Physiological and morphological responses to ... - Tree Physiology

Tree Physiology 25, 361–371 © 2005 Heron Publishing—Victoria, Canada

Physiological and morphological responses to water stress in two Acacia species from contrasting habitats D. O. OTIENO,1,2 M. W. T. SCHMIDT,1 S. ADIKU3 and J. TENHUNEN1 1

Department of Plant Ecology, University of Bayreuth, P.O. Box 95440, Bayreuth, Germany

2

Corresponding author ([email protected])

3

Department of Science, Faculty of Agriculture, University of Ghana, Accra, Ghana

Received February 20, 2004; accepted August 22, 2004; published online January 4, 2005

Summary Container-grown seedlings of Acacia tortilis Forsk. Hayne and A. xanthophloea Benth. were watered either every other day (well watered) or every 7 days (water-stressed) for 1 year in a greenhouse. Total plant dry mass (Tdm), carbon allocation and water relations were measured monthly. Differences in leaf area (LA) accounted for differences in Tdm between the species, and between well-watered and waterstressed plants. Reduction in LA as a result of water stress was attributed to reduced leaf initiation, leaf growth rate and leaf size. When subjected to prolonged water stress, Acacia xanthophloea wilted more rapidly than A. tortilis and, unlike A. tortilis, lost both leaves and branches. These differences between species were attributed to differences in the allocation of carbon between leaves and roots and in the ability to adjust osmotically. Rapid recovery in A. xanthophloea following the prolonged water-stress treatment was attributed to high cell wall elasticity. Previous exposure to water stress contributed to water-stress resistance and improved recovery after stress. Keywords: biomass allocation, cell wall elasticity, drought stress, osmotic adjustment, savanna, transpiration.

Introduction Soil water availability is a key factor in the growth, development, species composition and distribution of savanna trees (Noy-Meir 1973, Reynolds et al. 2004). Thus, understanding soil water uptake patterns by trees growing in the savanna, and the associated shoot responses to water loss during drought, will help explain differences among species in productivity, survival and distribution, and could shed more light on the functioning of these dryland ecosystems. Information about root distribution and knowledge of the basic mechanisms of soil water extraction and transport by tree species provide one basis for assessing differences among species in habitat preferences and ecological potentials. Extensive deep-rooting systems with a large surface area over which water absorption can take place facilitate soil water uptake; and in trees adapted to arid habitats, both the relative allocation of photosynthates to roots and the absolute rate of root growth rate tend to be high (Scholz et al. 2002). Extensive root devel-

opment allows extraction of water from a large volume of soil or from a deep water table (Jones 1992, Jackson et al. 2000). As water becomes limiting, certain trees show a decrease in cell sap osmotic potential, thus increasing the water potential gradient between soil and roots, thereby allowing water uptake to continue despite declining soil water content (Tyree and Jarvis 1982). However, tree performance in dry habitats cannot be evaluated without considering constraints within the plant that influence carbon gain (Ehleringer 1994). For example, trees with an effective water supply system may lack specific adaptations for controlling water loss, resulting in low tissue water status that affects plant performance (Kramer 1980, Levitt 1980). Low maximum stomatal conductance and high stomatal sensitivity to changes in water status may be required to maintain leaf water potential (ΨL) above a critical threshold and to avoid xylem cavitation (Tyree and Sperry 1989, Jones and Sutherland 1991). In certain tree species, however, stomatal conductance declines long before there is a noticeable change in soil water content, imposing an early restriction to CO2 uptake (Sperry 2000). Optimally, stomatal regulation of water loss should balance transpiration with water supply to the leaves so that a dangerous decrease in ΨL is avoided without unnecessary restriction to carbon gain (Meinzer 2002). Differences among species in the diurnal fluctuations in ΨL may reveal differences in soil water uptake or the flow of water between roots and shoots, or both, which in turn influences stomatal responses (Jones and Sutherland 1991). Differences in stomatal behavior depend therefore not only on differences in sensitivity to environmental factors associated with the development of water deficit, but also on root system development (Larcher 2003). Such differences in stomatal sensitivity between species, expressed during the development of drought, would serve to limit transpiration and compensate for differences in vulnerability to xylem cavitation (Tyree and Sperry 1988, Jones and Sutherland 1991). Because gaseous exchange, primary productivity and plant fitness are related (Ehleringer 1994, Saliendra et al. 1995), monitoring transpiration through sap flux measurements, coupled with instantaneous measurements of gas exchange activity during the de-

