Differential tissue-specific expression of NtAQP1 in Arabidopsis thaliana reveals a role for this protein in stomatal and mesophyll conductance of CO2 under standard and salt-stress conditions

The regulation of plant hydraulic conductance and gas conductance involves a number of different morphological, physiological and molecular mechanisms working in harmony. At the molecular level, aquaporins play a key role in the transport of water, as well as CO₂, through cell membranes. Yet, their tissue-related function, which controls whole-plant gas exchange and water relations, is less understood. In this study, we examined the tissue-specific effects of the stress-induced tobacco Aquaporin1 (NtAQP1), which functions as both a water and CO₂ channel, on whole-plant behavior. In tobacco and tomato plants, constitutive overexpression of NtAQP1 increased net photosynthesis (A _N), mesophyll CO₂ conductance (g _m) and stomatal conductance (g _s) and, under stress, increased root hydraulic conductivity (L _pr) as well. Our results revealed that NtAQP1 that is specifically expressed in the mesophyll tissue plays an important role in increasing both A _N and g _m. Moreover, targeting NtAQP1 expression to the cells of the vascular envelope significantly improved the plants’ stress response. Surprisingly, NtAQP1 expression in the guard cells did not have a significant effect under any of the tested conditions. The tissue-specific involvement of NtAQP1 in hydraulic and gas conductance via the interaction between the vasculature and the stomata is discussed.     

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.     

Aquaporin Tetramer Composition Modifies the Function of Tobacco Aquaporins

Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO₂-triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO₂-related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO₂-triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO₂-related transport processes, a structure built up by the tetramer is the basis of this function.     

Cell-specific expression of the mercury-insensitive plasma-membrane aquaporin NtAQP1 from Nicotiana tabacum

 The aquaporin NtAQP1 from Nicotiana tabacum L. is insensitive to heavy-metal ions. In addition to water, the transport of urea or glycerol is facilitated by this plasma-membrane-located water channel. Northern hybridization and whole-mount in situ hybridization revealed a high steady-state level of NtAQP1-RNA in roots, a decreased content in shoots and a low content in leaves. By immunolocalization with an antibody targeted to the N-terminus of the aquaporin, the localization of NtAQP1-protein at sites of expected high water transport rates from and to the apoplast or symplast could be demonstrated. The specific pattern of NtAQP1 distribution in petioles strongly indicates a transcellular movement of water. Received: 12 August 1999 / Accepted: 27 December 1999     

A major integral protein of the plant plasma membrane binds flavin

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.     

Plant water relations at elevated CO2 -- implications for water-limited environments

Long-term exposure of plants to elevated [CO2] leads to a number of growth and physiological effects, many of which are interpreted in the context of ameliorating the negative impacts of drought. However, despite considerable study, a clear picture in terms of the influence of elevated [CO2] on plant water relations and the role that these effects play in determining the response of plants to elevated [CO2] under water-limited conditions has been slow to emerge. In this paper, four areas of research are examined that represent critical, yet uncertain, themes related to the response of plants to elevated [CO2] and drought. These include (1) fine-root proliferation and implications for whole-plant water uptake; (2) enhanced water-use efficiency and consequences for drought tolerance; (3) reductions in stomatal conductance and impacts on leaf water potential; and (4) solute accumulation, osmotic adjustment and dehydration tolerance of leaves. A survey of the literature indicates that the growth of plants at elevated [CO2] can lead to conditions whereby plants maintain higher (less negative) leaf water potentials. The mechanisms that contribute to this effect are not fully known, although CO2-induced reductions in stomatal conductance, increases in whole-plant hydraulic conductance and osmotic adjustment may be important. Less understood are the interactive effects of elevated [CO2] and drought on fine-root production and water-use efficiency, and the contribution of these processes to plant growth in water-limited environments. Increases in water-use efficiency and reductions in water use can contribute to enhanced soil water content under elevated [CO2]. Herbaceous crops and grasslands are most responsive in this regard. The conservation of soil water at elevated [CO2] in other systems has been less studied, but in terms of maintaining growth or carbon gain during drought, the benefits of CO2-induced improvements in soil water content appear relatively minor. Nonetheless, because even small effects of elevated [CO2] on plant and soil water relations can have important implications for ecosystems, we conclude that this area of research deserves continued investigation. Future studies that focus on cellular mechanisms of plant response to elevated [CO2] and drought are needed, as are whole-plant investigations that emphasize the integration of processes throughout the soil--plant--atmosphere continuum. We suggest that the hydraulic principles that govern water transport provide an integrating framework that would allow CO2-induced changes in stomatal conductance, leaf water potential, root growth and other processes to be uniquely evaluated within the context of whole-plant hydraulic conductance and water transport efficiency.     

