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.     

Influence of saline stress on root hydraulic conductance and PIP expression in Arabidopsis

Measurements of the root hydraulic conductance (L0) of roots of Arabidopsis thaliana were carried out and the results were compared with the expression of aquaporins present in the plasma membrane of A. thaliana. L0 of plants treated with different NaCl concentrations was progressively reduced as NaCl concentration was increased compared to control plants. Also, L0 of plants treated with 60 mmol/L NaCl for different lengths of time was measured. Variations during the light period were seen, but only for the controls. A good correlation between mRNA expression and L0 was observed in both experiments. Control plants and plants treated with 60 mmol/L NaCl were incubated with Hg and then with DTT. For these plants, L0 and cell-to-cell pathway contributions to root water transport were determined. These results revealed that in control plants most water movement occurs via the cell-to-cell pathway, thus implying aquaporin involvement. But, in NaCl-stressed plants, the Hg-sensitive cell-to-cell pathway could be inhibited already by the effect of NaCl on water channels. Therefore, short periods of NaCl application to Arabidopsis plants are characterised by decreases in the L0 of roots, and are related to down-regulation of the expression of the PIP aquaporins. This finding indicates that the well known effect of salinity on L0 could involve regulation of aquaporin expression.     

Water-deficit tolerance in citrus is mediated by the down regulation of PIP gene expression in the roots

Water deficit (WD) is a growing problem in agriculture. In citrus crops, genetically-determined rootstock characteristics are important factors influencing plant responses to WD. Aquaporins are involved in regulating the water supply to the plant by mediating water flow through the cell membranes. Recent studies support a direct role for aquaporins in plant water relations and demonstrate their involvement in WD tolerance. This study investigates the relationship between photosynthetic and water-balance parameters with aquaporin expression levels and hydraulic conductance of roots (Kr) in conditions of moderate WD in citrus rootstocks. The plant materials used were the rootstocks Poncirus trifoliata (L.) Raf. (PT), Cleopatra mandarin (Citrus reshni Hort ex Tan.) (CM) and 030115 (a hybrid of the two former rootstocks), all grafted with the citrus variety ??Valencia Late?? (C. sinensis (L.) Osb). Plants were irrigated with two differents irrigation doses (normal irrigation and moderate WD) during 70 days and leaf water potential (??s), net CO₂ assimilation (A_CO2), transpiration, stomatal conductance (gs) and substomatal CO₂ concentration (Ci) were measured periodically under both irrigation conditions. Kr and PIP1 and PIP2 gene expression levels in fine roots of control plants and plants subjected to WD on day 43 of the experiment were determined. Under WD conditions, the hybrid 030115 drastically reduced aquaporin expression and Kr, accompanied by a loss of plant vigour but without reducing the net CO₂ assimilation (A_CO2). PT maintained the same aquaporin expression level and similar Kr under WD as under normal irrigation conditions, but suffered a sharp reduction in A_CO2. CM, which has lower Kr and aquaporin expression than PT under both normal irrigation conditions and WD, responded better to water stress conditions than PT. Low aquaporin levels, or down-regulated aquaporin expression, accompanied by decreased plant vigour led to decreased plasma membrane permeability, thereby facilitating water retention in the cells under water stress conditions. This may induce water stress tolerance in citrus rootstocks.     

Overexpression of PIP2;5 aquaporin alleviates effects of low root temperature on cell hydraulic conductivity and growth in Arabidopsis

The effects of low root temperature on growth and root cell water transport were compared between wild-type Arabidopsis (Arabidopsis thaliana) and plants overexpressing plasma membrane intrinsic protein 1;4 (PIP1;4) and PIP2;5. Descending root temperature from 25°C to 10°C quickly reduced cell hydraulic conductivity (L(p)) in wild-type plants but did not affect L(p) in plants overexpressing PIP1;4 and PIP2;5. Similarly, when the roots of wild-type plants were exposed to 10°C for 1 d, L(p) was lower compared with 25°C. However, there was no effect of low root temperature on L(p) in PIP1;4- and PIP2;5-overexpressing plants after 1 d of treatment. When the roots were exposed to 10°C for 5 d, L(p) was reduced in wild-type plants and in plants overexpressing PIP1;4, whereas there was still no effect in PIP2;5-overexpressing plants. These results suggest that the gating mechanism in PIP1;4 may be more sensitive to prolonged low temperature compared with PIP2;5. The reduction of L(p) at 10°C in roots of wild-type plants was partly restored to the preexposure level by 5 mm Ca(NO(3))(2) and protein phosphatase inhibitors (75 nm okadaic acid or 1 μm Na(3)VO(4)), suggesting that aquaporin phosphorylation/dephosphorylation processes were involved in this response. The temperature sensitivity of cell water transport in roots was reflected by a reduction in shoot and root growth rates in the wild-type and PIP1;4-overexpressing plants exposed to 10°C root temperature for 5 d. However, low root temperature had no effect on growth in plants overexpressing PIP2;5. These results provide strong evidence for a link between growth at low root temperature and aquaporin-mediated root water transport in Arabidopsis.     

