The arbuscular mycorrhizal symbiosis regulates aquaporins activity and improves root cell water permeability in maize plants subjected to water stress

Studies have suggested that increased root hydraulic conductivity in mycorrhizal roots could be the result of increased cell‐to‐cell water flux via aquaporins. This study aimed to elucidate if the key effect of the regulation of maize aquaporins by the arbuscular mycorrhizal (AM) symbiosis is the enhancement of root cell water transport capacity. Thus, water permeability coefficient (P_f) and cell hydraulic conductivity (L_pc) were measured in root protoplast and intact cortex cells of AM and non‐AM plants subjected or not to water stress. Results showed that cells from droughted‐AM roots maintained P_f and L_pc values of nonstressed plants, whereas in non‐AM roots, these values declined drastically as a consequence of water deficit. Interestingly, the phosphorylation status of PIP2 aquaporins increased in AM plants subjected to water deficit, and P_f values higher than 12 μm s^?1 were found only in protoplasts from AM roots, revealing the higher water permeability of AM root cells. In parallel, the AM symbiosis increased stomatal conductance, net photosynthesis, and related parameters, showing a higher photosynthetic capacity in these plants. This study demonstrates a better performance of AM root cells in water transport under water deficit, which is connected to the shoot physiological performance in terms of photosynthetic capacity.     

Differences in aquaporin levels among cell types of radish and measurement of osmotic water permeability of individual protoplasts

We investigated tissue- and cell-specific accumulation of radish aquaporin isoforms by immunocytochemical analysis. In taproots, the plasma membrane aquaporins (RsPIP1 and RsPIP2) were accumulated at high levels in the cambium, while the tonoplast aquaporin (RsTIP) was distributed in all tissues. The three isoforms were highly accumulated in the central cylinder of seedling roots and hypocotyls, and rich in the vascular tissue of the petiole of mature plants. The results suggest that RsPIP1 and RsPIP2 are abundant in the cells surrounding the sieve tube of the radish plant. The swelling rate of protoplasts in a hypotonic solution was determined individually by a newly established method to compare the osmotic water permeability of different cell types. All cells of the cortex and endodermis in seedlings showed a high water permeability of more than 300 microm s(-1). There was no marked difference in the values between the root endodermis and cortex protoplasts, although the RsPIP level was lower in the cortex than in the endodermis. This inconsistency suggests two possibilities: (1) a low level of aquaporin is enough for high water permeability and (2) the water channel activity of aquaporin in the tissues is regulated individually. The uneven distribution of aquaporins in tissues is discussed along with their physiological roles.     

Cytokinins as inhibitors of root growth

The elongation of roots of wheat ( Triticum aestivum L. cv. Diamant II), flax ( Linum usitatissimum L. cv. Concurrent) and cucumber ( Cucumis sativus L. cv. Favör) seedlings in the dark was strongly inhibited by various native and synthetic cytokinins (kinetin, benzyladenine, isopentenyladenine, zeatin and their corresponding 9-ribosides). An inhibition of 50% was obtained for wheat roots with 3 · 10⁻⁹ M zeatin and for flax roots with 6 · 10⁻⁹ M isopentenyladenine. The ribosides were in all cases less inhibitory. The inhibition was reversed by various types of 'antiauxins' and 'antiethylenes' (such as structural auxin analogues, uncouplers, specific inhibitors of ethylene synthesis, free radical scavengers, inhibitors of ethylene action). These substances as a rule counteract also inhibitions caused by auxins. Auxins and cytokinins stimulate ethylene production synergistically, and the similar inhibitory effects of these two types of hormone can be understood if it is assumed that their effect is at least partly mediated through ethylene. The cytokinins must be considered as possible natural inhibitors and regulators of root growth.     

