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.     

Arbuscular mycorrhizal symbiosis and methyl jasmonate avoid the inhibition of root hydraulic conductivity caused by drought

Hormonal regulation and symbiotic relationships provide benefits for plants to overcome stress conditions. The aim of this study was to elucidate the effects of exogenous methyl jasmonate (MeJA) application on root hydraulic conductivity (L) of Phaseolus vulgaris plants which established arbuscular mycorrhizal (AM) symbiosis under two water regimes (well-watered and drought conditions). The variation in endogenous contents of several hormones (MeJA, JA, abscisic acid (ABA), indol-3-acetic acid (IAA), salicylic acid (SA)) and the changes in aquaporin gene expression, protein abundance and phosphorylation state were analyzed. AM symbiosis decreased L under well-watered conditions, which was partially reverted by the MeJA treatment, apparently by a drop in root IAA contents. Also, AM symbiosis and MeJA prevented inhibition of L under drought conditions, most probably by a reduction in root SA contents. Additionally, the gene expression of two fungal aquaporins was upregulated under drought conditions, independently of the MeJA treatment. Plant aquaporin gene expression could not explain the behaviour of L. Conversely, evidence was found for the control of L by phosphorylation of aquaporins. Hence, MeJA addition modified the response of L to both AM symbiosis and drought, presumably by regulating the root contents of IAA and SA and the phosphorylation state of aquaporins.     

Roles of Morphology, Anatomy, and Aquaporins in Determining Contrasting Hydraulic Behavior of Roots

The contrasting hydraulic properties of wheat (Triticum aestivum), narrow-leafed lupin (Lupinus angustifolius), and yellow lupin (Lupinus luteus) roots were identified by integrating measurements of water flow across different structural levels of organization with anatomy and modeling. Anatomy played a major role in root hydraulics, influencing axial conductance (L_ax) and the distribution of water uptake along the root, with a more localized role for aquaporins (AQPs). Lupin roots had greater L_ax than wheat roots, due to greater xylem development. L_ax and root hydraulic conductance (L_r) were related to each other, such that both variables increased with distance from the root tip in lupin roots. L_ax and L_r were constant with distance from the tip in wheat roots. Despite these contrasting behaviors, the hydraulic conductivity of root cells (Lp_c) was similar for all species and increased from the root surface toward the endodermis. Lp_c was largely controlled by AQPs, as demonstrated by dramatic reductions in Lp_c by the AQP blocker mercury. Modeling the root as a series of concentric, cylindrical membranes, and the inhibition of AQP activity at the root level, indicated that water flow in lupin roots occurred primarily through the apoplast, without crossing membranes and without the involvement of AQPs. In contrast, water flow across wheat roots crossed mercury-sensitive AQPs in the endodermis, which significantly influenced L_r. This study demonstrates the importance of examining root morphology and anatomy in assessing the role of AQPs in root hydraulics.     

A putative role for TIP and PIP aquaporins in dynamics of leaf hydraulic and stomatal conductances in grapevine under water stress and re‐watering

We examined the role of aquaporins (AQPs) in regulating leaf hydraulic conductance (K_leaf) in Vitis vinifera L. (cv Chardonnay) by studying effects of AQP inhibitors, and AQP gene expression during water stress (WS) and recovery (REC). K_leaf was measured after 3 h of petiole perfusion with different solutions and to introduce inhibitors. The addition of 0.1 mm HgCl₂ to 15 mm KCl reduced K_leaf compared with perfusion in 15 mM KNO₃ or KCl, and these solutions were used for leaves from control, WS and REC plants. Perfusion for 3 h did not significantly alter stomatal conductance (g_s) though expression of VvTIP1;1 was increased. WS decreased K_leaf by about 30% and was correlated with g_s. The expression of VvTIP2;1 and VvPIP2;1 correlated with K_leaf, and VvTIP2;1 was highly correlated with g_s. There was no association between the expression of particular AQPs during WS and REC and inhibition of K_leaf by HgCl₂; however, HgCl₂ treatment itself increased expression of VvPIP2;3 and decreased expression of VvPIP2;1 . Inhibition by HgCl₂ of K_leaf only at early stages of WS and then after REC suggested that apoplasmic pathways become more important during WS. This was confirmed using fluorescent dyes confined to apoplasm or preferentially accumulated in symplasm.     

