向日葵根系水通道蛋白活性与苗龄关系的研究

利用压力室结合水通道蛋白抑制剂氯化汞(HgCl2)检测了不同苗龄(15d、25d和35d)向日葵根系水通道的活性,结果显示此生长期间根系导水率保持相对恒定,但0．1mmol／L氯化汞使所有苗龄根系的水流速率和根系导水率迅速降低,而降幅随根龄的增大而增大,表明向日葵根存在调节水分进入根系的水通道蛋白,其活性随根龄的增大而提高,质外体水流随根龄的增大而减小。结论是：在根系生长过程中,细胞到细胞途径水通道蛋白活性的提高可以补偿由于质外体途径导水度降低所致根系导水率的降低,从而维持根系导水率的相对稳定。     

蚕豆保卫细胞质膜水通道蛋白的鉴定及其在气孔运动中的作用

水通道或水通道蛋白是水分运动的主要通道.以RD28 cDNA和RD28抗体为探针证明了蚕豆(Vicia fabaL.)保卫细胞中存在水通道蛋白,并以气孔运动为指标,结合抗体和抑制剂处理证明水通道蛋白是水分运动的主要通道.研究表明编码质膜水通道蛋白的RD28转录体在叶片保卫细胞、叶肉细胞和维管束中高表达,尤以保卫细胞中最多;荧光免疫染色和Confocal显微镜观察表明,RD28抗体反应主要位于保卫细胞质膜.进一步采用RD28抗体和水通道蛋白抑制剂--HgCl2 (25μmol/L)处理可抑制壳梭孢素(FC)、光照诱导的气孔开放和原生质体体积膨胀以及ABA诱导的气孔关闭,但这种抑制作用可以被水通道抑制剂的逆转剂β-巯基乙醇(ME)逆转.表明蚕豆保卫细胞中存在水通道蛋白并参与蚕豆保卫细胞的运动过程.     

Aquaporin-facilitated water uptake in barley (Hordeum vulgare L.) roots

It is not known to what degree aquaporin-facilitated water uptake differs between root developmental regions and types of root. The aim of this study was to measure aquaporin-dependent water flow in the main types of root and root developmental regions of 14- to 17-d-old barley plants and to identify candidate aquaporins which mediate this flow. Water flow at root level was related to flow at cell and plant level. Plants were grown hydroponically. Hydraulic conductivity of cells and roots was determined with a pressure probe and through exudation, respectively, and whole-plant water flow (transpiration) determined gravimetrically in response to the commonly used aquaporin inhibitor HgCl(2). Expression of aquaporins was analysed by real-time PCR and in situ hybridization. Hydraulic conductivity of cortical cells in seminal roots was largest in lateral roots; it was smallest in the fully mature zone and intermediate in the not fully mature 'transition' zone along the main root axis. Adventitious roots displayed an even higher (3- to 4-fold) cortical cell hydraulic conductivity in the transition zone. This coincided with 3- to 4-fold higher expression of three aquaporins (HvPIP2;2, HvPIP2;5, HvTIP1:1). These were expressed (also) in cortical tissue. The largest inhibition of water flow (83-95%) in response to HgCl(2) was observed in cortical cells. Water flow through roots and plants was reduced less (40-74%). It is concluded that aquaporins contribute substantially to root water uptake in 14- to 17-d-old barley plants. Most water uptake occurs through lateral roots. HvPIP2;5, HvPIP2;2, and HvTIP1;1 are prime candidates to mediate water flow in cortical tissue.     

Water uptake and structural plasticity along roots of a desert succulent during prolonged drought

Desert succulents resume substantial water uptake within 1–2 d of the cessation of drought, but the changes in root structure and hydraulic conductivity underlying such recovery are largely unknown. In the monocotyledonous leaf succulent Agave deserti Engelm. substantial root mortality occurred only for lateral roots near the soil surface; nearly all main roots were alive at 180 d of drought. New main roots were initiated and grew up to 320 mm at soil water potentials lower than – 5·0 MPa, utilizing water from the shoot. The hydraulic conductivity of distal root regions decreased 62% by 45 d of drought and 70% thereafter. After 7 d of rewetting, root hydraulic conductivity was restored following 45 d of drought but not after 90 and 180 d. The production of new lateral roots and the renewed apical elongation of main roots occurred 7–11 d after rewetting following 180 d of drought. Hydraulic conductivity was higher in the distal region than at midroot and often increased again near the root base, where many endodermal cells lacked suberin lamellae. Suberization and xylem maturation were influenced by the availability of moisture, suggesting that developmental plasticity along a root allows A. deserti to capitalize on intermittent or heterogeneous supplies of water.     