Characterization of Aquaporins at the Plasma Membrane of Leaf Callus Protoplasts from Actinidia deliciosa cv. Hayward

The water transport activity of protoplasts from Actinidia deliciosa cv. Hayward was determined using a cell image system. The results showed that the protoplast volume increased swiftly when the protoplasts were placed in a hypotonic medium, and the volume increased with the increasing osmotic gradients. The P f values were 0.118×10-3, 0.121×10-3, and 0.133×10-3cm/s under the outward osmotic gradients of 75, 100, and 125 mmol/kg, respectively. The results also showed that the water transport activity of protoplasts could be inhibited by HgCl 2 and stimulated by amphotericin B. Moreover, it was found that ZnCl2 and ZnSO4 had a significant inhibitory effect on the water transport activity of the protoplasts from A. deliciosa var. deliciosa cv. Hayward. The results indicated that the protoplasts of A. deliciosa var. deliciosa cv. Hayward possessed the typical property of aquaporins, suggesting the presence of aquaporins at its plasma membranes.     

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.     

Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.     

Immunochemical analysis of aquaporin isoforms in Arabidopsis suspension-cultured cells

Aquaporins mediate the movement of water across biomembranes. Arabidopsis thaliana contains 35 aquaporins that belong to four subfamilies (PIP, TIP, SIP, and NIP). We investigated their expression profiles immunochemically in suspension-cultured Arabidopsis thaliana cells during growth and in response to salt and osmotic stresses. Protein amounts of all aquaporins were much lower in cultured cells than in the plant tissues. This is consistent with the low water permeability of protoplasts from cultured cells. After treatment with NaCl, the protein amounts of PIP2;1, PIP2;2, and PIP2;3 in the cells increased several-fold, and those of TIP1;1 and TIP1;2, 15- and 3-fold respectively. PIP1 did not change under the stress. Cell death began after 19 d in culture, accompanied by marked accumulation of PIPs and TIPs and a gradual decrease in SIPs. Our results suggest the followings: (i) Accumulation of aquaporin isoforms was individually regulated at low levels in single cells. (ii) At least PIP2;2, PIP2;3, TIP1;1, and TIP1;2 are stress-responsive aquaporins in suspension cells. (iii) A sudden increment of several members of PIP2 and TIP1 subfamilies might be related to cell death.     

Two different effects of calcium on aquaporins in salinity-stressed pepper plants

Two different effects of calcium were studied, respectively, in plasma membrane vesicles and in protoplasts isolated from roots of control pepper plants (Capsicum annuum L cv. California) or of plants treated with 50 mM NaCl, 10 mM CaCl(2) or 10 mM CaCl(2) + 50 mM NaCl. Under saline conditions, osmotic water permeability (P ( f )) values decreased in protoplasts and plasma membrane vesicles, and the same reduction was observed in the PIP1 aquaporin abundance, indicating inhibitory effects of NaCl on aquaporin functionality and protein abundance. The cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was reduced by salinity, as observed by confocal microscope analysis. Two different actions of Ca(2+) were observed. On the one hand, increase in free cytosolic calcium concentrations associated with stress perception may lead to aquaporin closure. On the other hand, when critical requirements of Ca(2+) were reduced (by salinity), and extra-calcium would lead to an upregulation of aquaporins, indicating that a positive role of calcium at whole plant level combined with an inhibitory mechanism at aquaporin level may work in the regulation of pepper root water transport under salt stress. However, a link between these observations and other cell signalling in relation to water channel gating remains to be established.     

Phosphate accumulation in leaves of wheat seedlings measured in leaf cell protoplasts isolated after root or foliar treatment with orthophosphate

Leaf cell protoplasts were isolated from wheat seedlings ( Triticum aestivum L. cv. Urquie) after orthophosphate (Pi) treatment of the plant to determine the capacity for intracellular phosphate accumulation. Seedlings were treated with Pi concentrations near the phytotoxic level to maximize the Pi concentration in the leaf prior to protoplast isolation 1 day later. Both foliar and root treatment of seedlings with Pi increased the phosphate content of leaf protoplasts by approximately 20 μmol (mg chlorophyll)⁻¹ over Pi levels in untreated controls. Phosphate-loaded protoplasts from treated seedlings had similar photosynthetic rates and starch content but 50% more soluble reducing sugar than protoplasts from untreated seedlings. Protoplast dark respiration decreased after treatments which increased protoplast potassium content. The results suggest that similar amounts of Pi can be accumulated by leaf cells of wheat after foliar or root application of Pi to the seedling without hindering Pi-sensitive processes such as photosynthesis and starch synthesis.     