Synergistic effects of drought and ascorbic acid on growth,mineral nutrients and oxidative defense system in canola (Brassica napus L.) plants

A study was conducted to find out the role of ascorbic acid (AsA) in modulating growth and different physio-biochemical attributes of canola plants under well-watered as well as water-deficit conditions. Drought stress imposed on 60 % field capacity significantly decreased the shoot and root fresh and dry weights, leaf chlorophyll contents, shoot and root P, root K⁺, and activity of CAT enzyme, while increased chlorophyll a/b contents, MDA, NPQ, leaf total phenolics, free proline and GB contents in both canola cultivars. Foliar-applied varying levels (50, 100 and 150 mg L^?1) of AsA enhanced shoot and root fresh and root dry weights, qN, NPQ, shoot and root P, AsA as well as the activity of POD enzyme particularly under drought stress conditions. Of both canola cultivars, cv. Dunkeld was higher in shoot fresh weights, ETR and F _v /F _m, MDA, proline and GB contents, and POD activity, however, cv. Cyclone in total phenolics and qN under well-watered and water-deficit conditions. Overall, the foliar-applied AsA had a positive effect, though not marked, on salt sensitive cv. Cyclone in terms of improved growth and other attributes, whereas exogenously applied AsA had a non-significant effect on relatively salt tolerant cv. Dunkeld.     

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

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

水分亏缺下紫花苜蓿和高粱根系水力学导度 与水分利用效率的关系

利用聚乙二醇(PEG-6000)模拟水分亏缺条件(胁迫水势-0.2MPa,胁迫48h),研究了变水条件下紫花苜蓿(品种:阿尔冈金和陇东)和高粱(品种:抗四)根系水力学导度(Lp_r)、根系活力、根叶相对含水量、水分利用效率等参数的动态变化,以期进一步明确植物水分吸收及散失过程调控的生理生态学基础。结果表明:水分亏缺限制了紫花苜蓿和高粱根系吸水,表现在Lp_r的下降和根系活力的降低;继而调控了其地上部反应,引起气孔导度、光合速率、叶片相对含水量和蒸腾速率等的下降,但限制性的提高了其水分利用效率,尤其在胁迫初期。恢复到正常供水条件后,Lp_r、根系活性、气孔导度等水分利用参数逐渐部分或完全恢复到了胁迫前水平,但恢复程度存在种间和品种间差异,并且根系吸水能力的恢复对于是植株地上部生长状态的恢复至关重要,尤其是水分恢复初期。紫花苜蓿根系中检测到水通道蛋白(AQPs)的存在,水分亏缺对紫花苜蓿Lp_r的影响认为主要是通过影响AQPs的活性实现的。比较紫花苜蓿和高粱水分吸收与利用状况在变水条件下的动态变化,认为紫花苜蓿幼苗对干旱逆境的适应能力相对弱于高粱,品种间陇东适应能力更强。     

The response of Arabidopsis root water transport to a challenging environment implicates reactive oxygen species- and phosphorylation-dependent internalization of aquaporins

