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.     

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.     

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...     

Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.     

Identification of the family of aquaporin genes and their expression in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>L.)

Background  

Cotton (Gossypium spp.) is produced in over 30 countries and represents the most important natural fiber in the world. One of the primary factors affecting both the quantity and quality of cotton production is water. A major facilitator of water movement through cell membranes of cotton and other plants are the aquaporin proteins. Aquaporin proteins are present as diverse forms in plants, where they function as transport systems for water and other small molecules. The plant aquaporins belong to the large major intrinsic protein (MIP) family. In higher plants, they consist of five subfamilies including plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP), and the recently discovered X intrinsic proteins (XIP). Although a great deal is known about aquaporins in plants, very little is known in cotton.     

Drought, salt and wounding stress induce the expression of the plasma membrane intrinsic protein 1 gene in poplar (Populus alba×P. tremula var. glandulosa)

Water uptake across cell membranes is a principal requirement for plant growth at both the cellular and whole-plant levels; water movement through plant membranes is regulated by aquaporins (AQPs) or major intrinsic proteins (MIPs). We examined the expression characteristics of the poplar plasma membrane intrinsic protein 1 gene (PatPIP1), a type of MIP, which was isolated from a suspension cell cDNA library of Populus alba×P. tremula var. glandulosa. Examination of protoplasts expressing the p35S-PatPIP1::sGFP fusion protein revealed that the protein was localized in the plasma membrane. Northern blot analysis revealed that the gene was strongly expressed in poplar roots and leaves. Gene expression was inducible by abiotic factors including drought, salinity, cold temperatures and wounding, and also by plant hormones including gibberellic acid, jasmonic acid and salicylic acid. Since we found that the PatPIP1 gene was strongly expressed in response to mannitol, NaCl, jasmonic acid and wounding, we propose that PatPIP1 plays an essential role in the defense of plants against water stress.     

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.     

Root-targeted biotechnology to mediate hormonal signalling and improve crop stress tolerance

Since plant root systems capture both water and nutrients essential for the formation of crop yield, there has been renewed biotechnological focus on root system improvement. Although water and nutrient uptake can be facilitated by membrane proteins known as aquaporins and nutrient transporters, respectively, there is a little evidence that root-localised overexpression of these proteins improves plant growth or stress tolerance. Recent work suggests that the major classes of phytohormones are involved not only in regulating aquaporin and nutrient transporter expression and activity, but also in sculpting root system architecture. Root-specific expression of plant and bacterial phytohormone-related genes, using either root-specific or root-inducible promoters or grafting non-transformed plants onto constitutive hormone producing rootstocks, has examined the role of root hormone production in mediating crop stress tolerance. Root-specific traits such as root system architecture, sensing of edaphic stress and root-to-shoot communication can be exploited to improve resource (water and nutrients) capture and plant development under resource-limited conditions. Thus, root system engineering provides new opportunities to maintain sustainable crop production under changing environmental conditions.     

植物质膜水通道蛋白转运及逆境胁迫响应的分子调控机制

质膜水通道蛋白即质膜内在蛋白(plasma membrane intrinsic proteins, PIPs),属于通道蛋白,定位在质膜上,是植物体内水分子、CO₂及其他一些小分子溶质跨细胞膜运输的通道。PIPs对运输基质具有高度选择性,在维持植物细胞的水分平衡过程中发挥重要作用。PIPs的表达、活性与定位不但受转录水平和翻译后水平的调控,而且受外界环境影响。研究表明在非生物胁迫下,PIPs表达模式和定位会发生改变。本文重点阐述了PIPs转运的分子机制、转录水平及翻译后水平的调控机制以及PIPs对非生物胁迫的响应机制,分析了目前关于PIPs的研究动态和值得探究的研究方向,以期帮助相关领域的科研人员对PIPs的研究进展有更深入地了解。     