Immunochemical Analysis of Aquaporin Isoforms in Arabidopsis Suspension-Cultured Cells

Aquaporins mediate the movement of water across biomembranes. Arabidopsis thaliana contains 35 aquaporins that belong to four subfamilies (PIP, TIP, SIP, and NIP). We investigated their expression profiles immunochemically in suspension-cultured Arabidopsis thaliana cells during growth and in response to salt and osmotic stresses. Protein amounts of all aquaporins were much lower in cultured cells than in the plant tissues. This is consistent with the low water permeability of protoplasts from cultured cells. After treatment with NaCl, the protein amounts of PIP2;1, PIP2;2, and PIP2;3 in the cells increased several-fold, and those of TIP1;1 and TIP1;2, 15- and 3-fold respectively. PIP1 did not change under the stress. Cell death began after 19 d in culture, accompanied by marked accumulation of PIPs and TIPs and a gradual decrease in SIPs. Our results suggest the followings: (i) Accumulation of aquaporin isoforms was individually regulated at low levels in single cells. (ii) At least PIP2;2, PIP2;3, TIP1;1, and TIP1;2 are stress-responsive aquaporins in suspension cells. (iii) A sudden increment of several members of PIP2 and TIP1 subfamilies might be related to cell death.     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

Effects of chlorosubstituted indoleacetic acids on root growth, ethylene and ATP formation

7-Chloroindoleacetic acid and dichloroindoleacetic acids with a Cl in the 7 position showed anti-auxinic activity and promoted root growth in wheat ( Triticum aestivum L. cv. Diamant II). In contrast, 4-, 5- and 6-chloroindoleatetic acids acted as strong auxins inhibiting the growth of wheat roots. Flax ( Linum usitatissimum L. cv. Concurrent) and cucumber ( Cucumis sativus L. cv. Favör) roots showed similar, but less clear-cut responses. 7-Chloroindoleacetic acid and 4,7-dichloroindoleacetic acid alleviated root growth inhibition in wheat caused by IAA, monochloroindoleacetic acids and benzyladenine. 2,4-D, 4- and 6-chloroindoleacetic acids strongly induced ethylene formation in cucumber seedlings; 4,7- and 6,7-dichloroindoleacetic acids did not, except at high concentrations. The more lipid-soluble dichloroindoleacetic acids were stronger inhibitors of ATP formation in cucumber mitochondria than monochloroindoleacetic acids, while IAA itself had only a very slight effect.     

Water permeability differs between growing and non-growing barley leaf tissues

A pressure probe technique and an osmotic swelling assay were used to compare water transport properties between growing and non-growing tissues of leaf three of barley. The epidermis was analysed in planta by pressure probe, whereas (predominantly) mesophyll protoplasts were analysed by osmotic swelling. Hydraulic conductivity (Lp) and, by implication, water permeability (Pf) of epidermal cells was 31% higher in the leaf elongation zone (Lp=0.5+/-0.2 microm s-1 MPa-1; Pf=65+/-25 microm s-1; means+/-SD of n=17 cells) than in the, non-growing, emerged leaf zone (Lp=0.4+/-0.1 microm s-1 MPa-1; Pf=50+/-15 microm s-1; n=24; P<0.05). Similarly, water permeability of mesophyll protoplasts was by 55% higher in the elongation compared with emerged leaf zone (Pf=13+/-1 microm s-1 compared with 8+/-1 microm s-1; n=57 and 36 protoplasts, respectively; P<0.01). Within the leaf elongation zone, a small population of larger-sized protoplasts could be distinguished. These protoplasts, which originated most likely from parenchymateous bundle sheath or midrib parenchyma cells, had a three-fold higher water permeability (P<0.001) as mesophyll protoplasts. The effect on Lp and Pf of known aquaporin inhibitors was tested with the pressure probe (Au+, Ag+, Hg2+, phloretin) and the osmotic swelling assay (phloretin). Only phloretin, when applied to protoplasts in the swelling assay caused an average decrease in Pf, but the effect varied between isolations. Technical approaches and cell-type and growth-specific differences in water transport properties are discussed.     