Aquaporins, which facilitate the diffusion of water across biological membranes, are key molecules for the regulation of water transport at the cell and organ levels. We recently reported that hydrogen peroxide (H₂O₂) acts as an intermediate in the regulation of Arabidopsis root water transport and aquaporins in response to NaCl and salicylic acid (SA).¹ Its action involves signaling pathways and an internalization of aquaporins from the cell surface. The present addendum connects these findings to another recent work which describes multiple phosphorylations in the C-terminus of aquaporins expressed in the Arabidopsis root plasma membrane.² A novel role for phosphorylation in the process of salt-induced relocalization of AtPIP2;1, one of the most abundant root aquaporins, was unraveled. Altogether, the data delineate reactive oxygen species (ROS)-dependent signaling mechanisms which, in response to a variety of abiotic and biotic stresses, can trigger phosphorylation-dependent PIP aquaporin intracellular trafficking and root water transport downregulation.Key words: reactive oxygen species, aquaporin, phosphorylation, cell signaling, stress, protein relocalization, root water transportPlants can regulate their water uptake capacity i.e. their root hydraulic conductivity (Lp_r) on a short term (minutes to hour) basis through regulation of plasma membrane (PM) aquaporins of the Plasma membrane Intrinsic Protein (PIP) subfamily.³ It has been known for a long time that salt stress (NaCl), as many other abiotic stresses such as cold, anoxia or nutrient deprivation, induces an inhibition of Lp_r in many plant species.³ In the recent study by Boursiac et al. (2008),¹ we identified SA as a new inhibitory increased the accumulation of ROS in roots, it was hypothesized that H₂O₂ or other ROS may have a central role in the regulation of root water transport in response to various biotic or abiotic stimuli. When Arabidopsis roots were treated with mM concentrations of exogenous H₂O₂, Lp_r was inhibited within minutes by up to 90%. These findings are consistent with previous reports showing that ROS can downregulate water transport in cucumber and maize roots or in the algae Chara corallina.^4–7 H₂O₂ and possibly other derived ROS may modulate the Lp_r through signaling mechanisms or by a direct oxidative gating of aquaporins. The latter hypothesis, which has been favored in previous studies by Steudle and colleagues,^6,7 was investigated by Boursiac et al., by functionally expressing aquaporins in Xenopus oocytes and by testing their sensitivity to external H₂O₂. The results show that Arabidopsis aquaporins are insensitive to direct oxidation by H₂O₂ or hydroxyl radicals. Thus, these and complementary pharmacological analyses on excised roots rather support a role for H₂O₂ as a second messenger that connects environmental stimulus perception to water transport regulation in plant roots. The additional finding that H₂O₂ can be transported by aquaporins^8,9 opens the possibility of intricate loop mechanisms whereby these proteins may interfere with their own regulation. For example, active PIP aquaporins could facilitate the diffusion within the cell of NADPH-oxidase derived apoplastic H₂O₂, which in turn would activate signaling pathways acting on PIP activity and/or subcellular localization.In a previous study, we monitored the subcellular localization of AtPIP1;2 and AtPIP2;1, two of the most abundant PIPs in roots, by expression in transgenic Arabidopsis of fusions with the green fluorescent protein (GFP).¹⁰ We observed that a 100 mM NaCl treatment induced in 2–4 hours an increased intracellular labeling which was interpreted as an intracellular relocalization of the two aquaporins.¹⁰ In our more recent study, both a 150 mM NaCl and a 0.5 mM SA treatments induced an intracellular labeling by GFP-PIP1;2 and PIP2;1-GFP fusions, with a “fuzzy” pattern or at the level of spherical bodies. Preventing the NaCl- or SA-dependent accumulation of ROS with exogenous catalase was able to almost completely counteract the effects of the two stimuli on the localization pattern of the PIP2;1-GFP fusion. In addition, the inhibition of Lp_r by SA was also counteracted at 33% by the catalase treatment. Altogether, the data stress the importance of an ROS-induced relocalization of aquaporins in the regulation of root water transport. Yet, we still miss quantitative data and complementary pharmacological evidence to determine the exact contribution of aquaporin relocalization with respect to other aquaporin regulatory mechanisms.Another recent work by our group has, however, provided deeper insights into the mechanisms of stress-induced relocalization of aquaporins in plants.² Our group identified by mass spectrometry multiple adjacent phosphorylation sites (up to 4 in the case of AtPIP2;4) in the C-terminus of aquaporins expressed at the root plasma membrane.