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OTIENO, SCHMIDT, ADIKU AND TENHUNEN

velopment of water stress, should provide reliable information about species performance and ecological potentials. In certain tree species, water loss is reduced through reduction of total leaf area (LA) (Munné-Bosch and Alegre 2004). Reduction in LA as soil water becomes limiting is achieved through reduction in leaf size, leaf rolling or leaf shedding, thus reducing the transpiring leaf surface, but with significant negative impact on carbon gain and overall plant productivity (Jones 1992). Thus, monitoring leaf phenology may provide valuable information about species fitness. The arid and semi-arid savannas of Kenya are dominated by Acacia species, most of which are drought tolerant (Oba et al. 2001). Knowledge is lacking on how different physiological and morphological mechanisms interact to bring about water stress tolerance as well as to maintain productivity during drought in these species. Acacia tortilis and A. xanthophloea have contrasting habitat preferences, with A. tortilis found in more xeric eastern and northern provinces of Kenya, whereas A. xanthophloea occurs in the mesic lowlands (Noad and Birnie 1989). These distribution patterns reflect differences in the ability to cope with water stress. Extrapolation of seedling characteristics to other life stages requires caution, since changes throughout development in the ability to access resources may cause plants at different stages of development to rely on different mechanisms for coping with stress (Cavender-Bares and Bazzaz 2000). Nevertheless, attributes possessed by seedlings may indicate the relative capacity of a species to survive and grow under drought conditions. Thus, controlled experiments may provide a basis for predicting mature tree responses to changes in the environment if scaling factors are applied that account for differences in response between seedlings and mature trees (Bazzaz et al. 1996). Seedlings of A. tortilis and A. xanthophloea were studied to test the hypothesis that distribution patterns of trees in the arid savanna are dependent on characteristics that improve soil water uptake, but limit the rate of soil water depletion. Most plant responses to water stress are elicited only after the plant experiences or senses impending water shortage (Ryel et al. 2004). Here, we test the hypothesis that mechanisms that lead to improved water uptake and conservation can be induced at early stages of tree development by subjecting seedlings to moderate water stress.

Materials and methods Plant culture and experimental design Seeds of Acacia tortilis and A. xanthophloea (Kibwezi provenance), previously obtained from the Kenya Forestry Research Institute (KEFRI), Nairobi, Kenya, were germinated on May 23, 2001, in a greenhouse at the University of Bayreuth, Germany. The seeds were pretreated by immersing them in water at 100 °C and leaving them to soak overnight as the water cooled. The imbibed seeds were sown in moist vermiculite and germinated at 27 °C. Most seeds germinated on the third day after sowing. The seedlings were transplanted to plastic pots (4 × 4 × 6 cm) and grown for 1 month, with regular watering, be-