植物水通道蛋白的干旱应答机制研究进展

干旱胁迫是严重影响全球作物生产的非生物胁迫之一,研究植物耐旱机制已成为一个重要领域。水通道蛋白是一类特异、高效转运水及其它小分子底物的膜通道蛋白,在植物中具有丰富的亚型,参与调节植物的水分吸收和运输。近10年来,水通道蛋白在植物不同生理过程中的作用,一直受到研究人员的关注,特别是在非生物胁迫方面,而研究表明水通道蛋白在干旱胁迫下对植物的耐旱性起着至关重要的作用,能维持细胞水分稳态和调控环境胁迫快速响应。水通道蛋白在植物耐旱过程中的调控机制及功能较复杂,而关于其应答机制和不同亚型功能性研究的报道甚少。该文综述了植物水通道蛋白的分类、结构、表达调控和活性调节,分别从植物水通道蛋白响应干旱表达调控机制、水通道蛋白基因表达的时空特异性、水通道蛋白基因的表达与蛋白丰度,水通道蛋白基因的耐旱转化四个方面阐明干旱胁迫下植物水通道蛋白的表达,重点阐述其参与植物干旱胁迫应答的作用机制,并提出水通道蛋白研究的主要方向。     

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.     

Beyond osmolytes and transcription factors: drought tolerance in plants <Emphasis Type="Italic">via</Emphasis> protective proteins and aquaporins

Mechanisms of drought tolerance have been studied by numerous groups, and a broad range of molecules have been identified to play important roles. A noteworthy response of stressed plants is the accumulation of novel protective proteins, including heat-shock proteins (HSPs) and late embryogenesis abundant (LEA) proteins. Identification of gene regulatory networks of these protective proteins in plants will allow a wide application of biotechnology for enhancement of drought tolerance and adaptation. Similarly, aquaporins are involved in the regulation of water transport, particularly under abiotic stresses. The molecular and functional characterization of protective proteins and aquaporins has revealed the significance of their regulation in response to abiotic stresses. Herein, we highlight new findings regarding the action mechanisms of these proteins. Finally, this review also surveys the current advances in engineering drought tolerant plants, particularly the engineering of protective proteins (sHSPs and LEA) and aquaporins for imparting drought stress tolerance in plants.     

The aquaporin gene family of the ectomycorrhizal fungus Laccaria bicolor: lessons for symbiotic functions

Soil humidity and bulk water transport are essential for nutrient mobilization. Ectomycorrhizal fungi, bridging soil and fine roots of woody plants, are capable of modulating both by being integrated into water movement driven by plant transpiration and the nocturnal hydraulic lift. Aquaporins are integral membrane proteins that function as gradient-driven water and/or solute channels. Seven aquaporins were identified in the genome of the ectomycorrhizal basidiomycete Laccaria bicolor and their role in fungal transfer processes was analyzed. Heterologous expression in Xenopus laevis oocytes revealed relevant water permeabilities for three aquaporins. In fungal mycelia, expression of the corresponding genes was high compared with other members of the gene family, indicating the significance of the respective proteins for plasma membrane water permeability. As growth temperature and ectomycorrhiza formation modified gene expression profiles of these water-conducting aquaporins, specific roles in those aspects of fungal physiology are suggested. Two aquaporins, which were highly expressed in ectomycorrhizas, conferred plasma membrane ammonia permeability in yeast. This indicates that these proteins are an integral part of ectomycorrhizal fungus-based plant nitrogen nutrition in symbiosis.     

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.     

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.     

The role of aquaporins in cellular and whole plant water balance

Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. More than 150 MIPs have been identified in organisms ranging from bacteria to animals and plants. In plants, aquaporins are present in the plasma membrane and in the vacuolar membrane where they are abundant constituents. Functional studies of aquaporins have hitherto mainly been performed by heterologous expression in Xenopus oocytes. A main issue is now to understand their role in the plant, where they are likely to be important both at the cellular and at the whole plant level. Plants contain a large number of aquaporin isoforms with distinct cell type- and tissue-specific expression patterns. Some of these are constitutively expressed, whereas the expression of others is regulated in response to environmental factors, such as drought and salinity. At the protein level, regulation of water transport activity by phosphorylation has been reported for some aquaporins.     

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.     

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C₄ grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.