A smoke-derived butenolide alleviates HgCl2 and ZnCl2 inhibition of water uptake during germination and subsequent growth of tomato--possible involvement of aquaporins

Aquaporins, concentrated in zones of cell division and enlargement, play a major role in governing the movement of water between neighboring cells during seed germination. The enhanced germination and growth found with smoke-water and butenolide could be the result of better water uptake, suggesting the involvement of aquaporins. The effects of butenolide, known aquaporin inhibitors (HgCl(2) and ZnCl(2)), along with several chemical agents known to reverse the inhibitory effects of mercuric chloride on the activity of aquaporins were tested. Seedlings raised in the presence of butenolide had higher moisture content (93%) compared to those imbibed in water only (85%). This suggests enhanced activity of aquaporins. The presence of aquaporin inhibitors (HgCl(2) and ZnCl(2)) reduced seedling water content and altered root development. The presence of HgCl(2) (10, 20 or 30muM) reduced the percentage imbibition of seeds by 11-12%. A corresponding gradual decline, from 17.5% (10muM) to 22.6% (30muM) (p0.05), in the root length was recorded. Addition of dithiothreitol (DTT, 500muM), beta-mercaptoethanol (ME, 250muM) and butenolide (0.1muM) along with the HgCl(2) overcame the observed inhibitory effects. The presence of ZnCl(2) (12.5 or 25muM) affected percentage imbibition as well as root length, which was reversed to some extent following addition of the butenolide. Though zinc chloride-mediated inhibition remained unaffected by the presence of DTT and ME, the butenolide reversed the effect. These results are interesting as they suggest additional avenues of research for uncovering the profound effect butenolide has on germination and growth.     

Effect of low root temperature on hydraulic conductivity of rice plants and the possible role of aquaporins

The role of root temperature T(R) in regulating the water-uptake capability of rice roots and the possible relationship with aquaporins were investigated. The root hydraulic conductivity Lp(r) decreased with decreasing T(R) in a measured temperature range between 10 degrees C and 35 degrees C. A single break point (T(RC) = 15 degrees C) was detected in the Arrhenius plot for steady-state Lp(r). The temperature dependency of Lp(r) represented by activation energy was low (28 kJ mol(-1)) above T(RC), but the value is slightly higher than that for the water viscosity. Addition of an aquaporin inhibitor, HgCl(2), into root medium reduced osmotic exudation by 97% at 25 degrees C, signifying that aquaporins play a major role in regulating water uptake. Below T(RC), Lp(r) declined precipitously with decreasing T(R) (E(a) = 204 kJ mol(-1)). When T(R) is higher than T(RC), the transient time for reaching the steady-state of Lp(r) after the immediate change in T(R) (from 25 degrees C) was estimated as 10 min, while it was prolonged up to 2-3 h when T(R) < T(RC). The Lp(r) was completely recovered to the initial levels when T(R) was returned back to 25 degrees C. Immunoblot analysis using specific antibodies for the major aquaporin members of PIPs and TIPs in rice roots revealed that there were no significant changes in the abundance of aquaporins during 5 h of low temperature treatment. Considering this result and the significant inhibition of water-uptake by the aquaporin inhibitor, we hypothesize that the decrease in Lp(r) when T(R) < T(RC) was regulated by the activity of aquaporins rather than their abundance.     