Relationship between hydraulic conductance and citrus dwarfing by the Flying Dragon rootstock (Poncirus trifoliata L. Raft var. monstruosa)

This work studied the hydraulic characteristics and physiological behavior of two trifoliate orange (Poncirus trifoliata L. Raft) varieties—Flying Dragon (FD) and Rubidoux (RT)—with contrasting size-controlling potential when used as rootstocks for citrus trees. Thus, Valencia orange scions growing on RT root system develop about 40 % more biomass than scions on FD. The anatomical study of xylem root tissue of both rootstocks showed that the number of vessels per cross-sectional area in RT almost doubled that found in FD, whereas diameter distribution did not vary significantly. Hydraulic resistance determined in rootstocks, and bud union segments were, respectively, 2- and 3.4-fold higher in trees on FD than in trees on RT. Root systems accounted for 46.5 and 55.2 % of whole-plant hydraulic resistance, whereas bud union segments represented 7.5 and 14.6 % of this parameter, the dwarfing rootstock (FD) having the highest values. Reduced hydraulic conductance in plants on FD rootstock diminished water potential in high evaporative demand periods, causing a reduction in stomatal conductance with respect to plants on RT. This leads to lower net photosynthetic CO₂ assimilation, which may affect biomass production. Translocation of ¹³C-labeled photoassimilates from leaves to roots was lower in plants on FD than in plants on RT, indicating that in the dwarfing rootstock (FD) there may be a vascular resistance to sucrose transport at the budding union level. Findings show that reduced hydraulic conductance may be the main cause of rootstock-induced dwarfing in citrus grafted onto FD.     

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.     

Fluoride inhibits root water transport and affects leaf expansion and gas exchange in aspen (Populus tremuloides) seedlings

The effects of sodium fluoride (0.3, 5 and 10 m M NaF) on root hydraulic conductivity, and gas exchange processes were examined in aspen ( Populus tremuloides Michx.) seedlings grown in solution culture. A long-term exposure of roots to NaF significantly decreased root hydraulic conductivity ( L _p) and stomatal conductance ( g _s). Root absorbed NaF significantly affected electrolyte leakage in leaf tissues and substantially restricted leaf expansion. NaF did not significantly affect leaf chlorophyll contents but decreased net photosynthesis ( P _n). A short-term exposure of excised roots to 5 m M NaF and KF significantly decreased root water flow ( Q _v) with a concomitant decline in root respiration and reduced g _s when applied through intact roots or excised stems. The same molar concentration of NaCl also decreased Q _v and g _s in intact seedlings, but to a lesser extent than NaF or KF, and did not significantly affect root respiration. The results suggest that fluoride metabolically inhibited Q _v or L _p, probably by affecting water channel activity. We suggest that the metabolic inhibition of L _p by root-absorbed fluoride affected gas exchange and leaf expansion in aspen seedlings.     

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.     

Exogenous ABA accentuates the differences in root hydraulic properties between mycorrhizal and non mycorrhizal maize plants through regulation of PIP aquaporins

The arbuscular mycorrhizal (AM) symbiosis has been shown to modulate the same physiological processes as the phytohormone abscisic acid (ABA) and to improve plant tolerance to water deficit. The aim of the present research was to evaluate the combined influence of AM symbiosis and exogenous ABA application on plant root hydraulic properties and on plasma-membrane intrinsic proteins (PIP) aquaporin gene expression and protein accumulation after both a drought and a recovery period. Results obtained showed that the application of exogenous ABA enhanced osmotic root hydraulic conductivity (L) in all plants, regardless of water conditions, and that AM plants showed lower L values than nonAM plants, a difference that was especially accentuated when plants were supplied with exogenous ABA. This effect was clearly correlated with the accumulation pattern of the different PIPs analyzed, since most showed reduced expression and protein levels in AM plants fed with ABA as compared to their nonAM counterparts. The possible involvement of plant PIP aquaporins in the differential regulation of L by ABA in AM and nonAM plants is further discussed.     

