Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Impairment of NtAQP1 gene expression in tobacco plants does not affect root colonisation pattern by arbuscular mycorrhizal fungi but decreases their symbiotic efficiency under drought

We investigated in two tobacco (Nicotiana tabacum) plant lines (wildtype or antisense mutant) whether impairment in expression of the plasma membrane aquaporin gene (NtAQP1) affects the arbuscular mycorrhizal (AM) fungal colonisation pattern or the symbiotic efficiency of AM fungi. These two objectives were investigated under well-watered and drought stress conditions. Both plant lines had a similar pattern of root colonisation under well-watered and drought stress conditions. In contrast, under drought stress, AM wildtype plants grew faster than mycorrhizal antisense plants. Plant gas exchange also appeared to depend on the expression of NtAQP1 and parallelled the determined growth increments. The implications of enhanced symplastic water transport via NtAQP1 for the efficiency of the AM symbiosis under drought stress conditions are further discussed.     

Involvement of a glucosinolate (sinigrin) in the regulation of water transport in Brassica oleracea grown under salt stress

Members of the Brassicaceae are known for their contents of nutrients and health‐promoting phytochemicals, including glucosinolates. The concentrations of these chemopreventive compounds (glucosinolate‐degradation products, the bioactive isothiocyanates) may be modified under salinity. In this work, the effect of the aliphatic glucosinolate sinigrin (2‐propenyl‐glucosinolate) on plant water balance, involving aquaporins, was explored under salt stress. For this purpose, water uptake and its transport through the plasma membrane were determined in plants after NaCl addition, when sinigrin was also supplied. We found higher hydraulic conductance (L₀) and water permeability (P_f) and increased abundance of PIP2 aquaporins after the direct administration of sinigrin, showing the ability of the roots to promote cellular water transport across the plasma membrane in spite of the stress conditions imposed. The higher content of the allyl‐isothiocyanate and the absence of sinigrin in the plant tissues suggest that the isothiocyanate is related to water balance; in fact, a direct effect of this nitro‐sulphate compound on water uptake is proposed. This work provides the first evidence that the addition of a glucosinolate can regulate aquaporins and water transport: this effect and the mechanism(s) involved merit further investigation.     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80 mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H⁺-ATPase. However, there was no effect of NaCl on water permeability (P_f) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H⁺-ATPase activity, thereby helping to reduce partially the Na⁺ concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H⁺-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.     

Significance of plasmalemma aquaporins for water‐transport in Arabidopsis thaliana

The plant plasma membrane intrinsic protein, PIP1b, facilitates water transport. These features were characterized in Xenopus oocytes and it has asked whether aquaporins are relevant for water transport in plants. In order to elucidate this uncertainty Arabidopsis thaliana was transformed with an anti-sense construct targeted to the PIP1b gene. Molecular analysis revealed that the anti-sense lines have reduced steady-state levels of PIP1b and the highly homologous PIP1a mRNA. The cell membrane water permeability was analyzed by swelling of protoplasts, which had been transferred into hypotonic conditions. The results indicate that the reduced expression of the specific aquaporins decreases the cellular osmotic water permeability coefficient approximately three times. The morphology and development of the anti-sense lines resembles that of control plants, with the exception of the root system, which is five times as abundant as that of control plants. Xylem pressure measurement suggests that the increase of root mass compensates the reduced cellular water permeability in order to ensure a sufficient water supply to the plant. The results obtained by this study, therefore, clearly demonstrate that aquaporins are important for plant water transport.     