fore transfer to larger containers (18 cm high and 14 cm in diameter). On September 13, 2001, seedlings were transferred to 0.027-m3 pots containing a 2:1 mix of forest soil and sand. The pots were arranged on a greenhouse bench in two blocks, each comprising 24 trees or pots per species, randomly arranged within the blocks. Differences between blocks in the watering treatment commenced on September 16, 2001, when all pots were watered to capacity. Subsequently, seedlings in one block (control) were watered every other day, whereas seedlings in the other (water-stressed) block were watered at progressively longer intervals, which reached 6 days without water on October 3, 2001, and then remained constant with watering every 7 days. The treatments were continued for 1 year, during which time greenhouse temperatures were 25–30 °C, and mean photosynthetic active radiation (PAR) was 500–800 µmol m – 2 s – 1 . Measurements Plant growth and morphology On a monthly basis, three randomly selected seedlings from each treatment per species were harvested and separated into leaves, stems and roots. Leaf area was measured with a portable leaf area meter (CI-202, CID, Camas, WA). Roots, stems and leaves were dried separately at 70–80 °C for 24 h and weighed. Total dry mass (Tdm), leaf dry mass (Ldm), root dry mass (Rdm), leaf area (LA) and total root:shoot dry mass ratio (R:S) were determined. Sap flux Sap flux was measured on 1-year-old plants in the greenhouse by the stem heat balance (SHB) method described by Sakuratani (1981, 1984). The gauges consisted of a flexible heater mounted on a cork and attached to the stem segment. Heat sensors (copper-constantan thermocouples) were mounted on the cork and insulators. The heating plate was continuously supplied with a 4.5 V DC current, of which one sixth was looped through the logger to record the current supplied to the heaters, as needed to calculate sap flow (F). Heat dissipation in the vertical (Qv) and radial (Qr) directions was sensed by thermocouple junctions on the mounting cork. The entire apparatus was insulated in thick polyvinyl foam and aluminum foil to prevent solar heating. Signals from the thermocouple junctions were automatically recorded by a data logger (Delta-T Devices, Burwell, U.K.). Data were collected every 5 min, and 30-min means stored. Sensors were installed on the main stems, 50 cm above the stem base on two trees per species in each treatment. The energy budget equation for the heated stem section was expressed as: Pin = Q r + Qv + Qflow

(1)

where Pin = electrical power to the heater (W) and Qflow = heat loss by convection by the sap (W). Sap flow (F) was calculated as:

F =

Qflow Cp ∆Tsap

TREE PHYSIOLOGY VOLUME 25, 2005


(2)

WATER STRESS RESPONSES IN ACACIA SEEDLINGS

where Cp = heat capacity of water (J g –1 °C – 1) and ∆Tsap = temperature difference between sap below and above the heater (°C). Diurnal changes in leaf water potential Diurnal courses of leaf water potential (ΨL) were determined 1 day after re-watering with a pressure chamber (PMS Instruments, Corvallis, OR) between 0800 and 1800 h. The days selected for measurements were those when re-watering for water-stressed and control plants coincided. Similar measurements were conducted on water-stressed plants on Day 6 of the drying cycle, when maximum water stress was attained. Due to the small size of Acacia leaves, measurements were made on young shoots bearing 2–3 leaves. Measurements were conducted between July and September when air temperatures and solar irradiances were near the maximum (25 °C and 1200 µmol m –2 s –1, respectively). Pressure–volume measurements Five young shoots per species in each treatment were excised under distilled deionized water early in the morning. The shoots were left to re-saturate in a dark chamber over a period of 24 h. During this period, they were wrapped with a plastic film to prevent evaporative water loss. After 24 h, the shoots were removed from water and their fresh saturated weights and water potentials (Ψ) determined. The shoots were then left to transpire freely under ambient conditions on a laboratory bench. At intervals (5–10 min), mass and Ψ of each shoot were measured. On each occasion, the pressure chamber (PMS Instruments) was pressurized slowly until a bubble of air or water appeared at the distal end of the cut shoot (Tyree and Jarvis 1982). Shoots were weighed immediately before and after each water potential determination and the weights used to calculate relative water content (RWC) according to Kramer (1980). Measurements were continued until the shoots were beyond the wilting point, i.e., there was no further change in mass. The shoots were then oven-dried at 80 °C for 24 h and weighed. To obtain pressure–volume (P–V) curves, reciprocals of tissue water potentials were plotted against relative water content RWC (%) for each shoot per species. Osmotic potential at full turgor (Π100 ), relative water content at the turgor loss point (RWCtlp) and water potential at the turgor loss point (Ψtlp) were derived from P–V curves by considering a regression line between the inverse of the final balancing pressure points and RWC (Tyree and Hammel 1972). Mean values for each species were statistically tested (t-test) for significant differences between treatments and species. Turgor potential was estimated as the difference between water potential (Ψ) and Π as described by Tyree and Jarvis (1982). Osmotic adjustment was calculated as the difference in mean Π100 between water-stressed plants and controls. Hydraulic conductance Leaf specific hydraulic conductance (K sL ) was estimated for each treatment per species from the reciprocals of total flow resistance (R). A simple Ohm’s Law analogy relating diurnal changes in E and ΨL was employed to estimate total flow resistance (R) from soil to leaf (Jones and Sutherland 1991).

