Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

The relations of stomatal closure and reopening to xylem ABA concentration and leaf water potential during soil drying and rewatering

Two tropical tree species, Acacia confusa and Leucaena leucocephala, were used to study the relationships among stomatal conductance, xylem ABA concentration and leaf water potential during a soil drying and rewatering cycle. Stomatal conductance of both A. confusa and L. leucocephala steadily decreased with the decreases in soil water content and pre-dawn leaf water potential. Upon rewatering, soil water content and pre-dawn leaf water potential rapidly returned to the control levels, whereas the reopening of stomata showed an obvious lag time. The length of this lag time was highly dependent not only upon the degree of water stress but also on plant species. The more severe the water stress, the longer the lag time. When A. confusa and L. leucocephala plants were exposed to the same degree of water stress (around –2.0 MPa in pre-dawn leaf water potential), the stomata of A. confusa reopened to the control level 6 days after rewatering. However, it took L. leucocephala about 14 days to reopen fully. A very similar response of leaf photosynthesis to soil water deficit was also observed for both species. Soil drying resulted in a significant increase in leaf and xylem ABA concentrations in both species. The more severe the water stress, the higher the leaf and xylem ABA concentrations. Both leaf ABA and xylem ABA returned to the control level following relief from water deficit and preceded the full recovery of stomata, suggesting that the lag phase of stomatal reopening was not controlled by leaf and/or xylem ABA. In contrast to drying the whole root system, drying half of the root system did not change the leaf water relations, but caused a significant increase in xylem ABA concentration, which could fully explain the decrease of stomatal conductance. After rewatering, the stomatal conductance of plants in which half of the roots were dried recovered more rapidly than those of whole-root dried plants, indicating that the leaf water deficit that occurred during the drying period was related to the post-stress stomatal inhibition. These results indicated that the decrease in stomatal conductance caused by water deficit was closely related to the increase in xylem ABA, but xylem ABA could not fully explain the reopening of stomata after relief of water stress, neither did the leaf ABA. Some unknown physiological and/or morphological processes in the guard cells may be related to the recovery process.     

Abscisic acid accumulation and cadmium tolerance in rice seedlings

Rice ( Oryza sativa L.) seeds were soaked for 18 h in distilled water in the absence (–PBZ) or presence (+PBZ, a triazole) of 100 mg l⁻¹ paclobutrazol and then air dried. These air-dried seeds were germinated in the dark and then cultivated in a Phytotron. Twelve-day-old –PBZ and +PBZ seedlings were treated or not with CdCl₂. Cd toxicity was judged by the decrease in biomass production, decrease in chlorophyll and protein content, increase in NH₄⁺ content and induction of oxidative stress. The results indicated that PBZ applied to seeds was able to protect rice seedlings from Cd toxicity. On treatment with CdCl₂, the abscisic acid (ABA) content increased in +PBZ leaves, but not in –PBZ leaves. The decrease in the transpiration rate of –PBZ seedlings by CdCl₂ was less than that of +PBZ seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone (Flu), reduced ABA accumulation, increased the transpiration rate and Cd content, and decreased the Cd tolerance of +PBZ seedlings. The effects of Flu on the Cd toxicity, transpiration rate and Cd content were reversed by the application of ABA. It seems that the PBZ-induced Cd tolerance of rice seedlings is mediated through an accumulation of ABA.     

What information is conveyed by an ABA signal from maize roots in drying field soil?

