Zinc treatment of hydroponically grown barley plants causes a reduction in root and cell hydraulic conductivity and isoform‐dependent decrease in aquaporin gene expression

The cellular and molecular basis of a reduction in root water uptake in plants exposed to heavy metals such as zinc (Zn) is poorly studied. The aim of the present study on hydroponically grown barley (Hordeum vulgare) was to test whether any reduction in root hydraulic conductivity (Lp) in response to Zn treatment is accompanied by a reduction in cell Lp and gene expression level of aquaporin (AQP) isoforms. Plants were grown in the presence of 0.25 μM, (control), 0.1 and 1 mM Zn in the root medium and analysed when they were 16–18 days old. Root and cell Lp was determined through exudation and cell pressure probe analyses, respectively, and gene expression of five candidate AQPs was analysed [real time quantitative polymerase chain reaction (PCR)]. Zinc treatments caused significant reductions (25–83%) in transpiration rate, root and shoot fresh weight, surface area and stomatal conductance. Zinc concentrations in tissues increased more than 100‐fold. Root Lp decreased by 24% (0.1 mM Zn) and 58% (1 mM Zn), while cell Lp decreased by 45 and 71%, respectively. Gene expression of AQPs decreased by 14–80%; decreases were statistically significant for HvPIP1;3, HvPIP2;4 and HvPIP2;5. Turgor in root cortex cells was not reduced by Zn treatments. It is concluded that reductions in plant water flow in response to Zn treatment are facilitated through decreases in root (Lp) and shoot (stomata) hydraulics. The decrease in root Lp is facilitated through reductions in cell Lp and AQP gene expression and may also reflect increased suberization in the endodermis.     

Nitrate induction of root hydraulic conductivity in maize is not correlated with aquaporin expression

Some plant species can increase the mass flow of water from the soil to the root surface in response to the appearance of nitrate in the rhizosphere by increasing root hydraulic conductivity. Such behavior can be seen as a powerful strategy to facilitate the uptake of nitrate in the patchy and dynamically changing soil environment. Despite the significance of such behavior, little is known about the dynamics and mechanism of this phenomenon. Here we examine root hydraulic response of nitrate starved Zea mays (L.) plants after a sudden exposure to 5 mM NO₃ ⁻ solution. In all cases the treatment resulted in a significant increase in pressure-induced (pressure gradient ~ 0.2 MPa) flow across the root system by ~50% within 4 h. Changes in osmotic gradient across the root were approximately 0.016 MPa (or 8.5%) and thus the results could only be explained by a true change in root hydraulic conductance. Anoxia treatment significantly reduced the effect of nitrate on xylem root hydraulic conductivity indicating an important role for aquaporins in this process. Despite a 1 h delay in the hydraulic response to nitrate treatment, we did not detect any change in the expression of six ZmPIP1 and seven ZmPIP2 genes, strongly suggesting that NO₃ ⁻ ions regulate root hydraulics at the protein level. Treatments with sodium tungstate (nitrate reductase inhibitor) aimed at resolving the information pathway regulating root hydraulic properties resulted in unexpected findings. Although this treatment blocked nitrate reductase activity and eliminated the nitrate-induced hydraulic response, it also produced changes in gene expression and nitrate uptake levels, precluding us from suggesting that nitrate acts on root hydraulic properties via the products of nitrate reductase.     

Nitrogen availability affects hydraulic conductivity of rice roots,possibly through changes in aquaporin gene expression

Background and aims

Nitrogen (N) availability affects water uptake from the roots, which decreases upon N deprivation and increases upon resupply. The aim of this study was to reveal possible mechanisms of regulation of water transport in roots through physiological and morphological adaptations to N availability.

Methods

The effects of continuous N deprivation and following resupply on root morphology, osmotic hydraulic conductivity, and expression of genes for aquaporins (water channels) were examined in rice (Oryza sativa L.) plants. The effect of local N availability was examined by using a split-root system.

Results

N deprivation decreased the expression of root-specific aquaporin genes, whereas N resupply increased their expression. Changes in aquaporin gene expression were correlated with changes in hydraulic conductivity. N deprivation increased dry matter allocation to the roots. In a split-root experiment, the expression of root-specific aquaporin genes was down-regulated in the N-deprived half, whereas it was up-regulated in the N-supplied half.

Conclusion

Our results suggest that expression of genes for root-specific aquaporins underlies the changes in conductivity during continuous N deprivation and resupply. Rice plants seem to adapt to N availability through coordinated adjustment of root proliferation and abundance of aquaporins.     

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.     