水曲柳幼苗根系在不同浓度NH4NO3溶液中水流导度的变化

水分吸收过程是根系重要的生理过程。水孔蛋白在根系水分径向运输中起着重要的作用,根系水流导度(L_p)的测定是研究水孔蛋白的重要途径。该研究采用压力流的方法,对相同生长条件下的水曲柳(Fraxinus mandshurica)幼苗根系进行研究,测定了根系在去离子水和不同浓度NH₄NO₃溶液中的L_p。结果表明:未经处理的水曲柳幼苗根系,L_p随NH₄NO₃浓度的增加而上升,而且NH₄NO₃溶液中的L_p比去离子水中的L_p平均高77%;经HgCl₂处理后,水曲柳幼苗根系的L_p仍然随NH₄NO₃浓度的增加而增大,但是根系L_p在去离子水下降了22%,而在NH₄NO₃溶液中下降了68%,与以前的研究相比发现,经HgCl₂处理后,以营养液为吸水基质的根系L_p的降低值普遍高于以去离子水为基质的试验。因此,基质中养分离子的存在对根系中水孔蛋白活性产生了重要的影响,进而影响根系水分的吸收过程。     

Roles of Aquaporins in Root Responses to Irrigation

Due to current environmental issues concerning the use of water for irrigation, the improvement of crop water-use efficiency and a reduction in water consumption has become a priority. New irrigation methods that reduce water use, while still maintaining production have been developed. To optimise these techniques knowledge of above- and below-ground plant physiological responses is necessary. During growth, plant roots are exposed to cycles of wetting and drying in normal rain-fed and irrigation situations. This review concentrates on the below-ground aspects, in particular the water permeability of roots. Significant research has been conducted on the root anatomy and hydraulic conductivity of desert plants subjected to wetting and drying. Major intrinsic proteins (MIPs), most of which show aquaporin (water-channel) activity are likely to be involved in balancing the water relations of the plants during water deficit. However, many MIPs seem to allow permeation of other small neutral solutes and some may allow permeation of ions under certain conditions. The ability of the plant to rapidly respond to rewetting may be important in maintaining productivity. It has been suggested that aquaporins may be involved in this rapid response. The down-regulation of the aquaporins during dry conditions can also limit water loss to the soil, and intrinsic sensitivity of aquaporins to water potential is shown here to be very strong in some cases (NOD26). However, the response of aquaporins in various plant species to water deficits has been quite varied. Another component of aquaporin regulation in response to various stresses (hypoxia/anoxia, salinity and chilling) may be related to redistribution of flow to more favourable regions of the soil. Some irrigation techniques may be triggering these responses. Diurnal fluctuations of root hydraulic conductance that is related to aquaporin expression seem to match the expected transpirational demands of the shoot, and it remains to be seen if shoot-to-root signalling may be important in regulation of root aquaporins. If so, canopy management typical of horticultural crops may impact on root hydraulic conductance. An understanding of the regulation of aquaporins may assist in the development of improved resistance to water stress and greater efficiency of water use by taking into account where and when roots best absorb water.     

The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots

When chilling-sensitive plants are chilled, root hydraulic conductance (L(o)) declines precipitously; L(o) also declines in chilling-tolerant plants, but it subsequently recovers, whereas in chilling-sensitive plants it does not. As a result, the chilling-sensitive plants dry out and may die. Using a chilling-sensitive and a chilling-tolerant maize genotype we investigated the effect of chilling on L(o), and its relationship to osmotic water permeability of isolated root cortex protoplasts, aquaporin gene expression, aquaporin abundance, and aquaporin phosphorylation, hydrogen peroxide (H(2)O(2)) accumulation in the roots and electrolyte leakage from the roots. Because chilling can cause H(2)O(2) accumulation we also determined the effects of a short H(2)O(2) treatment of the roots and examined the same parameters. We conclude from these studies that the recovery of L(o) during chilling in the chilling-tolerant genotype is made possible by avoiding or repairing membrane damage and by a greater abundance and/or activity of aquaporins. The same changes in aquaporins take place in the chilling-sensitive genotype, but we postulate that membrane damage prevents the L(o) recovery. It appears that the aquaporin response is necessary but not sufficient to respond to chilling injury. The plant must also be able to avoid the oxidative damage that accompanies chilling.     

Aquaporins account for variations in hydraulic conductance for metabolically active root regions of Agave deserti in wet, dry, and rewetted soil

The importance of aquaporins for root hydraulic conductance (L_P) was investigated along roots of the desert succulent Agave deserti in wet, dry and rewetted soil. Water channel activity was inferred from HgCl₂‐induced reductions of L_P that were reversible by 2‐mercaptoethanol. Under wet conditions, HgCl₂ reduced L_P for the distal root region by 50% and for the root region near the shoot base by 36% but did not affect L_P for the mid‐root region. For all root regions, L_P decreased by 30–60% during 10 d in drying soil and was not further reduced by HgCl₂. After soil rewetting, L_P increased to pre‐drying values and was again reduced by HgCl₂ for the distal and the basal root regions but not the mid‐root region. For the distal region, water channels in the epidermis/exodermis made a disproportionately large contribution to radial hydraulic conductance of the intact segment; for the basal region, water channel activity was highest in the cortex and endodermis. The role of water channels was greatest in tissues in which cells were metabolically active both in the distal root region, where new apical growth occurs in wet soil, and in the basal region, which is the most likely root region to intercept light rainfall.     