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Imposition of severe water stress Beginning August 25, 2002, water was withheld from sets of three plants per species of both control and water-stressed treatments until they failed to regain turgor overnight. Leaf water potential during severe water stress Leaf water potential (ΨL) was measured on Days 1, 5, 8, 12 and 14 during the period when water was withheld. Measurements of ΨL were conducted on the three replicate samples of each treatment per species. Leaf water potential was determined between 0600 and 0700 h, before any significant transpiration was realized, and also at midday when plants experience the lowest Ψ values. From every treatment per species, two shoots (2– 3 leaves) were obtained from well-exposed branches of the 15month-old plants for ΨL determination. Leaf transpiration and stomatal conductance during severe water stress Diurnal courses of leaf transpiration (E) and stomatal conductance (gs) were determined on duplicate leaves of well-exposed branches of the same plants as for the ΨL measurements. Measurements were conducted on Days 3, 4 and 5 during the time that water was withheld using a portable gas exchange system (LI-6400, Li-Cor, Lincoln, NE). Measurements were conducted under natural light conditions. After Day 5, leaves of control plants were wilted most of the day and no further gas exchange measurements could be made with them. Following measurements, the leaves were detached and their area determined with a portable leaf area meter (CI-202 CID). Leaf shedding During the severe stress treatment, the course of leaf shedding was monitored. Post-stress recovery Acacia xanthophloea plants subjected to severe water stress were re-watered to container capacity on September 7, 2002, and every 2 days thereafter, during which time their ΨL was monitored until they fully recovered, i.e., until their water potentials were about zero and they began to generate new leaves. Acacia tortilis plants subjected to severe stress were re-watered on September 11, 2002. This was the period when A. tortilis plants had attained ΨL similar to those previously attained by severely stressed A. xanthophloea plants and also when their leaves failed to regain turgor overnight. For both species and in each treatment after re-watering, two branches of approximately similar lengths, sizes, orientations and positions on the main stems were identified and marked. On a daily basis, the number of sprouting leaves on each of these branches was recorded in each treatment per species. Length, breadth, number and size of leaflets located on each side of the rachis were determined. The mean relative expansion rate during the period with optimum water supply was determined. Examination of leaf regeneration and leaf characteristics for controls of A. xanthophloea were conducted on the short stocks of the main stems, because the plants lost most of the aerial shoots during the severe water stress, leaving only a short (10–20-cm) portion of the stem alive. On two of the recovering plants of each species per treatment, sap flux was also monitored with stem heat balance (SHB) sensors as described above. For each species, the sensor