During two seasons, ABA concentrations were monitored in roots, leaves and xylem sap of field-grown maize. The water status of soil and plant was also measured. Plants were grown on plots with compacted or non-compacted soil, which were irrigated or remained unwatered. ABA concentration in the xylem sap before dawn and in the roots increases 25-fold and five-fold, respectively, as the soil dried, with a close correlation with the soil water status, but with no clear effect of the soil structure. In contrast to the results of several laboratory experiments, no appreciable increase in xylem [ABA] and reduction in stomatal conductance were observed with dehydration of the part of the root system located in soil upper layers. These responses only occurred when the water reserve of the whole soil profile was close to depletion and the transpiration declined. Xylem [ABA] measured during the day was appreciably higher in the compacted treatment than in non-compacted treatment, unlike that measured before dawn. Since a mechanical message is unlikely to undergo such day-night alterations, we suggest that this was due to a faster decrease in root water potential and water flux in the compacted treatment, linked to the root spatial arrangement. These results raise the possibility that ABA concentration in the xylem sap could be controlled by two coexisting mechanisms: (1) the rate of ABA synthesis in the roots linked to the soil or root water status, as shown in laboratory experiments; (2) the dilution of ABA in the water flow from roots, which could be an overriding mechanism in field conditions. This second mechanism would allow the plant to sense the water flux through the root system.     

Leaf xylem embolism, detected acoustically and by cryo-SEM, corresponds to decreases in leaf hydraulic conductance in four evergreen species

Hydraulic conductance of leaves ( K _leaf) typically decreases with increasing water stress. However, the extent to which the decrease in K _leaf is due to xylem cavitation, conduit deformation or changes in the extra-xylary pathway is unclear. We measured K _leaf concurrently with ultrasonic acoustic emission (UAE) in dehydrating leaves of two vessel-bearing and two tracheid-bearing species to determine whether declining K _leaf was associated with an accumulation of cavitation events. In addition, images of leaf internal structure were captured using cryo-scanning electron microscopy, which allowed detection of empty versus full and also deformed conduits. Overall, K _leaf decreased as leaf water potentials ( Ψ _L) became more negative. Values of K _leaf corresponding to bulk leaf turgor loss points ranged from 13 to 45% of their maximum. Additionally, Ψ _L corresponding to a 50% loss in conductivity and 50% accumulated UAE ranged from −1.5 to −2.4 MPa and from −1.1 to −2.8 MPa, respectively, across species. Decreases in K _leaf were closely associated with accumulated UAE and the percentage of empty conduits. The mean amplitude of UAEs was tightly correlated with mean conduit diameter ( R ² = 0.94, P  = 0.018). These results suggest that water stress-induced decreases in K _leaf in these species are directly related to xylem embolism.     

Decoupling the influence of leaf and root hydraulic conductances on stomatal conductance and its sensitivity to vapour pressure deficit as soil dries in a drained loblolly pine plantation

The study examined the relationships between whole tree hydraulic conductance ( K _tree) and the conductance in roots ( K _root) and leaves ( K _leaf) in loblolly pine trees. In addition, the role of seasonal variations in K _root and K _leaf in mediating stomatal control of transpiration and its response to vapour pressure deficit ( D ) as soil-dried was studied. Compared to trunk and branches, roots and leaves had the highest loss of conductivity and contributed to more than 75% of the total tree hydraulic resistance. Drought altered the partitioning of the resistance between roots and leaves. As soil moisture dropped below 50%, relative extractable water (REW), K _root declined faster than K _leaf. Although K _tree depended on soil moisture, its dynamics was tempered by the elongation of current-year needles that significantly increased K _leaf when REW was below 50%. After accounting for the effect of D on g _s, the seasonal decline in K _tree caused a 35% decrease in g _s and in its sensitivity to D , responses that were mainly driven by K _leaf under high REW and by K _root under low REW. We conclude that not only water stress but also leaf phenology affects the coordination between K _tree and g _s and the acclimation of trees to changing environmental conditions.     

Elevated CO2 enhances stomatal responses to osmotic stress and abscisic acid in Arabidopsis thaliana

A , carbon assimilation rate
ABA, abscisic acid
Ci , intercellular space CO₂ concentration
g , leaf conductance
WUE, water use efficiency