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.     

Role of a single aquaporin isoform in root water uptake

Aquaporins are ubiquitous channel proteins that facilitate the transport of water across cell membranes. Aquaporins show a typically high isoform multiplicity in plants, with 35 homologs in Arabidopsis. The integrated function of plant aquaporins and the function of each individual isoform remain poorly understood. Matrix-assisted laser desorption/ionization time-of-flight analyses suggested that Plasma Membrane Intrinsic Protein2;2 (PIP2;2) is one of the abundantly expressed aquaporin isoforms in Arabidopsis root plasma membranes. Two independent Arabidopsis knockout mutants of PIP2;2 were isolated using a PCR-based strategy from a library of plant lines mutagenized by the insertion of Agrobacterium tumefaciens T-DNA. Expression in transgenic Arabidopsis of a PIP2;2 promoter-beta-glucuronidase gene fusion indicated that PIP2;2 is expressed predominantly in roots, with a strong expression in the cortex, endodermis, and stele. The hydraulic conductivity of root cortex cells, as measured with a cell pressure probe, was reduced by 25 to 30% in the two allelic PIP2;2 mutants compared with the wild type. In addition, free exudation measurements revealed a 14% decrease, with respect to wild-type values, in the osmotic hydraulic conductivity of roots excised from the two PIP2;2 mutants. Together, our data provide evidence for the contribution of a single aquaporin gene to root water uptake and identify PIP2;2 as an aquaporin specialized in osmotic fluid transport. PIP2;2 has a close homolog, PIP2;3, showing 96.8% amino acid identity. The phenotype of PIP2;2 mutants demonstrates that, despite their high homology and isoform multiplicity, plant aquaporins have evolved with nonredundant functions.     

Heterogeneity in Water Availability Alters Cellular Development and Hydraulic Conductivity along Roots of a Desert Succulent

Plants of the desert succulent Agave deserti were grown in partitionedcontainers to determine whether heterogeneity in soil moistureleads to differences in cellular development and hydraulic conductivityalong individual roots. Roots from containers with a dry distalcompartment (furthest from the shoot), a wet middle compartment,and a dry proximal compartment had distal regions (includingthe root tips) that were more suberized and lignified in theendodermis and adjacent cell layers than were root regions fromthe wet middle compartment. Proximal root regions about 40 mmfrom the succulent shoot base were also relatively unsuberized,suggesting that both external and internal supplies of waterdelayed tissue maturation. Root segments from wet middle compartmentsand from dry proximal compartments had higher hydraulic conductivitythan did the more suberized root segments from dry distal compartments.Unlike distal root segments from wet compartments, segmentsfrom dry compartments suffered no decrease in hydraulic conductivityafter immersion in mercuric chloride, suggesting that aquaporinactivity diminished for roots during drought. The possible closureof water channels could help limit root water loss to a dryingsoil. The delayed development of suberized cell layers may allowroot regions to maximize water uptake from wet soil patches(such as under rocks), and the relatively immature, absorptiveroot region near the base of the shoot may help A. deserti capturewater from a briefly wetted surface soil. Copyright 2000 Annalsof Botany Company Agave deserti, root plasticity, water uptake, aquaporins, suberization, endodermis, divided pots.     