Influence of low air humidity and low root temperature on water uptake, growth and aquaporin expression in rice plants

The effects of low air humidity and low root temperature (LRT) on water uptake, growth and aquaporin gene expression were investigated in rice plants. The daily transpiration of the plants grown at low humidity was 1.5- to 2-fold higher than that at high humidity. LRT at 13°C reduced transpiration, and the extent was larger at lower humidity. LRT also reduced total dry matter production and leaf area expansion, and the extent was again larger at lower humidity. These observations suggest that the suppression of plant growth by LRT is associated with water stress due to decreased water uptake ability of the root. On the other hand, the net assimilation rate was not affected by low humidity and LRT, and water use efficiency was larger for LRT. We found that low humidity induced coordinated up-regulation of many PIP and TIP aquaporin genes in both the leaves and the roots. Expression levels of two root-specific aquaporin genes, OsPIP2;4 and OsPIP2;5, were increased significantly after 6 and 13 d of LRT exposure. Taken together, we discuss the possibility that aquaporins are part of an integrated response of this crop to low air humidity and LRT.     

Flooding, gas exchange and hydraulic root conductivity of highbush blueberry

Highbush blueberry plants ( Vaccinium corymbosum L. cv. Bluecrop) growing in containers were flooded in the laboratory for various durations to determine the effect of flooding on carbon assimilation, photosynthetic response to varying CO₂ and O₂ concentrations and apparent quantum yield as measured in an open flow gas analysis system. Hydraulic conductivity of the root was also measured using a pressure chamber. Root conductivity was lower and the effect of increasing CO₂ levels on carbon assimilation less for flooded than unflooded plants after short-(i-2 days), intermediate-(10–14 days) and long-term (35–40 days) flooding. A reduction in O₂ levels surrounding the leaves from 21 to 2% for unflooded plants increased carbon assimilation by 33% and carboxylation efficiency from 0.012 to 0.021 mol CO₂ fixed (mol CO₂)⁻¹. Carboxylation efficiency of flooded plants, however, was unaffected by a decrease in percentage O₂, averaging 0.005 mol CO₂ fixed (mol CO₂)⁻¹. Apparent quantum yield decreased from 2.2 × 10⁻¹ mol of CO₂ fixed (mol light)⁻¹ for unflooded plants to 2.0 × 10⁻³ and 9.0 × 10⁻⁴ for intermediate- and long-term flooding durations, respectively. Shortterm flooding reduced carbon assimilation via a decrease in stomatal conductance, while longer flooding durations also decreased the carboxylation efficiency of the leaf.     

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.     

Rhizosphere humic acid interacts with root cell walls to reduce hydraulic conductivity and plant development

Humic acids are ubiquitous, organic-end-products of the chemical and microbial degradation of dead biota in soils throughout the world. Humic acids can be transported in soil water as heterogeneous, supra-molecular, colloidal-agglomerates. Humic acid accumulation in the rhizosphere of transpiring plants may chemically stimulate development by increasing root availability of mineral nutrients and/or growth regulatory biomolecules. This report introduces novel, physical mechanisms by which humic acid can also reduce plant development. Effects of humic acid addition to the root media of intact maize plants (Zea mays L.) on their growth, transpiration and resistance to water deficits were assayed, as were the effects of external humic acid on the hydraulic conductivity of excised primary-roots. Humic acid reduced shoot growth, transpiration and resistance to water stress but not root growth. Root hydraulic conductivity was reduced by up to 44% via a time-, concentration- and size-dependent fouling mechanism resulting from humic acid accumulation at root cell-walls. Thus, humic acid is shown, apparently for the first time, to be able to exert novel physical effects in addition to its known chemical effects on plant development.     

A technique to measure root tip hydraulic conductivity and root water potential simultaneously

A procedure for the simultaneous measurement of hydraulic conductivityand xylem water potential of roots is presented. Roots remainintact and attached to the transpiring plant during measurement.The rate of water uptake by roots is measured at different waterpotential gradients along the root radial axis, obtained byplacing them in solutions with different osmotic potentials.Hydraulic conductivity and xylem water potential are calculatedby regression analysis of the relationship between water uptakerate and osmotic potential of the bathing solution, assumingthat xylem water potential and reflection coefficient remainconstant during measurement. Results for tomato plants experiencingdrought are presented and discussed. Key words: Root, hydraulic conductivity, water potential     

Transpiration from shoots triggers diurnal changes in root aquaporin expression

Root hydraulic conductivity (Lp(r)) and aquaporin amounts change diurnally. Previously, these changes were considered to be spontaneously driven by a circadian rhythm. Here, we evaluated the new hypothesis that diurnal changes could be triggered and enhanced by transpirational demand from shoots. When rice plants were grown under a 12h light/12h dark regime, Lp(r) was low in the dark and high in the light period. Root aquaporin mRNA levels also changed diurnally, but the amplitudes differed among aquaporin isoforms. Aquaporins, such as OsPIP2;1, showed moderate changes, whereas root-specific aquaporins, such as OsPIP2;5, showed temporal and dramatic induction around 2h after light initiation. When darkness was extended for 12h after the usual dark period, no such induction was observed. Furthermore, plants under 100% relative humidity (RH) showed no induction even in the presence of light. These results suggest that transpirational demand triggers a dramatic increase in gene expressions such as OsPIP2;5. Immunocytochemistry showed that OsPIP2;5 accumulated on the proximal end of the endodermis and of the cell surface around xylem. The strong induction by transpirational demand and the polar localization suggest that OsPIP2;5 contributes to fine adjustment of radial water transport in roots to sustain high Lp(r) during the day.     