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guages were installed on the main stems, on the last day of severe water stress. The sensors were installed 50 cm above the stem base on each of the measured plants. Resumption of sap flow was monitored during the period when the plants were recovering and new leaves being formed. Results Plant growth In the control treatment, A. xanthophloea and A. tortilis had mean Tdm values of 403 ± 6.1 and 243 ± 0.74 g per plant, respectively, at the end of the experiment (Table 1). The waterstress treatment reduced dry mass by 45% in A. xanthophloea and 40% in A. tortilis. Significant differences (P < 0.05) between treatments and species occurred 6 months after imposition of water stress. In the control treatment, leaf areas at the end of the experiment were 0.57 and 0.34 m 2 in A. xanthophloea and A. tortilis plants, respectively (Table 1). Water stress resulted in a 26% reduction in LA in A. xanthophloea versus a reduction of only 15% in A. tortilis. The water-stress treatment caused significant leaf senescence and shedding, and a reduction in leaf initiation and expansion as the pots dried, which contributed to the reduction in total LA in the water-stressed plants. The relationship between LA and Tdm was analyzed by log plots of mean values of accumulated LA versus Tdm. There was a linear relationship irrespective of species between log LA and log Tdm for both water-stressed and control plants (Figure 1), suggesting the dependence of Tdm on LA development. There was a significant change (P < 0.001) in the slope and intercept of the relationship when seedlings were water stressed. Carbon partitioning The water-stress treatment significantly increased R:S ratio of A. tortilis to 0.67 (± 0.04) at the end of the experiment compared with 0.52 (± 0.01) in the controls. Irrespective of treatment, the R:S ratio of A. tortilis increased two- and threefold over the course of the experiment in control and water-stressed plants, respectively. The R:S ratios of water-stressed A. tortilis were probably even higher than measured, as this species developed many fine roots as water stress increased, some of which were lost when the roots were harvested. No significant difference in R:S ratio was observed between controls and wa-

Figure 1. Transformed plot of total dry mass (Tdm ) versus leaf area (LA) of (A) controls and (B) stressed A. xanthophloea (䊏, 䊐; thick lines) and A. tortilis (䊉, 䊊; thin lines). Controls were watered every other day, while stressed seedlings were watered every 7 days.

ter-stressed A. xanthophloea (Table 1) seedlings. Acacia xanthophloea showed a decline in R:S ratio over the course of the experiment of 14 and 3% in controls and water-stressed plants, respectively. Root dry mass to leaf area (Rdm:LA) ratio increased in both species with time. Water-stressed A. tortilis had higher Rdm:LA ratio (38%) than controls, but no significant difference was

Table 1. A summary of growth parameters (root dry mass (Rdm ), leaf area (LA) and total dry mass (Tdm) in control and water-stressed Acacia xanthophloea and Acacia tortilis seedlings. Root:shoot (R:S) ratio, and root dry mass to leaf area (Rdm:LA), ratios are also shown. Data are from the final harvest made 15 months after germination. Values are means (± SD, n = 3). Significant differences are indicated by asterisks: * = P < 0.05; and ** = P < 0.001. Parameter

Tdm (g) LA (m2) Rdm:LA ratio R:S ratio

A. xanthophloea

A. tortilis

Water-stressed

Control

Water-stressed

Control

222 ± 2.56 * 0.42 ± 0.04 * 0.011 * 0.19 ± 0.002

403 ± 6.1 0.57 ± 0.08 0.001 0.22 ± 0.001

147 ± 0.75 ** 0.29 ± 0.009 ** 0.013 ** 0.67 ± 0.04 **

243 ± 0.74 0.34 ± 0.04 0.008 0.52 ± 0.01 *




seen in R:S ratio between controls and water-stressed A. xanthophloea. The increase in Rdm:LA ratio observed in A. xanthophloea was mainly attributable to leaf shedding. The species differed significantly in their rooting patterns. Acacia tortilis tended to develop a stronger tap root system and rooting depth increased significantly in the water-stressed plants, whereas roots of A. xanthophloea were more fibrous and lacked a well-defined tap root. Sap flux and leaf water potential Water stress reduced sap flux by 54 and 44% in A. xanthophloea and A. tortilis, respectively (Figure 2). Control plants of A. xanthophloea showed higher (mean diurnal maximum = 110 g h –1) sap fluxes than A. tortilis (mean diurnal maximum = 80 g h –1). Expressing sap flux per unit LA (Figure 2B) revealed no significant differences (P < 0.05) between controls of A. tortilis and A. xanthophloea. The water-stress treatment significantly reduced whole-plant sap flux, with rates declining by half at the end of the water-stress cycle (not shown). Sap flux increased more rapidly following re-watering of waterstressed plants of A. xanthophloea than of A. tortilis. Stem water storage must have been small, because of the small size of the plants (Sakuratani 1984); therefore, we assumed that sap flux was equivalent to transpirational water loss. Increasing transpiration early in the day led to a decline in ΨL (Figure 3). Controls of A. xanthophloea, which had a high transpiration rate, underwent a steeper drop in ΨL than con-