On the pH regulation of plant aquaporins

The majority of plants are unable to evade unfavorable conditions such as flooding, salinity, or drought. Therefore, a fine-tuned water homeostasis appears to be of crucial importance for plant survival, and it was assumed that aquaporins play a significant role in these processes. Regulation of plant aquaporin conductivity was suggested to be achieved by a gating mechanism that involves protein phosphorylation under drought stress conditions and protonation after cytosolic acidification during flooding. The effect of protein phosphorylation or protonation of aquaporins was studied on two plasma membrane intrinsic proteins, NtPIP2;1 and NtAQP1 from tobacco, which were heterologously expressed in yeast. Our results on mutated aquaporins with serine-to-alanine exchange indicate that phosphorylation of the two key serine residues did not affect the pH-dependent modification of water permeability. Protonation on a conserved histidine residue decreased water conductivity of NtPIP2;1. Although cells expressing NtPIP2;1 with a replacement of the histidine by an alanine were found to be pH-insensitive with regard to water permeability, these maintain high water transport rates, similar to those obtained under acidic conditions. The data clearly support the role of histidine at 196 as a component of pH-dependent modification of aquaporin-facilitated water transport. The predictions of combined effects from phosphorylation at conserved serines and histidine protonation were not supported by the results of functional analysis. The obtained results challenge the gating model as a general regulation mechanism for plant plasma membrane aquaporins.     

Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana

Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils.     

Mechanisms of water transport mediated by PIP aquaporins and their regulation via phosphorylation events under salinity stress in barley roots

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed.     

Root aquaporins contribute to whole plant water fluxes under drought stress in rice (Oryza sativa L.)

Aquaporin activity and root anatomy may affect root hydraulic properties under drought stress. To better understand the function of aquaporins in rice root water fluxes under drought, we studied the root hydraulic conductivity (Lpr) and root sap exudation rate (Sr) in the presence or absence of an aquaporin inhibitor (azide) under well‐watered conditions and following drought stress in six diverse rice varieties. Varieties varied in Lpr and Sr under both conditions. The contribution of aquaporins to Lpr was generally high (up to 79% under well‐watered conditions and 85% under drought stress) and differentially regulated under drought. Aquaporin contribution to Sr increased in most varieties after drought, suggesting a crucial role for aquaporins in osmotic water fluxes during drought and recovery. Furthermore, root plasma membrane aquaporin (PIP) expression and root anatomical properties were correlated with hydraulic traits. Three chromosome regions highly correlated with hydraulic traits of the OryzaSNP panel were identified, but did not co‐locate with known aquaporins. These results therefore highlight the importance of aquaporins in the rice root radial water pathway, but emphasize the complex range of additional mechanisms related to root water fluxes and drought response.     

PIP1 plasma membrane aquaporins in tobacco: from cellular effects to function in plants

The molecular functions of several aquaporins are well characterized (e.g., by analysis of aquaporin-expressing Xenopus oocytes). However, their significance in the physiology of water transport in multicellular organisms remains uncertain. The tobacco plasma membrane aquaporin NtAQP1 was used to elucidate this issue. By comparing antisense plants that were inhibited in NtAQP1 expression with control plants, we found evidence for NtAQP1 function in cellular and whole-plant water relations. The consequences of a decrease in cellular water permeability were determined by measurement of transpiration rate and stem and leaf water potential as well as growth experiments under extreme soil water depletion. Plants impaired in NtAQP1 expression showed reduced root hydraulic conductivity and lower water stress resistance. In conclusion, our results emphasize the importance of symplastic aquaporin-mediated water transport in whole-plant water relations.     

植物水分传输过程中的调控机制研究进展

农田土壤水分的转化利用与调控是以土壤-植物-大气连续体(SPAC)为基础,以植物为核心,其中水分在植物体内的传输与调控研究一直是国际学术研究的前沿性热点课题。本文概述了植物水分传输的驱动力和传输途径,重点从植物的气孔调节、水容调节、渗透调节、水孔蛋白调节、贮水调节、气穴和栓塞调节等方面综述了植物水分传输过程中的调控机制研究进展。通过对植物存在优化调控水分平衡的潜在能力的研究,不仅可充实SPAC系统水分传输理论,而且有助于明确植物对环境的适应机制和高效用水的潜力及其节水调控的效应,对指导干旱半干旱地区农业生产提供理论依据。     

植物抗旱和耐重金属基因工程研究进展

干旱和重金属污染严重影响植物的生长发育.植物耐逆相关基因的克隆和功能鉴定研究,为通过基因工程途径提高植物的抗逆性奠定了理论基础.水分亏缺、高盐、低温和重金属胁迫都能诱导LEA(late embryogenesis abundant protein)基因的表达.转基因研究表明,LEA蛋白具有抗旱保护作用、离子结合特性以及抗氧化活性;水孔蛋白存在于细胞膜和液泡膜上,在细胞乃至整个植物体水分吸收和运输过程中发挥重要作用.干旱和盐胁迫促进水孔蛋白基因转录物的积累.过量表达水孔蛋白可增强水分吸收和运输,提高植物的抗旱能力.金属转运蛋白参与重金属离子的吸收、运输和累积等过程.这些蛋白基因在改良草坪草植物的抗旱节水和耐重金属能力等方面具有潜在的应用价值.     