Root system hydraulic conductivity in species with contrasting root anatomy

Previous research suggested that the hydraulic properties of root systems of intact plants could be described by two parameters: the hydraulic conductivity (Lpr) or the slope of the flow-density/water potential gradient relationship, and the offset or minimum water potential gradient required to induce flow. In this study Lpr and offset were correlated with anatomical features of the root radial path in plants with contrasting root anatomy. Two woody and three herbaceous species were examined which exhibit a range of root anatomical features: Asparagus densiflorus (Kunth) Jessop (asparagus), Dendrobium superbum Rchb. f. (dendrobium), Glycine max (L.) Merr. (soybean), Prunus persica (L.) Batsch. (peach), Citrus aurantium L. (sour orange). Lpr varied about 8-fold, and the offset varied about 6-fold among the five species. Lpr was inversely related to root diameter (r²0.39) and cortex width (r²0.55), suggesting that species with thinner roots or roots with a thin cortex had the highest Lpr. Further observations suggested that the cortex width was a stronger determinant of Lpr than root diameter. However, the offset was not correlated with root diameter, stele diameter or cortex width, but was >2-fold higher in species having an exodermis in the root radial path (sour orange, asparagus, and dendrobium) compared to those lacking an exodermis (peach and soybean). The data on root Lr obtained were similar to those given in the literature for both intact plants and excised roots which have been measured with different techniques. It is concluded that Lpr and offset, which describe the flow-water potential relationship for intact root systems, are related to differences in the root cortex; specifically, its thickness and the presence/absence of a suberized exodermis. Hence, these anatomical differences may, in part, cause the variability in root hydralic properties that exists among plant species.     

Role of aquaporin activity in regulating deep and shallow root hydraulic conductance during extreme drought

Key message

Deep root hydraulic conductance is upregulated during severe drought and is associated with upregulation in aquaporin activity.

Abstract

In 2011, Texas experienced the worst single-year drought in its recorded history and, based on tree-ring data, likely its worst in the past millennium. In the Edwards Plateau of Texas, rainfall was 58 % lower and the mean daily maximum temperatures were >5 °C higher than long-term means in June through September, resulting in extensive tree mortality. To better understand the balance of deep and shallow root functioning for water supply, we measured root hydraulic conductance (K _R) in deep (~20 m) and shallow (5–10 cm) roots of Quercus fusiformis at four time points in the field in 2011. Deep roots of Q. fusiformis obtained water from a perennial underground (18–20 m) stream that was present even during the drought. As the drought progressed, deep root K _R increased 2.6-fold from early season values and shallow root K _R decreased by 50 % between April and September. Inhibitor studies revealed that aquaporin contribution to K _R increased in deep roots and decreased in shallow roots as the drought progressed. Deep root aquaporin activity was upregulated during peak drought, likely driven by increased summer evaporative demand and the need to compensate for declining shallow root K _R. A whole-tree hydraulic transport model predicted that trees with greater proportions of deep roots would have as much as five times greater transpiration during drought periods and could sustain transpiration during droughts without experiencing total hydraulic failure. Our results suggest that trees shift their dependence on deep roots versus shallow roots during drought periods, and that upregulation of aquaporin activity accounts for at least part of this increase.     

Involvement of root ABA and hydraulic conductivity in the control of water relations in wheat plants exposed to increased evaporative demand

We studied the possible involvement of ABA in the control of water relations under conditions of increased evaporative demand. Warming the air by 3°C increased stomatal conductance and raised transpiration rates of hydroponically grown Triticum durum plants while bringing about a temporary loss of relative water content (RWC) and immediate cessation of leaf extension. However, both RWC and extension growth recovered within 30 min although transpiration remained high. The restoration of leaf hydration and growth were enabled by increased root hydraulic conductivity after increasing the air temperature. The use of mercuric chloride (an inhibitor of water channels) to interfere with the rise on root hydraulic conductivity hindered the restoration of extension growth. Air warming increased ABA content in roots and decreased it in shoots. We propose this redistribution of ABA in favour of the roots which increased the root hydraulic conductivity sufficiently to permit rapid recovery of shoot hydration and leaf elongation rates without the involvement of stomatal closure. This proposal is based on known ability of ABA to increase hydraulic conductivity confirmed in these experiments by measuring the effect of exogenous ABA on osmotically driven flow of xylem sap from the roots. Accumulation of root ABA was mainly the outcome of increased export from the shoots. When phloem transport in air-warmed plants was inhibited by cooling the shoot base this prevented ABA enrichment of the roots and favoured an accumulation of ABA in the shoot. As a consequence, stomata closed.     