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trols of A. tortilis, reaching a mean diurnal minimum value of –2.4 MPa (Figure 3A). Declines in ΨL were less in waterstressed plants than in control plants and recovered rapidly at the end of the day. Withholding water significantly reduced soil water availability, leading to low ΨL. Acacia xanthophloea, however, experienced much lower ΨL throughout the day than A. tortilis (Figure 3C). A strong inhibition to the decline in water potential was observed for both species after midday when soil water was limiting. Hydraulic conductance Mean leaf specific hydraulic conductances (K sL ) for controls were 4.73 and 3.48 mmol MPa –1 m –2 s –1 in A. tortilis and A. xanthophloea, respectively. Mean values estimated for water-stressed plants were 4.45 and 3.13 mmol MPa –1 m –2 s –1 in A. tortilis and A. xanthophloea, respectively. In both treatments, A. tortilis exhibited greater hydraulic conductance than A. xanthophloea. There was no significant (P < 0.05) difference in K sL between water-stressed and control plants. Tissue water relations Parameters derived from P–V curves are shown in Table 2. Osmotic potential at full turgor (Π100 ) of A. tortilis was significantly lower in water-stressed plants than in control plants. It was also lower than in A. xanthophloea. Acacia tortilis had a mean osmotic adjustment of 0.48 MPa. A slight but smaller (0.16 MPa) osmotic adjustment also occurred in the water-

Figure 2. Mean whole-tree sap flow (A) and whole-tree sap flow expressed per unit leaf area (B), in 1-year-old control and water-stressed Acacia xanthophloea and Acacia tortilis seedlings. Symbols: 䊏 = A. xanthophloea (control); 䊐 = A. xanthophloea (waterstressed); 䊉 = A. tortilis (control); 䊊 = A. tortilis (water-stressed). Data represent means of two plants from each treatment per species.



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both species had a higher relative water content at the turgor loss point (RWCtlp) than water-stressed plants. Bulk modulus of elasticity (ε) was significantly greater in water-stressed plants than in control plants of A. xanthophloea, but not of A. tortilis. Severe water stress Prolonged withholding of water resulted in a faster decline in the water status of plants that were previously well watered (control) than of those that had been previously water stressed (Figure 5). For example, leaves of control plants of A. xanthophloea failed to regain turgor overnight after only 3 days without watering, whereas leaves of previously water-stressed plants maintained turgor overnight for 8 days without re-watering. Similarly, in A. tortilis, previously water-stressed plants survived 12 days without water before losing turgor overnight, compared with 7 days in control plants. Controls of A. xanthophloea lost turgor by midday (Table 2) after the second day of the severe water-stress treatment, attaining a mean minimum water potential of –1.5 MPa during the day (not shown). After reaching the Ψtlp, further declines in ΨL were accompanied by leaf senescence and branch desiccation, which was more severe in control plants (data not shown). Previous water-stress treatment prolonged the time before reaching Ψtlp to 5 days. Control and previously water-stressed A. tortilis plants reached Ψtlp (1.1 ± 0.11 and 1.4 ± 0.10 MPa, respectively, c.f. Table 2) after 7 and 11 days without re-watering, respectively. Recovery after watering was resumed occurred more slowly in A. tortilis than in A. xanthophloea. Leaf transpiration, stomatal conductance and water potential

Figure 3. Diurnal changes in leaf water potential (ΨL ) of control (䊏, 䊉) and water-stressed (䊐, 䊊) (A) A. xanthophloea and (B) A. tortilis seedlings. Plants were watered to container capacity the previous night before measurement the following day. (C) Diurnal pattern of (A) and (B) on Day 6 of water stress. Error bars show deviation from the mean (n = 3).