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...     

Effects of water deficit on photosynthetic rate and osmotic adjustment in tetraploid wheats

Osmotic adjustment, accumulation of soluble saccharides, and photosynthetic gas exchange were studied in five durum wheat (Triticum turgidum L. var. durum) and one wild emmer wheat (Triticum turgidum L. var. dicoccoïdes) cultivars of contrasting drought tolerance and yield stability. Soil water contents (SWC) were 100, 31, 20, and 12 % of maximum capillary capacity. Under mild water stress (SWC 31 to 20 %), osmotic adjustment capacity and high accumulation of saccharides were found in cv. Cham1, a high yielding and drought tolerant cultivar, and in var. dicoccoïdes, while lowest values were noted in the durum wheat landraces Oued-Zenati and Jennah-Khotifa. Under more severe water stress (SWC 12 %), the cv. Cham1 maintained higher net photosynthetic rate (PN) than other genotypes. The observed changes in the ratio intercellular/ambient CO2 concentration (ci/ca) indicated that under mild and severe water stress, the decrease in PN was mainly due to stomatal and non-stomatal factors, respectively.     

小麦与谷子不对称体细胞杂交

以不同剂量的紫外线(UV)照射谷子(Setariaitalica cy. Shanxi)原生质体为供体与普通小麦(Triticum aestivum)济南177和99P的原生质体用PEG法诱导融合.利用同工酶,RAPD和染色体分析再生的克隆及白化苗.从小麦济南177与谷子融合再生的86个克隆中,24个被鉴定为杂种克隆;而小麦99P与谷子融合再生的67个克隆中,27个被鉴定为杂种克隆.虽然用作融合材料的双亲培养细胞均丧失再生能力,但是部分来源于低剂量UV处理的杂种克隆再生了绿点、根和白化苗.证明小麦体细胞杂交中的双亲再生能力互补现象也存在于远缘族间融合组合中.讨论了小麦远缘族间融合未能再生绿色植株的原因.     

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.     

Plasma membrane aquaporins play a significant role during recovery from water deficit

The role of plasma membrane aquaporins (PIPs) in water relations of Arabidopsis was studied by examining plants with reduced expression of PIP1 and PIP2 aquaporins, produced by crossing two different antisense lines. Compared with controls, the double antisense (dAS) plants had reduced amounts of PIP1 and PIP2 aquaporins, and the osmotic hydraulic conductivity of isolated root and leaf protoplasts was reduced 5- to 30-fold. The dAS plants had a 3-fold decrease in the root hydraulic conductivity expressed on a root dry mass basis, but a compensating 2.5-fold increase in the root to leaf dry mass ratio. The leaf hydraulic conductance expressed on a leaf area basis was similar for the dAS compared with the control plants. As a result, the hydraulic conductance of the whole plant was unchanged. Under sufficient and under water-deficient conditions, stomatal conductance, transpiration rate, plant hydraulic conductance, leaf water potential, osmotic pressure, and turgor pressure were similar for the dAS compared with the control plants. However, after 4 d of rewatering following 8 d of drying, the control plants recovered their hydraulic conductance and their transpiration rates faster than the dAS plants. Moreover, after rewatering, the leaf water potential was significantly higher for the control than for the dAS plants. From these results, we conclude that the PIPs play an important role in the recovery of Arabidopsis from the water-deficient condition.