Coulter counter cell size determination of protoplasts fromArabidopsis thaliana PIP1b aquaporin antisense lines under iso- and hypo-osmotic conditions

Summary Water uptake ofArabidopsis thaliana protoplasts was measured after transfer into hypo-osmotic conditions. The time-dependent swelling of protoplast populations was monitored by a Coulter counter device. In order to ascertain the contribution of the plasma membrane intrinsic protein 1b (PIP1b) to the membrane's water permeability, protoplasts of five different plant lines that were transformed with a PIP1b antisense construct were compared to controls. The size distribution of 5 independent protoplast preparations provided similar results for control and antisense lines under iso-osmolar conditions. After transfer into hypo-osmotic conditions, a time difference for the swelling of protoplasts from the different sources was observed. The sizes of control protoplasts changed in less than 20 s, which indicates high water influx rates. In contrast, the protoplast populations obtained from 5 different antisense plants took about 75 s to reach a steady-state cell size distribution. The difference in time by a factor of about 3 confirms the significance of the aquaporin PIP1b for the water permeability of plant plasma membranes and the cellular water transport.     

Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses

Water transport across cellular membranes is regulated by a family of water channel proteins known as aquaporins (AQPs). As most abiotic stresses like suboptimal temperatures, drought or salinity result in cellular dehydration, it is imperative to study the cause–effect relationship between AQPs and the cellular consequences of abiotic stress stimuli. Although plant cells have a high isoform diversity of AQPs, the individual and integrated roles of individual AQPs in optimal and suboptimal physiological conditions remain unclear. Herein, we have identified a plasma membrane intrinsic protein gene (MusaPIP1;2) from banana and characterized it by overexpression in transgenic banana plants. Cellular localization assay performed using MusaPIP1;2::GFP fusion protein indicated that MusaPIP1;2 translocated to plasma membrane in transformed banana cells. Transgenic banana plants overexpressing MusaPIP1;2 constitutively displayed better abiotic stress survival characteristics. The transgenic lines had lower malondialdehyde levels, elevated proline and relative water content and higher photosynthetic efficiency as compared to equivalent controls under different abiotic stress conditions. Greenhouse‐maintained hardened transgenic plants showed faster recovery towards normal growth and development after cessation of abiotic stress stimuli, thereby underlining the importance of these plants in actual environmental conditions wherein the stress stimuli is often transient but severe. Further, transgenic plants where the overexpression of MusaPIP1;2 was made conditional by tagging it with a stress‐inducible native dehydrin promoter also showed similar stress tolerance characteristics in in vitro and in vivo assays. Plants developed in this study could potentially enable banana cultivation in areas where adverse environmental conditions hitherto preclude commercial banana cultivation.     

PIP Aquaporin Gene Expression in Arbuscular Mycorrhizal Glycine max and Lactuca sativa Plants in Relation to Drought Stress Tolerance

Although the discovery of aquaporins in plants has resulted in a paradigm shift in the understanding of plant water relations, the relationship between aquaporins and plant responses to drought still remains elusive. Moreover, the contribution of aquaporin genes to the enhanced tolerance to drought in arbuscular mycorrhisal (AM) plants has never been investigated. Therefore, we studied, at a molecular level, whether the expression of aquaporin-encoding genes in roots is altered by the AM symbiosis as a mechanism to enhance host plant tolerance to water deficit. In this study, genes encoding plasma membrane aquaporins (PIPs) from soybean and lettuce were cloned and their expression pattern studied in AM and nonAM plants cultivated under well-watered or drought stressed conditions. Results showed that AM plants responded to drought stress by down-regulating the expression of the PIP genes studied and anticipating its down-regulation as compared to nonAM plants. The possible physiological implications of this down-regulation of PIP genes as a mechanism to decrease membrane water permeability and to allow cellular water conservation is further discussed.     

