plant water flux and, consequently, greater gas exchange and growth rates under mild osmotic stress conditions. The results also provide evidence for the functional significance of PIP2;5 aquaporin in water transport and its strong link to growth processes in poplar.     

High Expression of the Tonoplast Aquaporin ZmTIP1 in Epidermal and Conducting Tissues of Maize

Aquaporins are integral membrane proteins of the tonoplast and the plasma membrane that facilitate the passage of water through these membranes. Because of their potentially important role in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important. We used in situ hybridization to examine the expression pattern of the tonoplast aquaporin ZmTIP1 in different organs of maize (Zea mays L.). This tonoplast water channel is highly expressed in the root epidermis, the root endodermis, the small parenchyma cells surrounding mature xylem vessels in the root and the stem, phloem companion cells and a ring of cells around the phloem strand in the stem and the leaf sheath, and the basal endosperm transfer cells in developing kernels. We postulate that the high level of expression of ZmTIP1 in these tissues facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required.     

Irrigation effects on daily and seasonal variations of trunk sap flow and leaf water relations in olive trees

Irrigation effects on whole-plant sap flow and leaf-level water relations were characterised throughout a growing season in an experimental olive (Olea europaea L.) orchard. Atmospheric evaporative demand and soil moisture conditions for irrigated and non-irrigated olive trees were also monitored. Whole-plant water use in field-grown irrigated and rain fed olive trees was determined using a xylem sap flow method (compensation heat-pulse velocity). Foliage gas exchange and water potentials were determined throughout the experimental period. Physiological parameters responded diurnally and seasonally to variations in tree water status, soil moisture conditions and atmospheric evaporative demand. There was a considerable degree of agreement between daily transpiration deduced from heat-pulse velocity and that determined by calibration using the Penman–Monteith equation in the field. Summer drought caused decreasing leaf gas exchange and water potentials, and a progressive increase in hydraulic conductance (stronger in non-irrigated than irrigated trees), probably attributable to modifications in hydraulic properties at the soil-root interface. Negligible hysteresis, attributable to low plant capacitance, was observed in the relationship between leaf water potential and sap flow. A proportional decrease in maximum daily leaf conductance with increasing vapour pressure deficit was observed, while mean daytime canopy stomatal conductance decreased with the season. As a result, plant water use was limited and excessive drought stress prevented. Non-irrigated olive trees recovered after the summer drought, showing a physiological behaviour similar to that of irrigated trees. In addition to physiological and environmental factors, there are endogenous keys (chemical signals) influencing leaf level parameters. Olive trees are confirmed to be economical and sparing users of soil water, with an efficient xylem sap transport, maintenance of significant gas exchange and transpiration, even during drought stress.     

Aquaporin Content in Cell Membranes of Mesembryanthemum crystallinum as Affected by Plant Transition from C3 to CAM Type of Photosynthesis

Western-blot analysis was used to determine the contents of aquaporin isoforms MIP A, MIP B, and MIP C in cell membranes isolated from roots and leaves of Mesembryanthemum crystallinum plants with C₃ and Crassulacean acid metabolism (CAM) types of photosynthesis. These membrane preparations were also used to assess osmotic water permeability; to this end, the rate of osmotic vesicle shrinking was registered as the light scattering intensity by the method of stopped flow. The cell membranes represented by the plasmalemma and the tonoplast-enriched fraction were obtained by separating the microsomes in a two-phase polymer system. Plant transition from C₃ to CAM-photosynthesis occurred in the course of plant development or was induced by salinization. All three isoforms under study were found in the plasma membranes of roots and leaves of the C₃ plants, whereas in the CAM plants, independent of the transition-inducing factor, the aquaporin contents notably decreased in the leaf membranes and remained unchanged in the roots. In the membranes isolated from roots and leaves of the C₃ plants, the values of osmotic water permeability exceeded two–threefold the corresponding indices characteristic of the CAM plants. The authors believe that aquaporin isoforms in M. crystallinum are under the organ- and tissue-specific control.     

Abscisic Acid is not the only stomatal inhibitor in the transpiration stream of wheat plants

Xylem sap was collected from the transpiration stream of wheat (Triticum aestivum L.) plants and assayed for the presence of an inhibitor of transpiration using leaves detached from well-watered plants. Transpiration of detached leaves was reduced by nearly 60% by sap collected from plants in drying soil, and to a lesser extent (about 25%) by sap from plants in well-watered soil. As the soil dried the abscisic acid (ABA) concentration in the sap increased by about 50 times to 5 × 10⁻⁸ molar. However, the ABA in the sap did not cause its inhibitory activity. Synthetic ABA of one hundred times this concentration was needed to reduce transpiration rates of detached leaves to the same extent. Furthermore, inhibitory activity of the sap was retained after its passage through an immunoaffinity column to remove ABA. Xylem sap was also collected by applying pressure to the roots of plants whose leaf water status was kept high as the soil dried. Sap collected from these plants reduced transpiration to a lesser extent than sap from nonpressurised plants. This suggests that the inhibitory activity was triggered partly by leaf water deficit and partly by root water deficit.     