Expression and inhibition of aquaporins in germinating Arabidopsis seeds

Extensive and kinetically well-defined water exchanges occur during germination of seeds. A putative role for aquaporins in this process was investigated in Arabidopsis. Macro-arrays carrying aquaporin gene-specific tags and antibodies raised against aquaporin subclasses revealed two distinct aquaporin expression programs between dry seeds and young seedlings. High expression levels of a restricted number of tonoplast intrinsic protein (TIP) isoforms (TIP3;1 and/or TIP3;2, and TIP5;1) together with a low expression of all 13 plasma membrane aquaporin (PIP) isoforms was observed in dry and germinating materials. In contrast, prevalent expression of aquaporins of the TIP1, TIP2 and PIP subgroups was induced during seedling establishment. Mercury (5 microM HgCl(2)), a general blocker of aquaporins in various organisms, reduced the speed of seed germination and induced a true delay in maternal seed coat (testa) rupture and radicle emergence, by 8-9 and 25-30 h, respectively. Most importantly, mercury did not alter seed lot homogeneity nor the seed germination developmental sequence, and its effects were largely reversed by addition of 2 mM dithiothreitol, suggesting that these effects were primarily due to oxidation of cell components, possibly aquaporins, without irreversible alteration of cell integrity. Measurements of water uptake in control and mercury-treated seeds suggested that aquaporin functions are not involved in early seed imbibition (phase I) but would rather be associated with a delayed initiation of phase III, i.e. water uptake accompanying expansion and growth of the embryo. A possible role for aquaporins in germinating seeds and more generally in plant tissue growth is discussed.     

Low air humidity increases leaf-specific hydraulic conductance of Arabidopsis thaliana (L.) Heynh (Brassicaceae)

The typical isohydric plant response to low relative humidity involves stomatal closure, followed by long-term responses like adjustment of shoot-to-root ratios. Little information is available on the early responses of the root system to exposure of shoots to low humidity, nor is it clear to what extent responses of Arabidopsis thaliana conform to the isohydric model. In this study, A. thaliana plants grown hydroponically at high humidity were exposed to two constant relative humidities, 17% and 77%, while the root system remained in aerated nutrient solution. Leaf conductance (g(s)), transpiration, water potential (Psi(l)), osmotic potential, and whole plant hydraulic conductance (K) were determined for the following time intervals: 0-10, 10-20, and 20-40 min, and 0-5, 5-10, and 24-29 h. At low relative humidity, no change in g(s) was detected. Psi(l) decreased by 0.28 MPa during the first 5 h and then remained stable. During the first hour, leaf-specific K averaged 1.6 x 10(-5) kg MPa(-1) m(-2) s(-1) at high humidity. At low humidity it increased >3-fold to 5.8 x 10(-5) kg MPa(-1) m(-2) s(-1). Similar significant differences in K were observed during all time periods. Low concentration mercury amendments in the hydroponic solution (5 microM and 10 microM HgCl(2)) had no discernible influence, but pre-exposure to 50 microM HgCl(2) reduced K differences between humidity treatments. As HgCl(2) is known to be a potent inhibitor of aquaporin function, this suggests that aquaporins may have played a role in the fast hydraulic response of plants transferred to low humidity. The rapid hydraulic response and the influence of mercury raise the possibility that an alternative response to atmospheric dryness is increased K modulated by aquaporins.     

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.     

Effects of light intensity and duration on leaf hydraulic conductance and distribution of resistance in shoots of silver birch (Betula pendula)

Variation in leaf hydraulic conductance (K(L)) and distribution of resistance in response to light intensity and duration were examined in shoots of silver birch (Betula pendula Roth). K(L) was determined on detached shoots using the evaporative flux method (transpiration was measured with a porometer and water potential drop with a pressure chamber). Although K(L) depended on light duration per se, its dynamics was largely determined by leaf temperature (T(L)). Both upper-crown leaves and branches developed in well-illuminated environment exhibited higher hydraulic efficiency compared with the lower crown, accounting for vertical trends of apparent soil-to-leaf hydraulic conductance in canopy of silver birch revealed in our previous studies. K(L) varied significantly with light intensity, the highest values for both shade and sun foliage were recorded at photosynthetic photon flux density of 330 micromol m(-2) s(-1). Light responses of K(L) were associated evidently with an irradiance-mediated effect on extravascular tissues involving regulation of cell membrane aquaporins. Effects of irradiance on K(L) resulted in changes of Psi(L), bringing about considerable alteration in partitioning of the resistance between leaves and branch (leafless shoot stem): the contribution of leaves to the shoot total resistance decreased from 94% at -1.0 MPa to 75% at -0.2 MPa. Treatment with HgCl2 decreased hydraulic conductance of both leaves and branches, implying that condition of bordered pit membranes or shoot living tissues may be involved in responses of xylem conductance to Hg2+.