Carbon dioxide and abscisic acid (ABA) are two major signals triggering stomatal closure. Their putative interaction in stomatal regulation was investigated in well-watered air-grown or double CO₂-grown Arabidopsis thaliana plants, using gas exchange and epidermal strip experiments. With plants grown in normal air, a doubling of the CO₂ concentration resulted in a rapid and transient drop in leaf conductance followed by recovery to the pre-treatment level after about two photoperiods. Despite the fact that plants placed in air or in double CO₂ for 2 d exhibited similar levels of leaf conductance, their stomatal responses to an osmotic stress (0·16–0·24 MPa) were different. The decrease in leaf conductance in response to the osmotic stress was strongly enhanced at elevated CO₂. Similarly, the drop in leaf conductance triggered by 1 μ M ABA applied at the root level was stronger at double CO₂. Identical experiments were performed with plants fully grown at double CO₂. Levels of leaf conductance and carbon assimilation rate measured at double CO₂ were similar for air-grown and elevated CO₂-grown plants. An enhanced response to ABA was still observed at high CO₂ in pre-conditioned plants. It is concluded that: (i) in the absence of stress, elevated CO₂ slightly affects leaf conductance in A. thaliana ; (ii) there is a strong interaction in stomatal responses to CO₂ and ABA which is not modified by growth at elevated CO₂.     

Physiological response of Pinus halepensis needles under ozone and water stress conditions

The aim of this study was to evaluate how physiological processes of potted Pinus halepensis plants, grown under controlled conditions, were affected by ozone (O₃) and/or water stress, integrating the gas exchange and biochemical data with fluorescence OJIP polyphasic transient data. Plants submitted to only water stress (T₁) and with ozone (T₃) showed a strong decrease in stomatal conductance and gas exchange, coinciding with a reduction of maximum yield of photochemistry ( φ _po) and very negative values of leaf water potential. Simultaneously, a great increase of both PSII antenna size, indicated by absorption per reaction centre, and electron transport per reaction centre were found. The reduction of photosynthesis in the O₃-treated plants (T₂) by a slowing down of the Calvin cycle was supported by the increase of related fluorescence parameters such as relative variable fluorescence, heat de-excitation constant, energy de-excitation by spillover, and the decrease of φ _po. We suggest an antagonistic effect between the two stresses to explain the delayed ozone-induced decrease of stomatal conductance values for T₃ with respect to T₁ plants, by an alteration of the physiological mechanisms of stomatal opening, which involve the increase of intra-cellular free-calcium induced by ABA under co-occurring water shortage. We emphasise the importance of considering the intensity of the individual stress factor in studies concerning the interaction of stresses.     

Water stress, carbon dioxide, and light effects on sucrosephosphate synthase activity in Phaseolus vulgaris

The characteristics of sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity in leaves of Phaseolus vulgaris L. cv. Linden was studied in plants subjected to water stress and various CO₂ and light treatments. When water was withheld for 3 days causing mild water stress (–0.9 MPa), the activity of SPS measured in crude extracts was reduced ca 50%. The effect of water stress was most evident when the enzyme was assayed with saturating amounts of its substrates fructose 6-phosphate and UDP glucose. Placing a water-stressed plant in an atmosphere containing 1% CO₂ reversed the effect of water stress on SPS activity over 5 h even though the water stress was not relieved. Holding unstressed leaves in low CO₂ partial pressure reduced the extractable activity of SPS. After 1 h of low CO₂ treatment the effect of low CO₂ could be reversed by 20 min of 5% CO₂. However, after 24 h of low CO₂ treatment, less SPS activity was recovered by the 20 min treatment. The cytosolic protein synthesis inhibitor cycloheximide prevented the slow recovery of SPS activity, but did not affect the rapid recovery of SPS. We conclude that the effect of water stress on SPS activity was a consequence of the inhibition of photosynthesis caused by stomatal closure. Responses of Phaseolus vulgaris SPS to light were similar to the response to low CO₂ in that the effects were most pronounced under V_max assay conditions. This is the first report of this type of light response of SPS in a dicotyledonous species.     