Participation of Plant Hormones in Growth Resumption of Wheat Shoots Following Short-Term NaCl Treatment

The effects of sodium-chloride salinity on the leaf elongation rate, transpiration rate, cell sap osmolality, and phytohormone content in 7-day-old shoots of durum wheat (Triticum durum L.) were studied. Leaf growth was suppressed under the salinity stress and resumed 1 h after NaCl removal. The resumption of leaf growth coincided with a decrease in the transpiration rate due to the rapid ABA accumulation in the differentiation leaf zone. The increased IAA concentration in the growing leaf zone promoted the formation of the attraction signal. The authors concluded that the changes in phytohormonal status in wheat plants occurred already following short-term (up to 1 h) salinity and were directed to the maintenance of plant growth under these conditions.     

Drought, abscisic acid and transpiration rate effects on the regulation of PIP aquaporin gene expression and abundance in Phaseolus vulgaris plants

BACKGROUND AND AIMS: Drought causes a decline of root hydraulic conductance, which aside from embolisms, is governed ultimately by aquaporins. Multiple factors probably regulate aquaporin expression, abundance and activity in leaf and root tissues during drought; among these are the leaf transpiration rate, leaf water status, abscisic acid (ABA) and soil water content. Here a study is made of how these factors could influence the response of aquaporin to drought. METHODS: Three plasma membrane intrinsic proteins (PIPs) or aquaporins were cloned from Phaseolus vulgaris plants and their expression was analysed after 4 d of water deprivation and also 1 d after re-watering. The effects of ABA and of methotrexate (MTX), an inhibitor of stomatal opening, on gene expression and protein abundance were also analysed. Protein abundance was examined using antibodies against PIP1 and PIP2 aquaporins. At the same time, root hydraulic conductance (L), transpiration rate, leaf water status and ABA tissue concentration were measured. KEY RESULTS: None of the treatments (drought, ABA or MTX) changed the leaf water status or tissue ABA concentration. The three treatments caused a decline in the transpiration rate and raised PVPIP2;1 gene expression and PIP1 protein abundance in the leaves. In the roots, only the drought treatment raised the expression of the three PIP genes examined, while at the same time diminishing PIP2 protein abundance and L. On the other hand, ABA raised both root PIP1 protein abundance and L. CONCLUSIONS: The rise of PvPIP2;1 gene expression and PIP1 protein abundance in the leaves of P. vulgaris plants subjected to drought was correlated with a decline in the transpiration rate. At the same time, the increase in the expression of the three PIP genes examined caused by drought and the decline of PIP2 protein abundance in the root tissues were not correlated with any of the parameters measured.     

Response of three broccoli cultivars to salt stress, in relation to water status and expression of two leaf aquaporins

The aim of this study was to compare differences in water relations in the leaves of three broccoli cultivars and differential induction of the expression of PIP2 aquaporin isoforms under salt stress. Although broccoli is known to be moderately tolerant to salinity, scarce information exists about the involvement of leaf aquaporins in its adaptation to salinity. Thus, leaf water relations, leaf cell hydraulic conductivity (Lp_c), gas exchange parameters and the PIP2 expression pattern were determined for short- (15 h) and long- (15 days) term NaCl treatments. In the long term, the lower half-time of water exchange in the cells of cv. Naxos, compared with Parthenon and Chronos, and its increased PIP2 abundance may have contributed to its Lp_c maintenance. This unmodified Lp_c in cv. Naxos under prolonged salinity may have diluted NaCl in the leaves, as suggested by lower Na⁺ concentrations in the leaf sap. By contrast, the increase in the half-time of water exchange and the lower PIP2 abundance in cvs. Chronos and Parthenon would have contributed to the reduced Lp_c values. In cv. Parthenon, there were no differences between the ε values of control and salt-stressed plants; in consequence, cell turgor was enhanced. Also, the increases in BoPIP2;2 and BoPIP2;3 expression in cv. Chronos for the short-term NaCl treatment suggest that these isoforms are involved in osmotic regulation as downstream factors in this cultivar, in fact, in the short-term, Chronos had a significantly reduced osmotic potential and higher PIP2 isoforms expression.     

Modification of leaf apoplastic pH in relation to stomatal sensitivity to root-sourced abscisic acid signals

The confocal microscope was used to determine the pH of the leaf apoplast and the pH of microvolumes of xylem sap. We quantified variation in leaf apoplast and sap pH in relation to changes in edaphic and atmospheric conditions that impacted on stomatal sensitivity to a root-sourced abscisic acid signal. Several plant species showed significant changes in the pH of both xylem sap and the apoplast of the shoot in response to environmental perturbation. Xylem sap leaving the root was generally more acidic than sap in the midrib and the apoplast of the leaf. Increasing the transpiration rate of both intact plants and detached plant parts resulted in more acidic leaf apoplast pHs. Experiments with inhibitors suggested that protons are removed from xylem sap as it moves up the plant, thereby alkalinizing the sap. The more rapid the transpiration rate and the shorter the time that the sap resided in the xylem/apoplastic pathway, the smaller the impact of proton removal on sap pH. Sap pH of sunflower (Helianthus annuus) and Commelina communis did not change significantly as soil dried, while pH of tomato (Lycopersicon esculentum) sap increased as water availability in the soil declined. Increasing the availability of nitrate to roots also significantly alkalinized the xylem sap of tomato plants. This nitrogen treatment had the effect of enhancing the sensitivity of the stomatal response to soil drying. These responses were interpreted as an effect of nitrate addition on sap pH and closure of stomata via an abscisic acid-based mechanism.