The Nicotiana tabacum plasma membrane aquaporin NtAQP1 is mercury-insensitive and permeable for glycerol

A new aquaporin from Nicotiana tabacum (cv. Samsun) was characterized. It shares sequence homology to the Arabidopsis thaliana PIP1 protein family. By two-phase partitioning and immunoblot analysis, plasma membrane localization could be demonstrated. The corresponding mRNA is highly abundant in roots and flowers, while it is rarely expressed in leaves and stems. Functional expression in Xenopus oocytes revealed that NtAQP1 can mediate glycerol transport in addition to water flow. However, NtAQP1 is impermeable for Na+, K+ and Cl- ions. The water permeability and selectivity could not be modulated by addition of mercurials or the activity of cAMP-dependent protein kinases.     

The plasma membrane aquaporin NtAQP1 is a key component of the leaf unfolding mechanism in tobacco

Epinastic leaf movement of tobacco is based on differential growth of the upper and lower leaf surface and is distinct from the motor organ-driven mechanism of nyctinastic leaf movement of, for example, mimosa species. The epinastic leaf movement of tobacco is observed not only under diurnal light regimes but also in continuous light, indicating a control by light and the circadian clock. As the transport of water across membranes by aquaporins is an important component of rapid plant cell elongation, the role of the tobacco aquaporin Nt aquaporin (AQP)1 in the epinastic response was studied in detail. In planta NtAQP1-luciferase (LUC) activity studies, Northern and Western blot analyses demonstrated a diurnal and circadian oscillation in the expression of this plasma membrane intrinsic protein (PIP)1-type aquaporin in leaf petioles, exhibiting peaks of expression coinciding with leaf unfolding. Cellular water permeability of protoplasts isolated from leaf petioles was found to be high in the morning, i.e. during the unfolding reaction, and low in the evening. Moreover, diurnal epinastic leaf movement was shown to be reduced in transgenic tobacco lines with an impaired expression of NtAQP1. It is concluded that the cyclic expression of PIP1-aquaporin represents an important component of the leaf movement mechanism.     

Estimation of whole-plant transpiration of bananas using sap flow measurements

Banana, one of the largest rhizomatous herbs in the world, is the fourth most important global food crop. It has a high water requirement, but the whole-plant water use in the field has not been determined satisfactorily. In this study, whole-plant water use in potted and field-grown banana plants (Musa 'Cavendish' cv. Williams) was successfully determined using a xylem sap flow method. This was achieved using Granier sensor probes implanted into the central cylinder of the banana corm. The whole-plant water use in field-grown bananas was 9-10 l plant(-1) d(-1). The values of daily total sap flow in potted plants correlated closely with gravimetric measurements (r(2)=0.92) and with changes in soil water status (r(2)=0.77). In well-watered, mature, field-grown plants, hourly sap flow also closely correlated with changes in solar radiation, vapour pressure deficit and evapotranspiration. The study indicates that sap flow measurement is a sensitive and accurate method for determining whole-plant water use in bananas under potted as well as field conditions.     

Phosphorus deprivation effects on water relations of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plant via reducing plasma membrane permeability

Plants grown in phosphorus-deprived solutions often exhibit disruption of water transport due to reduction in root hydraulic conductivity (Lp_r). To uncover the relationship between root Lp_r and water permeability coefficient (P_f) of plasma membrane and the role of aquaporins, we evaluated P_f of plasma membrane and also PIP-type aquaporin gene expression in tobacco (Nicotiana tabacum L.) plant roots after seven days P-deprivation. The results showed significant reduction in sap flow rate (J_v) and osmotic root hydraulic conductivity (L_pr-o) in P-deprived roots. These effects were reversed 24 h after P-resupplying. Interestingly, the P_f of root protoplasts was 57% lower in P-deprived plants compared with P-sufficient ones. The expression of NtPIP1;1 and NtPIP2;1 aquaporins did not change significantly in P-deprived plants compared with P-sufficient ones, but the copy number of NtAQP1 increased significantly in P-deprived plants. P-deprivation did not change Lpr-o significantly in antisense NtAQP1 plants. Taken together, these findings suggest that P-deprivation may play an important role in modulation of root hydraulic conductivity by affecting P_f in transcellular pathway of water flow across roots and aquaporins. Finally, we concluded that dominant water transport pathway under P-deprivation was transcellular one.     