² Phosphorylation of AtPIP2;1, which shows a simpler profile with only two sites at Ser280 and Ser283, was studied in closer detail by site-directed mutagenesis and expression in transgenic Arabidopsis of GFP-PIP2;1 fusions. A Ser283Ala mutation, which mimics a constitutively dephosphorylated Ser283, induced a marked intracellular accumulation of GFP-PIP2;1 in resting conditions. Because no phenotype was observed after a Ser280Ala mutation, the data suggest a specific role for Ser283 phosphorylation in the proper targeting of the protein. When plants were treated by 100 mM NaCl for 2 to 4 hours, the wild type (WT) and Ser280Ala mutant forms of GFP-PIP2;1 showed similar intracellular staining, in both “fuzzy” structures or spherical bodies. On the contrary, the Ser283Ala mutant did not label any spherical body. Interestingly, a Ser283Asp mutation that mimics a constitutively phosphorylated Ser283 resulted in a salt-induced labeling of spherical bodies similar to the one observed with WT GFP-PIP2;1 whereas no “fuzzy” staining was observed. Therefore, the phosphorylation status of Ser283 seems to determine the redistribution of AtPIP2;1 towards fuzzy structures (non-phosphorylated Ser283) or spherical bodies (phosphorylated Ser283). Although the nature of these intracellular structures remains to be identified, we now consider the possibility that the spherical bodies correspond to the late endosome/prevacuolar compartment that orientates aquaporins towards a degradation pathway whereas the fuzzy structures may act as a storage compartment for subsequent relocalization of PIP aquaporins to the PM, and rapid recovery of the PM water permeability. Although we favor the idea that the intracellular labeling shown by GFP-PIP2;1 in response to salt originates from aquaporins relocalized from the PM, newly synthesized proteins may also contribute to this pattern.Prak et al., also developed an absolute quantification method to show that the phosphorylation profile of AtPIP2;1 at the root plasma membrane was altered upon 100 mM NaCl and 2 mM H₂O₂ treatments. Whereas NaCl decreased the abundance of phosphorylated Ser283, H₂O₂ enhanced the overall phosphorylation of the AtPIP2;1 C-terminus. These observations add another level of complexity to the mechanisms of stimulus-induced and phosphorylation- dependent relocalisation of plant aquaporins uncovered in our group. Although one of the primary effects of NaCl is undoubtedly an accumulation of ROS, the difference in phosphorylation patterns observed in response to H₂O₂ and NaCl treatments may come from quantitative and kinetic differences in ROS patterns between the two treatments or from additional regulations activated by salt.We note that phosphorylation of PIP aquaporins had already been investigated in detail.^11–13 In particular, studies with spinach SoPIP2;1 has pointed to two phosphorylation sites, Ser115 in the first cytoplasmic loop (loop B) and Ser274 at the C-terminus, as important for modulating the water transport activity of this aquaporin after expression in Xenopus oocytes. A role for these two sites in aquaporin gating was also deduced from the atomic structure of SoPIP2;1.¹⁴ Whereas Ser280 in AtPIP2;1 corresponds to Ser274 in SoPIP2;1, the functional role of sites equivalent to Ser283 in AtPIP2;1 had not been considered previously in any other PIP. To our knowledge, the study by Prak et al., provides the first evidence in plants for a role of phosphorylation on the relocalization of aquaporins and highlights the importance of multiple phosphorylations sites in the C-terminus of aquaporins, as has been recently shown in human Aquaporin-2.^15,16Overall, the advance provided by our two recent studies delineates a working model (Fig. 1), whereby multiple abiotic and biotic stresses, which all induce an accumulation of ROS, activate common signaling pathways to downregulate root water transport. We have provided evidence that some of these pathways are calcium- and/ or protein kinase-dependent. One regulatory mechanism triggered by these pathways is the relocalization of aquaporins into intracellular “fuzzy” structures or bigger spherical bodies. For AtPIP2;1, the sorting between these structures is determined in part by the phosphorylation status of Ser283, which ultimately may control the cellular fate of the protein for degradation or remobilization to the PM. A coming challenge will be to determine how this and other cellular mechanisms quantitatively contribute to the integrated regulation of water transport at the cell and tissue (whole root) levels. Another avenue for future research will be to identify the molecular components involved in upstream ROS-dependent cell signaling and aquaporin phosphorylation. These studies will tell us how the regulation of root water uptake in parallel to the regulation of transpiration allows the plant to preserve its water status when it is continuously challenged by multiple stresses.Open in a separate windowFigure 1Tentative model of regulation of root hydraulic conductivity (Lp_r) through reactive oxygen species (ROS) signaling. Multiple biotic and abiotic stimuli such as NaCl or salicylic acid can induce an intra- and/or extracellular accumulation of ROS by acting on their production, degradation or transport. The stimulus-induced ROS in turn activate signaling pathways involving protein kinases and cytosolic calcium. These events result in changes in the phosphorylation and subcellular localization patterns of plasma membrane (PM) aquaporins (PIPs). In particular, endocytosis can direct PIPs towards various intracellular compartments for subsequent recycling at the PM or degradation. Phosphorylation can interfere with this routing process, but also determines the intrinsic water transport activity (gating) of PM localized PIPs. The possibility exists that signaling components directly act on PIP gating, recycling or degradation through phosphorylation- and endocytosis-independent pathways (not shown). In addition, transport of H₂O₂ by PIP aquaporins may provide retroactive effects of aquaporins on upstream signaling events. Aquaporin activity at the PM determines root cell water permeability, which contributes to most of Lp_r in Arabidopsis. The overall scheme shows how stress-induced ROS signaling results in an inhibition of PIP aquaporin activity and, as a consequence, in an overall downregulation of Lp_r.     