Diurnal patterns of leaf transpiration (E) and gs in control and water-stressed A. xanthophloea and A. tortilis measured on Day 3 of the severe water-stress treatment are shown in Figure 6. Maximum E and gs declined more than twofold in control plants compared with water-stressed plants of both species. Acacia xanthophloea was affected most, showing depressed gs and E around midday. Stomatal conductance peaked in the morning (1000 h), reaching maximum values of 250 and 50 mmol m – 2 s –1 for water-stressed and control plants, respectively, but rapidly declined to near-zero before noon and only resumed later in the day. A similar pattern was exhibited by E. Previously water-stressed plants of A. tortilis maintained high E and gs (3 mmol m – 2 s –1 and 220 mmol m – 2 s –1, respectively) and A. tortilis control plants showed no midday depression in either E or gs. Post-stress recovery

stressed A. xanthophloea. Water potential at the turgor loss point was higher in the controls of both species than in the water-stress treatment. However, Ψtlp of water-stressed A. tortilis was significantly lower than that of control plants. It was also lower than that of water-stressed A. xanthophloea. There was a strong relationship (r 2 = 0.96) between Π100 and Ψtlp, irrespective of species and treatments (Figure 4). Control plants of

Plant recovery after the severe water-stress treatment was associated with leaf initiation, leaf growth and LA development. New shoots and leaves developed rapidly after re-watering. Previously water-stressed A. tortilis retained leaves during the severe stress treatment, and although most of the leaves looked wilted, they continued to transpire, as shown in Figure 7. After re-watering, leaf initiation was much slower in previously wa-




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Table 2. Water potential at the turgor loss point (Ψtlp), osmotic potential at full turgor (Π100 MPa), bulk modulus of elasticity (ε) and relative water content at the turgor loss point (RWCtlp) in control and water-stressed A. xanthophloea and A. tortilis plants. Values are means (± SD). Significant difference P < 0.05 between means is indicated by (*) and P < 0.001 by (**). A. xanthophloea

Ψtlp (–MPa) ε (MPa) Π100 (–MPa) RWCtlp (%)

A. tortilis

Water-stressed

Control

n

Water-stressed

Control

n

1.24 ± 0.071 9.91 ± 1.32) ** 0.97 ± 0.12 85 ± 0.8 *

1.1 ± 0.16 18.7 ± 2.05 0.81 ± 0.18 88 ± 0.8

9 9 9 9

1.4 ± 0.101 ** 11.2 ± 2.34 1.33 ± 0.16 ** 85 ± 0.5 *

1.1 ± 0.11 10.4 ± 3.11 0.85 ± 0.06 87 ±1

10 10 10 10

Discussion

Figure 4. Relationship between osmotic potential at turgor loss point (Πtlp) and water potential at turgor loss point (Ψtlp) of control and water-stressed A. xanthophloea and A. tortilis plants. Deviations from the means are shown by error bars (n > 8).

ter-stressed plants than in control plants (Figure 8). Leaf initiation was also slower in A. tortilis than in A. xanthophloea. The severe water-stress treatment reduced mean leaf area by about 60 and 80% relative to control plants in previously waterstressed A. xanthophloea and A. tortilis, respectively.

The water-stress treatment resulted in significant decreases in Tdm and LA, and a shift in carbon allocation. Leaf area was most sensitive to water stress, with significant differences between treatments appearing earlier after imposition of stress than differences in other morphological characteristics. Water stress reduced LA through reduced leaf initiation, leaf size and leaf production rate in both species. By the end of the experiment, water stress had caused 45 and 40% declines in Tdm in A. xanthophloea and A. tortilis, respectively. After 15 months of growth, a major decline in growth in both species, as a result of water stress, was mediated through LA reduction (Figure 1) and probably through reduced CO2 assimilation as well, because the rate of stomatal conductance also declined with water stress (Figure 6). Water stress affected Tdm accumulation in the fast-growing A. xanthophloea more than in A. tortilis, and this was in agreement with the magnitude of decline in LA caused by water stress. The results are consistent with other studies. For example, a fast-growing Eucalyptus provenance with higher LA and sufficient water supply exhibited a large proportional decrease in mean leaf size, LA and plant biomass under drought conditions and this reduction in growth was due to reduced foliage area (Osorio et al. 1998, Pita and Pardos 2001). For A. xanthophloea, a 45% reduction in Tdm caused by water stress corresponded closely with a 41% decline in LA, whereas in A. tortilis, a 32% reduction in LA as a result of water stress was associated with a 40% reduction in Tdm. The dif-