Accumulation rate of ABA in detached maize roots correlates with root water potential regardless of age and branching order

How much ABA can be supplied by the roots is a key issue for modelling the ABA-mediated influence of drought on shoot physiology. We quantified accumulation rates of ABA ( S _ABA) in maize roots that were detached from well-watered plants and dehydrated to various extents by air-drying. S _ABA was estimated from changes in ABA content in root segments incubated at constant relative water content (RWC). Categories of root segments, differing in age and branching order, were compared (root branches, and nodal roots subdivided into root tips, subapical unbranched sections, and mature sections). All categories of roots accumulated ABA, including turgid and mature tissues containing no apex. S _ABA measured in turgid roots changed with root age and among root categories. This variability was largely accounted for by differences in water content among different categories of turgid roots. The response of S _ABA to changes in root water potential ( Ψ _root) induced by dehydration was common to root tips, nodal roots and branches of several ages, while this was not the case if root dehydration was expressed in terms of RWC. Differences among root categories in the response of S _ABA to RWC were due to different RWC values among categories at a given Ψ _root, and not to differences in the response of S _ABA to Ψ _root.     

Effect of ABA on the water relations of winter-wheat cultivars varying in drought resistance

Water and osmotic potentials were measured in leaves of a drought-sensitive ('Ponca') and a drought-resistant ('KanKing') cultivar of winter wheat ( Triticum aestivum L . em. Thell.) to determine if the potentials of the drought-sensitive cultivar could be made similar to those of the drought-resistant cultivar through application of abscisic acid (ABA). Stomatal resistance was also measured. Plants were sprayed with ABA and grown in soil, which was watered or allowed to dry. In well-watered plants, ABA closed the stomata of both cultivars. Stomatal resistance of plants grown without added water and with ABA was less than that of plants grown without added water and without ABA. Under ample water supply, ABA decreased water and osmotic potentials of the drought-sensitive cultivar (Ponca), but had no effect on these potentials in the drought-resistant cultivar (KanKing). Under water-deprived conditions, ABA increased water and osmotic potentials of Ponca, but did not change these potentials in KanKing. The overall effect of ABA was to decrease the differences in the water and osmotic potentials between the two cultivars.     

Development and accumulation of ABA in fluridone-treated and drought-stressed Vicia faba plants under different light conditions

The effects of fluridone on guard cell morphology, chloroplast ultrastructure and accumulation of drought stress-induced abscisic acid (ABA) were studied in Vicia faba L. plants grown under different light conditions. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. The appearance of defective and undeveloped stomata, and chloroplasts with a destroyed thylakoid membrane system was found in fluridone-treated plants grown at a photosynthetic photon flux (PPF) of 600 μmol m^-2 s^-1. Plants grown at a PPF of 40 μmol m^-2 s^-1 had diminished levels of ABA after imposition of dehydration. Fluridone treatment reduced the level of ABA in both unstressed and dehydrated leaves. Accumulation of ABA in the control plants was considerably reduced when they were exposed to dark periods of 24, 48 and 72 h just before imposition of the stress. Twenty-four hours after the dark treatment dehydration of the leaves resulted in a 3-fold decrease in the level of stress-induced ABA, and 72 h after dark treatment the amount of stress-induced ABA approximated the prestressed values. Fluridone-treated plants failed to accumulate ABA under water stress. In addition to functionally active chloroplasts, well-developed and functional stomata are required for drought stress to elicit a rise in ABA.     

Genetic variability and heritability of drought-induced abscisic acid accumulation in spring wheat

Abstract. Twenty cultivars of spring wheat were examined for variation in abscisic acid (ABA) accumulation following partial dehydration of excised leaves. A 3-fold range of ABA concentration was obtained.
A cross between two cultivars which differed in drought-induced ABA accumulation was used to study the heritability of ABA accumulation and to develop lines differing in their capacity to accumulate ABA. Broad sense heritability was 0–32 between the F₂ and F₃ generations and 0–70 between the F₃ and F₄ generations. Apparent homozygosity for ABA accumulation was achieved in several selections at the F₄. The possible significance for drought resistance of differences in capacity to accumulate ABA is discussed.     