Elevated CO2 alleviates negative effects of salinity on broccoli (Brassica oleracea L. var Italica) plants by modulating water balance through aquaporins abundance

In the global change scenario, increased CO₂ may favour water use efficiency (WUE) by plants. By contrast, in arid and semiarid areas, salinity may reduce water uptake from soils. However, an elevated WUE does not ensure a reduced water uptake and upon salinity this fact may constitute an advantage for plant tolerance. In this work, we aimed to determine the combined effects of enhanced [CO₂] and salinity on the plant water status, in relation to the regulation of PIP aquaporins, in the root and leaf tissues of broccoli plants (Brassica oleracea L. var Italica), under these two environmental factors. Thus, different salinity concentrations (0, 60 and 90 mM NaCl) were applied under ambient (380 ppm) and elevated (800 ppm) [CO₂]. Under non-salinised conditions, stomatal conductance (G_s) and transpiration rate (E) decreased with rising [CO₂] whereas water potential (Ψ_ω) was maintained stable, which caused a reduction in the root hydraulic conductance (L₀). In addition, PIP1 and PIP2 abundance in the roots was decreased compared to ambient [CO₂]. Under salinity, the greater stomatal closure observed at elevated [CO₂] – compared to that at ambient [CO₂] – caused a greater reduction in G_s and E and allowed plants to maintain their water balance. In addition, a lower decrease in L₀ under salt stress was observed at elevated [CO₂], when comparing with the decrease at ambient [CO₂]. Modifications in PIP1 and PIP2 abundance or their functionality in the roots is discussed. In fact, an improved water status of the broccoli plants treated with 90 mM NaCl and elevated [CO₂], evidenced by a higher Ψ_ω, was observed together with higher photosynthetic rate and water use efficiency. These factors conferred on the salinised broccoli plants greater leaf area and biomass at elevated [CO₂], in comparison with ambient [CO₂]. We can conclude that, under elevated [CO₂] and salt stress, the water flow is influenced by the tight control of the aquaporins in the roots and leaves of broccoli plants and that increased PIP1 and PIP2 abundance in these organs provides a mechanism of tolerance that maintains the plant water status.     

Rapid shoot‐to‐root signalling regulates root hydraulic conductance via aquaporins

We investigated how root hydraulic conductance (normalized to root dry weight, L_o) is regulated by the shoot. Shoot topping (about 30% reduction in leaf area) reduced L_o of grapevine (Vitis vinifera L.), soybean (Glycine max L.) and maize (Zea mays L.) by 50 to 60%. More detailed investigations with soybean and grapevine showed that the reduction in L_o was not correlated with the reduction in leaf area, and shading or cutting single leaves had a similar effect. Percentage reduction in L_o was largest when initial L_o was high in soybean. Inhibition of L_o by weak acid (low pH) was smaller after shoot damage or leaf shading. The half time of reduction in L_o was approximately 5 min after total shoot decapitation. These characteristics indicate involvement of aquaporins. We excluded phloem‐borne signals and auxin‐mediated signals. Xylem‐mediated hydraulic signals are possible since turgor rapidly decreased within root cortex cells after shoot topping. There was a significant reduction in the expression of several aquaporins in the plasma membrane intrinsic protein (PIP) family of both grapevine and soybean. In soybean, there was a five‐ to 10‐fold reduction in GmPIP1;6 expression over 0.5–1 h which was sustained over the period of reduced L_o.     

QTLs for the elongation of axile and lateral roots of maize in response to low water potential

Changes in root architecture and the maintenance of root growth in drying soil are key traits for the adaptation of maize (Zea mays L.) to drought environments. The goal of this study was to map quantitative trait loci (QTLs) for root growth and its response to dehydration in a population of 208 recombinant inbred lines from the International Maize and Wheat Improvement Center (CIMMYT). The parents, Ac7643 and Ac7729/TZSRW, are known to be drought-tolerant and drought-sensitive, respectively. Roots were grown in pouches under well-watered conditions or at low water potential induced by the osmolyte polyethylene glycol (PEG 8000). Axile root length (L _Ax) increased linearly, while lateral root length (L _Lat) increased exponentially over time. Thirteen QTLs were identified for six seedling traits: elongation rates of axile roots (ER_Ax), the rate constant of lateral root elongation (k _Lat), the final respective lengths (L _Ax and L _Lat), and the ratios k _Lat/ER_Ax and L _Lat/L _Ax. While QTLs for lateral root traits were constitutively expressed, most QTLs for axile root traits responded to water stress. For axile roots, common QTLs existed for ER_Ax and L _Ax. Quantitative trait loci for the elongation rates of axile roots responded more clearly to water stress compared to root length. Two major QTLs were detected: a QTL for general vigor in bin 2.02, affecting most of the traits, and a QTL for the constitutive increase in k _Lat and k _Lat/ER_Ax in bins 6.04–6.05. The latter co-located with a major QTL for the anthesis-silking interval (ASI) reported in published field experiments, suggesting an involvement of root morphology in drought tolerance. Rapid seedling tests are feasible for elucidating the genetic response of root growth to low water potential. Some loci may even have pleiotropic effects on yield-related traits under drought stress.     