Relationships between xylem anatomy,root hydraulic conductivity,leaf/root ratio and transpiration in citrus trees on different rootstocks

The aim of the study was to determine the extent in which leaf and whole plant transpiration (Tp) were influenced by root hydraulic conductance (K_r), leaf to root ratio and leaf mass. Also, the relationships between the anatomic characteristics of roots and K_r were investigated. To this end, 9‐month‐old seedlings of the citrus rootstocks Cleopatra mandarin (CM), Poncirus trifoliata (PT), and their hybrids Forner‐Alcaide no 5 (FA‐5) and Forner‐Alcaide no 13 (FA‐13) and 15‐month‐old trees of Valencia orange budded on these four rootstocks were tested. The hybrid FA‐13 and PT had higher values of K_r and leaf transpiration rates (E) than FA‐5 and CM. There was a positive curvilinear correlation between E and K_r. Furthermore, E levels in the different types of plants decreased with increased leaf/root (L/R) ratios. Pruning of the roots and defoliation confirmed that transpiration rates were strongly influenced by the L/R ratio. However, variations in E because of differences in L/R ratios were less pronounced in trees budded on FA‐13 and PT than on the other two rootstocks. In addition, there was a positive correlation between Tp and leaf biomass, although differences between rootstocks may be attributed to differences in K_r. The average lumen diameter of xylem vessels was greater in rootstocks with high K_r. Size of epidermal and hypodermal cells of fibrous roots may also restrict K_r.     

Seminal roots of wild and cultivated barley differentially respond to osmotic stress in gene expression,suberization, and hydraulic conductivity

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5-fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA-sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit.     

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.     

Review article. Apoplastic barriers in roots: chemical composition of endodermal and hypodermal cell walls

Based on the characterization of the chemical composition of endodermal and hypodermal cell walls isolated from seven monocotyledonous and three dicotyledonous plant species, a model of the composition of apoplastic barriers in roots is proposed. Depending on the species, endodermal and hypodermal cell walls of roots contained varying amounts of the biopolymers suberin, lignin, cell wall proteins, and carbohydrates. Although analysis of the chemical composition of these apoplastic barriers of roots is now possible, it is pointed out that conclusions from these data concerning the functional properties of these cell walls can not easily be drawn. However, in analogy to suberized periderms it is argued that the suberin should play a role in establishing an apoplastic transport barrier in roots, albeit not a perfect barrier. Furthermore, due to the combined occurrence of suberin, lignin and cell wall proteins it is argued that endodermal and hypodermal cell walls also have an important function as barriers towards pathogens. Finally, it is pointed out that additional experimental approaches combining the investigation of transport properties and of the chemical composition of apoplastic transport barriers in roots are necessary before the function of endodermal and hypodermal cell walls in roots can be fully understood.     

Effects of salinity on growth,membrane permeability and root hydraulic conductivity in three saltbush species

The species of the genus Atriplex have been introduced in West Asia and North Africa to determine their adaptability for use as fodder species. These halophytes are well adapted to extreme environmental conditions and may possess interesting properties for soil rehabilitation. The effect of NaCl stress on growth, water relation and mineral nutrition were investigated in three xero-halophyte species of Atriplex used for rehabilitation of arid steppe in Algeria. Atriplex halimus, Atriplex canescens and Atriplex nummularia, were cultivated in hydroponic conditions and treated with increasing doses of NaCl (0–300 mM). All species showed positive plant growth for low and moderate levels of salinity. A. halimus had higher dry weight production than A. nummularia and A. canescens in high salinity concentration. Increasing concentration of salinity induced decrease in chlorophyll content (Chl a and b) and root hydraulic conductivity (L₀) in all species, especially in A. canescens. All three species showed marked increase in electrolyte leakage across the salinity gradient. In addition all species were able to accumulate a large quantity of sodium (Na), chloride (Cl) and proline and to maintain higher relative water content, which was probably associated with a greater capacity for osmotic adjustment, whereas potassium (K) and calcium (Ca) decreased with increase salinity. The data suggest that salt tolerance strategies in all Atriplex species could involve a delicate balance among ion accumulation, osmotic adjustment, production of osmotica and maintenance of relative water content and growth.