Effect of water stress on some oxidative enzymes and senescence in Vigna seedlings

Seedlings of Vigna catjang Endl. were subjected to water stress for 6, S and 10 days by withholding water to investigate the activities of some oxidative enzymes and the pattern of senescence in leaves of 17-day-old seedlings undergoing water stress. Increasing duration of stress produced a proportional increase in the activities of IAA-oxidase, AA-oxidase, peroxidase and glycolate oxidase but decreased catalase activity and the contents of both chlorophyll and protein, hastening senescence. Leaf water potential and relative water content were also lowered with incresing duration of stress. Permeability was increased in leaf tissue undergoing water stress for 8 days. Seed treatment with CaCl₂ (10⁻² and 10⁻¹⁴ M ) for 6 h improved the water status of leaves, decreased tissue permeability, activities of oxidative enzymes, decline of chlorophyll and protein contents and delayed senescence compared to untreated water stressed plants.     

Xylem ABA controls the stomatal conductance of field-grown maize subjected to soil compaction or soil drying

Stomatal conductance of individual leaves was measured in a maize field, together with leaf water potential, leaf turgor, xylem ABA concentration and leaf ABA concentration in the same leaves. Stomatal conductance showed a tight relationship with xylem ABA, but not with the current leaf water status or with the concentration of ABA in the bulk leaf. The relationship between stomatal conductance and xylem [ABA] was common for variations in xylem [ABA] linked to the decline with time of the soil water reserve, to simultaneous differences between plants grown on compacted, non-compacted and irrigated soil, and to plant-to-plant variability. Therefore, this relationship is unlikely to be fortuitous or due to synchronous variations. These results suggest that increased concentration of ABA in the xylem sap in response to stress can control the gas exchange of plants under field conditions.     

Changes in water relations and in some physiological functions of bean under very light osmotic shock induced by polyethylene glycol

Bean plantlets ( Phaseolus vulgaris L. cv. Topcrop) were stressed at the age of 16–18 days by gradual (2–8%) or abrupt addition of 6% (w/v) polyethylene glycol Mw 6000 (PEG 6000) to Hoagland solution. Leaf conductance, photosynthesis, internal CO₂ partial pressure (C_i), relative water content (RWC), water content/dry weight (H₂O/DW), apoplastic PEG concentrations and weight of leaves, stems and roots were determined. Leaf conductance, photosynthesis and C_i were determined on non-detached primary leaves, and leaf potentials (water, osmotic and turgor potentials) were investigated in freshly detached (non-rehydrated) primary leaves, both in treated and control plants; RWC and osmotic potential were also assessed at the null turgor point. Low PEG 6000 concentrations induced early and evident decrease in leaf conductance and photosynthesis, whereas C_i decreased only moderately and tended to recover during advanced stress. There were moderate though significant decreases in RWC and H₂O/DW, no change or increases in water potential, no significant changes in osmotic potential and a moderate but significant increase in turgor potential. Even when referred to null turgor point, RWC significantly decreased and osmotic potential was unchanged. It was concluded that apoplastic PEG 6000 accumulation at evaporating sites would account for the early decrease in conductance which would also justify the unchanged or the prevalent increase in water potential and turgor potential. The subsequent PEG diffusion and concentration in the leaf apoplastic water would have induced the RWC and H₂O/DW decrease and the final turgor flexion documented.     

Stomatal responses of pearl millet (Pennisetum americanum [L.] Leeke) to leaf water status and environmental factors in the field