Physiological foundation for the differences of long-term water use efficiency among Populus deltoides clones

With randomized complete block design, long-term water use efficiency (WUE_L), photosynthetic parameters (net photosynthetic rate Pn, transpiration rate Tr, stomatal conductance Gs, instantaneous water use efficiency WUE_i, maximum photosynthetic rate P max, light saturation pointLSP, carboxylic efficiency Ce), stomatal properties (frequency, size, superior/inferior stoma amount ratio) and root/shoot ratio of twelve Populus deltoids clones were studied under water stress in a greenhouse. Four water treatments were designed, namely, well-watered condition, slight water stress, moderate water stress, and severe water stress. Although the volumes of irrigated water for different treatments were the same, the irrigation intervals were strictly controlled. The results showed that long-term water use efficiency (WUE_L) differences among tested clones were obvious and became greater with the increase in water stress. J₂, J₆, J₇, J₈, and J₉ were excellent clones with high WUE_L. Stomatal frequency, stomatal size, Gs, Tr, Pn, and root/shoot ratio of J₂et al were moderate, whereas the superior/inferior ratio, P_max, LSP, Ce of J2et al were higher than other clones. The interrelations between WUE_L and the physiological parameters were analyzed. The results revealed that the WUE_L differences were induced by a series of physiological parameters, and clones with higher WUE_L always had strong photosynthetic capacity, higher WUE_i, and optimum root/shoot ratio.     

Different ability of three Mediterranean oak species to tolerate progressive water stress

Inter-comparisons in the gas exchange patterns and root characteristics under both well-watered and drought conditions were done in three-years-old seedlings of three oak species (Quercus cerris L., Q. frainetto Ten., and Q. ilex L.) growing in controlled environment. Well-watered Q. cerris had greater physiological performances than other oaks, but under drought it was not able to face the water stress showing also structural modifications such as reduction of root length and average diameter. On the other hand, Q. ilex maintained root growth both in drought or well-watered soils. Moreover, it was able to keep open stomata also under water stress, although stomatal conductance (g _s) was low. Q. frainetto had an intermediate position in regard to its physiological and root structural characteristics between Q. cerris and Q. ilex under drought stress. For all oaks the relationship between g _s and the ratio of sub-stomatal and ambient CO₂ concentration (C _i/C _a) highlighted the dynamic adaptation of g _s to the increase of hydraulic resistances of leaf, stem, and roots portions, more evident during the air humidity change and progressive soil dehydration. This suggests a well-triggered above-and under-ground mechanism to endure the drought stress.     

Maize root responses to drought stress depend on root class and axial position

In this study we tested the hypotheses that root classes would exhibit distinctive anatomical and architectural responses to drought stress, and that those responses would vary along the root axes. The root systems of four maize (Zea mays L.) sweet corn genotypes designated SC1, SC2, SC3 and SC4 were phenotyped under well-watered and drought treatments in greenhouse mesocosms, permitting increasing stratification of moisture availability as the drought progressed. Anatomical and architectural responses to drought were evaluated for each root class. Lignin distribution was assessed by image processing of UV-illuminated root cross-sections acquired by laser ablation tomography. The two cultivars with less biomass reduction under drought, SC3 and SC4, substantially enhanced lateral root development along the apical segments of axial roots when plants were grown with drought stress. These segments grew into the deeper part of the mesocosm where more moisture was available. Apical segments of the axial and large lateral roots from drought-stressed plants were thicker and had greater theoretical axial water conductance than basal segments, especially in SC3 and SC4. Basal segments of crown roots of SC3 and SC4 showed increased lignification of the stele under drought. Root anatomical and architectural responses to drought are complex and vary among cultivars and root classes, and along root axes. Drought-induced proliferation of lateral roots on apical segments of axial roots would be expected to enhance deep water acquisition, while lignification of axial roots could help preserve axial water transport.

     

Aquaporin-Mediated Reduction in Maize Root Hydraulic Conductivity Impacts Cell Turgor and Leaf Elongation Even without Changing Transpiration