Advances in functional regulation mechanisms of plant aquaporins: Their diversity,gene expression,localization, structure and roles in plant soil-water relations (Review)

Aquaporins are important molecules that control the moisture level of cells and water flow in plants. Plant aquaporins are present in various tissues, and play roles in water transport, cell differentiation and cell enlargement involved in plant growth and water relations. The insights into aquaporins’ diversity, structure, expression, post-translational modification, permeability properties, subcellular location, etc., from considerable studies, can lead to an understanding of basic features of the water transport mechanism and increased illumination into plant water relations. Recent important advances in determining the structure and activity of different aquaporins give further details on the mechanism of functional regulation. Therefore, the current paper mainly focuses on aquaporin structure-function relationships, in order to understand the function and regulation of aquaporins at the cellular level and in the whole plant subjected to various environmental conditions. As a result, the straightforward view is that most aquaporins in plants are to regulate water flow mainly at cellular scale, which is the most widespread general interpretation of the physiological and functional assays in plants.     

Advances in functional regulation mechanisms of plant aquaporins: their diversity, gene expression, localization, structure and roles in plant soil-water relations (Review)

Aquaporins are important molecules that control the moisture level of cells and water flow in plants. Plant aquaporins are present in various tissues, and play roles in water transport, cell differentiation and cell enlargement involved in plant growth and water relations. The insights into aquaporins' diversity, structure, expression, post-translational modification, permeability properties, subcellular location, etc., from considerable studies, can lead to an understanding of basic features of the water transport mechanism and increased illumination into plant water relations. Recent important advances in determining the structure and activity of different aquaporins give further details on the mechanism of functional regulation. Therefore, the current paper mainly focuses on aquaporin structure-function relationships, in order to understand the function and regulation of aquaporins at the cellular level and in the whole plant subjected to various environmental conditions. As a result, the straightforward view is that most aquaporins in plants are to regulate water flow mainly at cellular scale, which is the most widespread general interpretation of the physiological and functional assays in plants.     

Heavy browsing affects the hydraulic capacity of Ceanothus rigidus (Rhamnaceae)

Defoliation by herbivores can reduce carbon assimilation, change plant water relations, and even shift the biotic structure of plant communities. In this study, we took advantage of a long-term deer exclosure experiment to examine the consequences of persistent deer herbivory on plant water relations and the xylem structure–function relationships in Ceanothus rigidus, a maritime chaparral shrub in coastal California. Browsed plants had thicker stems with many intertwined short distal twigs, and significantly higher sapwood-to-leaf area ratios than their non-browsed counterparts. Leaf area-specific hydraulic conductivity was similar in both browsed and non-browsed plants, but xylem area-specific conductivity was significantly lower in the browsed plants. Vessel diameters were equivalent in both plant groups, but the number of vessels on a transverse area basis was nearly 40 % lower in the browsed plants, accounting for their lower transport efficiency. Mid-day in situ water potentials and losses of hydraulic conductivity due to embolism were similar in both groups of plants but stomatal conductance was higher in the browsed shrubs in the early part of the growing season. We discuss our findings in the context of whole-plant ecophysiology, and explore the consequences of herbivory on hormonal signals, wood anatomy, and xylem function.     

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.     

Why Canny's theory doesn't hold water

A critique of Martin Canny's theory of water transport supported by tissue pressure is given with reference to basic principles of cellular water relations and biomechanics. It is shown that the application of tissue pressure in Canny's theory is neither internally consistent nor compatible with basic biophysics. Canny's translation of tissue pressure into an altered steady-state pressure in the xylem conduits has no defensible mechanism, relying instead on untenable action-at-a-distance and poor definitions. Tissue pressure itself, as defined by Canny and illustrated by the example of a turgid leaf, may well exist. However, it cannot function in whole-plant processes as envisioned by Canny, nor can it exist in the magnitude his theory would require. Rigid outer tissue layers containing internal pressures of the magnitude postulated by Canny would require a tensile strength quite incompatible with the observed biological materials. A simple application of La Place's Law illustrates that this is an issue of scale and that the turgor generated by osmotic potentials must be balanced primarily at the cellular level, and not the tissue level.     

植物水孔蛋白最新研究进展

水孔蛋白(aquaporin,AQP)是高效转运水分子的膜内在蛋白,具有丰富的多样性,在调控植物的水分关系中有重要作用．介绍了AQP的分类、结构特征及其在植物生长发育过程中的多种生理功能和AQP活性的各种调控方式．综述了水分胁迫和盐胁迫等逆境条件及脱落酸、赤霉素和乙烯等植物激素对AQP基因表达调控等方面的研究进展．