Abstract. Factors affecting stomatal conductance (g₁) of pearl millet ( Pennisetum americanum [L.] Leeke), cultivar BJ 104, were examined in the field in India during the dry season.
Diurnal changes in g₁ were evaluated for upper expanded leaves at flowering on two occasions using plants subjected to varying degrees of water stress. Except for the most severely stressed treatment, diurnal changes in g₁ closely matched changes in irradiance ( I ), the promotive effect of which largely overcame opposing influences on g₁ of increasing atmospheric vapour pressure deficit, and decreasing leaf water and turgor potentials (Ψ, Ψ_p).
Two main effects of water stress on g₁ were evident: (i) a decrease in the amplitude of the mid-day peak in g₁, and (ii) a decrease in the time over which high g₁ was maintained, resulting in early (mid-day) closure and hysteresis in the relationship between g₁ and I .
Leaf conductance was greatest for upper leaves and decreased down the canopy. At equivalent depths in the canopy g₁ was higher in flowering than in photoperiodically-retarded plants of the same age. The magnitude of water stress-induced stomatal closure increased down the plant, and was more marked in retarded than in flowering plants.
Within individual stress treatments Ψ of upper leaves decreased linearly as transpiration flux increased. It is concluded that stomatal behaviour of upper leaves of pearl millet at flowering largely operates to maximize assimilation rather than to minimize water loss.     

Transport constraints on water use by the Great Basin shrub, Artemisia tridentata

As soil and plant water status decline, decreases in hydraulic conductance can limit a plant's ability to maintain gas exchange. We investigated hydraulic limitations for Artemisia tridentata during summer drought. Water use was quantified by measurements of soil and plant water potential ( Ψ ), transpiration and leaf area. Hydraulic transport capacity was quantified by vulnerability to water stress-induced cavitation for root and stem xylem, and moisture release characteristics for soil. These data were used to predict the maximum possible steady-state transpiration rate ( E _crit) and minimum leaf xylem pressure ( Ψ _crit). Transpiration and leaf area declined by ~ 80 and 50%, respectively, as soil Ψ decreased to –2·6 MPa during drought. Leaf-specific hydraulic conductance also decreased by 70%, with most of the decline predicted in the rhizosphere and root system. Root conductance was projected to be the most limiting, decreasing to zero to cause hydraulic failure if E _crit was exceeded. The basis for this prediction was that roots were more vulnerable to xylem cavitation than stems (99% cavitation at –4·0 versus –7·8 MPa, respectively). The decline in water use during drought was necessary to maintain E and Ψ within the limits defined by E _crit and Ψ _crit.     

Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings

Vigna cutjang Endl. cv. Pusa Barsati seedlings, subjected to increasing degrees of water stress (−0.5, −1.0, −1,5 MPa), produced an approximately proportional increase in glycolate oxidase activity, hydrogen peroxide (H₂O₂) and proline content but a decrease in catalase activity, ascorbic acid and protein content. Leaf water potential (leaf ψ) and relative water content (RWC) were also lowered with increasing stress. Pretreatment with l -cysteine and reduced glutathione (10-3 M) decreased glycolate oxidase activity, H₂O₂ content, ascorbic acid oxidase activity, proline content and also slightly improved the water status of leaves stressed (−1.0 MPa) for 2 days. Pretreatment of non-stressed seedlings with these antioxidants had little or no effect. These studies indicate that treatment with antioxidants makes the plant tolerant against water stress by modulating the endogenous levels of H₂O₂ and ascorbic acid in stressed tissue.     

Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativd) plants

Susceptibility of alfalfa ( Medicago saliva L. cv. Aragón) nodules and leaves to water stress has been investigated. Nodule acetylene reduction activity (ARA), leaf CO₂ exchange rate (CER) as well as soluble protein, proline and total soluble sugar (TSS) contents were determined during drought. Water status was estimated as water potential (Ψ_w) and Relative water content (RWC) of the respective tissues. Maximum rates of ARA required higher Ψ_w than CER. Nodules had lower RWC for a given Ψ_w than leaves. Water stress reduced soluble protein content in both tissues; however, the decline in soluble protein content was detected at greater Ψ_w in nodules than in leaves. Proline and TSS increased in leaves and nodules, and again the threshold Ψ_w triggering such accumulation was higher in nodule tissues. Oior results suggest that alfalfa nodules are more susceptible to water shortage than leaves. Effects of accumulated TSS and proline upon leaf and nodule physiology are discussed in relation to protein stability (proline), pH control (proline) and osmotic adjustment (proiine and TSS). The TSS accumulation induced by water stress suggests that substrate shortage would not be the primary effect of drought on nodule activity.