Root hydraulic conductivity in plants (Lp_r) exhibits large variations in response to abiotic stimuli. In this study, we investigated the impact of dynamic, aquaporin-mediated changes of Lp_r on leaf growth, water potential, and water flux throughout the plant. For this, we manipulated Lp_r by subjecting roots to four independent treatments, with aquaporin inhibitors applied either to transpiring maize (Zea mays) plants grown in hydroponics or to detopped root systems for estimation of Lp_r. The treatments were acid load at pH 6.0 and 5.0 and hydrogen peroxide and anoxia applied for 1 to 2 h and subsequently reversed. First, we established that acid load affected cell hydraulic conductivity in maize root cortex. Lp_r was reduced by all treatments by 31% to 63%, with half-times of about 15 min, and partly recovered when treatments were reversed. Cell turgor measured in the elongating zone of leaves decreased synchronously with Lp_r, and leaf elongation rate closely followed these changes across all treatments in a dose-dependent manner. Leaf and xylem water potentials also followed changes in Lp_r. Stomatal conductance and rates of transpiration and water uptake were not affected by Lp_r reduction under low evaporative demand. Increased evaporative demand, when combined with acid load at pH 6.0, induced stomatal closure and amplified all other responses without altering their synchrony. Root pressurization reversed the impact of acid load or anoxia on leaf elongation rate and water potential, further indicating that changes in turgor mediated the response of leaf growth to reductions in Lp_r.Leaf growth is an essential process for crop production and is subject to large temporal fluctuations with environmental conditions. There is accumulating evidence that a large part of the changes observed in leaf growth depends on water transport within the plant (Sperry et al., 1998; Bouchabke et al., 2006). It has also been shown that changes in leaf water potential induced by root pressurization can trigger rapid variations of leaf elongation rate in wheat (Triticum aestivum) and barley (Hordeum vulgare; Passioura and Munns, 2000). This raises the question of whether and to what extent low hydraulic conductivity within the plant can limit leaf growth.After the stomata, the root system represents the largest resistance to water flow in the soil-plant atmosphere continuum (Steudle and Peterson, 1998). Root hydraulic conductivity (Lp_r) is affected by environmental stimuli such as drought, salinity, anoxia, low temperature, and nutrient availability (Zhang and Tyerman, 1991; Azaizeh et al., 1992; Birner and Steudle, 1993; Boursiac et al., 2005; Vandeleur et al., 2009). This ability to respond rapidly to fluctuating conditions suggests that Lp_r may participate in plant adaptation to diverse environments (Steudle, 2000). Aquaporins, a large family of water channel proteins located in plasma and intracellular membranes, are the main determinants of water flow across plant cells and tissues (Javot et al., 2003; Maurel et al., 2008). The dynamic changes in Lp_r in response to chemical or environmental stimuli may result from modifications of aquaporin abundance or activity (Carvajal et al., 1996; Tournaire-Roux et al., 2003; Boursiac et al., 2005). In particular, aquaporin regulation by phosphorylation, protonation, and relocalization in intracellular compartments has been reported in response to extracellular stimuli (Guenther et al., 2003; Tournaire-Roux et al., 2003; Vera-Estrella et al., 2004; Boursiac et al., 2008).The first insights into the involvement of aquaporins in physiological processes such as cell enlargement, tissue differentiation, and organ movement have been obtained at the cell or tissue level (Hukin et al., 2002; Moshelion et al., 2002; Wei et al., 2007). It is still unknown to what extent changes in root aquaporin activity impact integrated physiological processes such as shoot growth of intact plants. The importance of aquaporins in controlling physiological processes in adult, transpiring plants is assumed to be rather limited; this is because the proportion of water transport controlled by aquaporins is believed to be much lower than that in slowly transpiring plants (Steudle and Frensch, 1996; Steudle and Peterson, 1998).The manipulation of aquaporin activity offers the possibility to address this question. A classical approach is to alter the expression of aquaporin genes. Down-regulation of genes encoding aquaporins of the Plasma membrane Intrinsic Protein1 (PIP1) and PIP2 subfamilies in Arabidopsis and NtAQP1 in tobacco (Nicotiana tabacum) reduced the ability of these plants to recover after a water deficit treatment (Martre et al., 2002; Siefritz et al., 2002). However, genetic studies have been hindered by phenotypic compensation due to the functional redundancy of aquaporin isoforms in plants (Hachez et al., 2006b). The use of aquaporin inhibitors is a useful tool to investigate the role of root aquaporins in controlling leaf growth. Mercuric chloride (HgCl₂), which blocks aquaporins by binding of Hg²⁺ ions to Cys residues, has been widely used to evaluate the contribution of aquaporins to root water transport (Maggio and Joly, 1995; Carvajal et al., 1996). For instance, Lu and Neumann (1999) have observed that root exposure to 0.5 mm HgCl₂ immediately inhibited leaf growth in water-stressed rice (Oryza sativa) seedlings, thereby suggesting a role for aquaporins in controlling leaf growth. However, the signaling mechanisms involved in leaf growth inhibition remained unclear. Side effects of HgCl₂ application, such as the reduction of membrane potential in root cortex cells or an impaired cell respiration, restrict its usefulness in physiological studies (Wan and Zwiazek, 1999; Zhang and Tyerman, 1999). Manipulating the root environment is an alternative strategy to efficiently alter Lp_r and to assess the significance of such changes on leaf and/or shoot growth. The effects of varying Lp_r by root chilling or anoxia are correlated to leaf growth responses (Malone, 1993; Else et al., 1995, 2001), but the interpretation of these results remains controversial in the absence of precise hydraulic measurements.The goal of this study was to determine whether alterations of root aquaporin activity can influence leaf growth in intact, adult plants via effects on Lp_r and cell turgor in the leaf elongation zone. In this work, we compared three chemical treatments that target aquaporin inactivation in roots via different mechanisms. Each of them could exert side effects, but provided that all treatments resulted in common responses, the role of aquaporins on leaf growth could be established. We have followed, with a high temporal definition, the consequences of experimentally induced changes in Lp_r on water flux, leaf water potential, and leaf elongation rate under different scenarios (three evaporative demands and pressurized or nonpressurized root systems). In addition, we have measured cell turgor in growing leaves using a pressure probe to investigate whether cell turgor responds to changes in root hydraulic conductivity and whether such changes could account for the control of leaf growth.The first treatment used to alter Lp_r was acid loading of the solution surrounding the roots, which causes cytosolic acidification in root cortex cells. This triggers the closure of aquaporins due to the protonation of a conserved His residue (Tournaire-Roux et al., 2003). The second treatment was hydrogen peroxide (H₂O₂) application to the roots, which results in the inhibition of Lp_r in maize (Zea mays) by oxidative gating of aquaporins and/or their internalization (Ye et al., 2004; Aroca et al., 2005; Ye and Steudle, 2006; Boursiac et al., 2008). The third treatment was anoxia, an environmental stress that induces an inhibition of Lp_r in a large array of species through proton-induced closure of aquaporins (Zhang and Tyerman, 1991; Birner and Steudle, 1993; Else et al., 1995; Tournaire-Roux et al., 2003).     