Regulation of a plant aquaporin by a Casparian strip membrane domain protein‐like

The absorption of soil water by roots allows plants to maintain their water status. At the endodermis, water transport can be affected by initial formation of a Casparian strip and further deposition of suberin lamellas and regulated by the function of aquaporins. Four Casparian strip membrane domain protein‐like (CASPL; CASPL1B1, CASPL1B2, CASPL1D1, and CASPL1D2) were previously shown to interact with PIP2;1. The present work shows that CASPL1B1, CASPL1B2, and CASPL1D2 are exclusively expressed in suberized endodermal cells, suggesting a cell‐specific role in suberization and/or water transport regulation. When compared with wild‐type plants, and by contrast to caspl1b1*caspl1b2 double loss of function, caspl1d1*caspl1d2 double mutants showed, in some control or NaCl stress experiments and not upon abscisic acid (ABA) treatment, a weak enlargement of the continuous suberization zone. None of the mutants showed root hydraulic conductivity (Lp_r) phenotype, whether in control, NaCl, or ABA treatment conditions. The data suggest a slight negative role for CASPL1D1 and CASPL1D2 in suberization under control or salt stress conditions, with no major impact on whole root transport functions. At the molecular level, CASPL1B1 was able to physically interact with PIP2;1 and potentially could influence the regulation of aquaporins by acting on their phosphorylated form.     

水孔蛋白在细胞延长、盐胁迫和光合作用中的作用

水孔蛋白属于一个高度保守的、能够进行跨生物膜水分运输的通道蛋白MIP家族。水孔蛋白作为膜水通道,在控制细胞和组织的水含量中扮演重要角色。本研究的重点是属于PIP亚家族的GhPIP1;2和属于TIP亚家族的γTIP1在植物细胞延长中的作用。使用特异基因探针的Northern杂交和实时荧光PCR技术证明GhPIP1;2和GhγTIP1主要在棉花纤维延长过程中显著表达,且最高表达量在开花后5d。在细胞延长过程中,GhPIP1;2和GhγTIP1表达显著,表明它们在促使水流迅速进入液泡这一过程中扮演重要角色。而且也研究了盐胁迫植物中钙离子对水孔蛋白的影响。分别或一起用NaCl或CaCl2处理原生质体或细胞质膜。结果发现在盐胁迫条件下,水渗透率值在原生质体和质膜颗粒中都下降了,同时PIP1水孔蛋白的含量也下降了,表明NaCl对水孔蛋白的功能和含量有抑制作用。同时也观察了Ca2+的两种不同的作用。感知胁迫的胞质中游离钙离子浓度的增加可能导致水孔蛋白的关闭。而过剩的钙离子将导致水孔蛋白的上游调控。同时实验已经证明大麦的一类水孔蛋白-HvPIP2;1有更高的水和CO2转移率。本研究的目标是确定负责转运水和CO2的关键水孔蛋白...     

How does arbuscular mycorrhizal symbiosis regulate root hydraulic properties and plasma membrane aquaporins in Phaseolus vulgaris under drought, cold or salinity stresses?

Here, we evaluated how the arbuscular mycorrhizal (AM) symbiosis regulates root hydraulic properties and root plasma membrane aquaporins (PIP) under different stresses sharing a common osmotic component. Phaseolus vulgaris plants were inoculated or not with the AM fungus Glomus intraradices, and subjected to drought, cold or salinity. Stress effects on root hydraulic conductance (L), PIP gene expression and protein abundance were evaluated. Under control conditions, L in AM plants was about half that in nonAM plants. However, L was decreased as a result of the three stresses in nonAM plants, while it was almost unchanged in AM plants. At the same time, PIP2 protein abundance and phosphorylation state presented the same trend as L. Finally, the expression of each PIP gene responded differently to each stress and was dependent on the AM fungal presence. Differential expression of the PIP genes studied under each stress depending on the AM fungal presence may indicate a specific function and regulation by the AM symbiosis of each gene under the specific conditions of each stress tested.