Over-expression of PIP2;5 aquaporin alleviates gas exchange and growth inhibition in poplars exposed to mild osmotic stress with polyethylene glycol

The effects of mild osmotic stress conditions on aquaporin-mediated water transport are not well understood. In the present study, mild osmotic stress treatments with 20 and 50 g L^?1 polyethylene glycol 6000 (PEG) in Hoagland’s mineral solution were applied for 3 weeks under controlled environmental conditions to transgenic Populus tremula × Populus alba plants constitutively over-expressing a Populus PIP2;5 aquaporin and compared with the wild-type plants. The PEG treatments resulted in growth reductions and triggered changes in net photosynthesis, transpiration, stomatal conductance and root hydraulic conductivity in the wild-type plants. However, height growth, leaf area, gas exchange, and root hydraulic conductivity were less affected by the PEG treatments in PIP2;5-over-expressing poplar lines. These results suggest that water transport across the PIP2;5 aquaporin is an important process contributing to tolerance of mild osmotic stress in poplar. Greater membrane abundance of PIP2;5 was most likely the factor that was responsible for higher root hydraulic conductivity leading to improved plant water flux and, consequently, greater gas exchange and growth rates under mild osmotic stress conditions. The results also provide evidence for the functional significance of PIP2;5 aquaporin in water transport and its strong link to growth processes in poplar.     

Effect of genotype and graft type on the hydraulic characteristics and water relations of grafted melon

Abstract

From the measurements of the profiles of hydraulic conductance and water potential from soil through to the leaf system in fully established melon plants, the limits to water flow set by coupling of hydraulic conductance (k) with water relation parameters was evaluated in the laboratory using high pressure flow device (HPFM) and evaporative flux method (EF). The rootstock Arava was grafted onto self, and onto two genotypes (AR57 and AR82) using side and V graft types, and there was an ungrafted control. Hydraulic transport efficiency was estimated from measurements of evaporative flux (transpiration rate) and leaf water potential (ψL) measured between pre-dawn and sunset during the growth cycle. Measured parameters to characterize the hydraulic efficiency (architecture) of the vascular system of melon were normalized to areas of leaves and stem cross section; this enabled the examination of their physiological and ecological functions. The effects of rootstock genotype were more marked on graft union and scion water relations. Differences in the magnitudes of water relation parameters of hydraulic conductance, water potential (lwp) and evaporative water loss (EF) were detected. AR/RS82 side grafted exhibited high EF and Kh despite its lower leaf water potential compared to AR/RS57 V grafted. Self grafting (Arava/Arava grafts) in melon seems to improve water relations and xylem water transport efficiency. Parameters describing the hydraulic efficiency (architecture) of vascular system of melon plants were described in relation to plant attributes. The expression of hydraulic conductance of the root and shoot system relative to plant attributes did not eliminate differences in the magnitudes of conductance elements in tomato and melon. Differences obtained among the different melon grafts in whole plant leaf and stem area specific hydraulic conductance (Kl) indicate the carbon efficiency and hence the cost of resource allocation to areas of root surface and leaves. The role of plant water relations in root-shoot communications and whole plant regulation of water flux are inferred from this study.     

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.     

The effectiveness of grafting to improve salt tolerance in tomato when an ‘excluder’ genotype is used as scion

If the main effect of long-term exposure of tomato plants to salinity is the accumulation of toxic concentrations of Na⁺ and Cl⁻ in the leaves, then the selection of ‘excluder’ rootstocks should increase tolerance to salinity in grafted tomato plants, independently of the genotype used as the scion. The question addressed in this study is whether shoot genotypes with an ‘excluder’ character are able to increase their salt tolerance when grafted onto rootstocks of the same characteristics. Moneymaker (with excluder character) was grafted onto two root genotypes, Radja and Pera, selected for their very different ability to regulate the transport of saline ions to the shoot over time. Grafting onto either Pera or Radja improved fruit yield compared to the self-grafted plants of Moneymaker (M/M) when the plants were grown at 50 mM NaCl, whereas there was no effect of either rootstock or of grafting per se (M/M) on fruit yield in the absence of or at 25 mM NaCl. The relationship between the salt responses to mid- and long-term depended on the stress level; after 27 d of 150 mM NaCl treatment, both graft combinations enhanced similarly their salt tolerances as did in the long-term experiment. Moreover, the tolerance induced by rootstock was related to the low rates of saline ion accumulation in their leaves. However, the positive effect of rootstock was only observed with rootstock Pera when the grafted plants were grown at 50 mM NaCl (the same salt level used in the long-term experiment) for 35 d. According to the physiological changes induced by rootstock in the leaves, the different salt responses seem to be due to the fact that the osmotic effect predominated on the toxic effect under these last conditions. Consequently, in order to select rootstocks care must be taken in the timing of any selection process: the stress level and length of exposure to salinity must be sufficient for the true differences in salt tolerance for toxicity to be shown. Taken together, these results show the effectiveness of grafting to enhance fruit yield in tomato and provide evidence that the positive effect induced by rootstock is related to the re-establishment of ionic homeostasis.