Responses of transpiration and hydraulic conductance to root temperature in nitrogen- and phosphorus-deficient cotton seedlings

Suboptimal N or P availability and cool temperatures all decrease apparent hydraulic conductance (L) of cotton (Gossypium hirsutum L.) roots. The interaction between nutrient status and root temperature was tested in seedlings grown in nutrient solutions. The depression of L (calculated as the ratio of transpiration rate to absolute value of leaf water potential [Ψ_w]) by nutrient stress depended strongly on root temperature, and was minimized at high temperatures. In fully nourished plants, L was high at all temperatures ≥20°C, but it decreased greatly as root temperature approached the chilling threshold of 15°C. Decreasing temperature lowered Ψ_w first, followed by transpiration rate. In N- or P-deficient plants, L approached the value for fully nourished plants at root temperatures ≥30°C, but it decreased almost linearly with temperature as roots were cooled. Nutrient effects on L were mediated only by differences in transpiration, and Ψ_w was unaffected. The responses of Ψ_w and transpiration to root cooling and nutrient stress imply that if a messenger is transmitted from cooled roots to stomata, the messenger is effective only in nutrient-stressed plants.     

Regulation of a plant aquaporin by a Casparian strip membrane domain protein‐like

The absorption of soil water by roots allows plants to maintain their water status. At the endodermis, water transport can be affected by initial formation of a Casparian strip and further deposition of suberin lamellas and regulated by the function of aquaporins. Four Casparian strip membrane domain protein‐like (CASPL; CASPL1B1, CASPL1B2, CASPL1D1, and CASPL1D2) were previously shown to interact with PIP2;1. The present work shows that CASPL1B1, CASPL1B2, and CASPL1D2 are exclusively expressed in suberized endodermal cells, suggesting a cell‐specific role in suberization and/or water transport regulation. When compared with wild‐type plants, and by contrast to caspl1b1*caspl1b2 double loss of function, caspl1d1*caspl1d2 double mutants showed, in some control or NaCl stress experiments and not upon abscisic acid (ABA) treatment, a weak enlargement of the continuous suberization zone. None of the mutants showed root hydraulic conductivity (Lp_r) phenotype, whether in control, NaCl, or ABA treatment conditions. The data suggest a slight negative role for CASPL1D1 and CASPL1D2 in suberization under control or salt stress conditions, with no major impact on whole root transport functions. At the molecular level, CASPL1B1 was able to physically interact with PIP2;1 and potentially could influence the regulation of aquaporins by acting on their phosphorylated form.