Figure 5. Progressive changes in predawn water potential (Ψpd ) of control and previously water-stressed plants of A. xanthophloea and A. tortilis during a subsequent severe water stress. Plants were first watered to container capacity before withholding water until they were wilted overnight. Values are means of three plants per species per treatment. Deviations from the means are shown by error bars.



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Figure 6. Diurnal courses of leaf transpiration (E) and stomatal conductance (gs ) of A. xanthophloea (upper panel) and A. tortilis (lower panel) plants conducted on the third day after withholding water during the severe water-stress treatment. Seedlings were initially well watered (control) or subjected to moderate water stress (stressed). Plants were watered to container capacity before completely withholding water.

ference between species in the slope of the relationship between LA and Tdm (Figure 1) may be explained by differences in carbon partitioning between leaves and roots (Abrams

Figure 7. Recovery of sap flux in (A) water-stressed A. xanthophloea, (B) controls and (C) water-stressed A. tortilis after the plants were subjected to severe water stress beyond the wilting point and then re-watered to container capacity. Values are means of two plants per treatment. Values for controls of A. xanthophloea are missing (see text).

1994) or in osmotic adjustment (Munns 1988, Blake et al. 1991), either of which could improve soil water uptake at the expense of growth (Osonubi and Davies 1978, Abrams 1994). Acacia xanthophloea grew faster than A. tortilis, which we attribute to its ability to allocate a greater proportion of dry mass to LA, thereby maximizing carbon gain under favorable conditions. Control plants of A. xanthophloea with higher LA experienced greater water use compared to those of A. tortilis (100 and 80 g h –1, respectively; see Figure 2). On a long-term basis, reduction in transpiration as a result of the water-stress treatment was associated with LA reduction. However, mechanisms such as stomatal regulation, leaf folding, root characteristics and root-to-shoot water transport were involved in controlling the daily plant water budget. Under conditions of ample water supply, A. xanthophloea experienced higher transpiration rates and lower ΨL (–2.0 and –1.4 MPa, respectively) at the end of the day than A. tortilis, suggesting a poor balance between transpirational water loss and shoot water supply. At midday, when ΨL was low, plants showed strong regulation of water loss through reduced stomatal conductance, resisting further decline in ΨL (Figure 3). In A. xanthophloea, however, the resulting low transpiration rates were not accompanied by an immediate recovery of ΨL as observed in A. tortilis, suggesting an interrupted supply of water from roots (Tyree and Sperry 1989, Meinzer and Grantz 1990, Sperry and Pockman 1993, Sperry 2000). Hydraulic conductance is associated with water uptake at the root surfaces, root:leaf surface area ratio or inherent absorption capacity, root permeability and an effective water transport system (Tyree and Sperry 1988, Reich and Hinkley 1989, Ni and Pallardy 1990). Control plants of A. tortilis exhibited higher hydraulic conductance than control plants of A. xanthophloea (4.73 and 3.48 mmol MPa –1 m –2 s –1, respectively). At high soil water, soil resistance to water flow is small and any observed differences in hydraulic conductance should




Figure 8. Recovery and development of (A) leaf area and (B) leaf initiation of controls and previously water-stressed A. xanthophloea and A. tortilis after the plants were subjected to severe water stress beyond the wilting point and then re-watered to container capacity. Measurements were taken from four branches of two trees per treatment. Data for controls of A. xanthophloea are missing in B.

be largely attributed to differences in plant resistance (Ni and Pallardy 1990). From our results, it follows that A. tortilis has a more robust water-conducting system than A. xanthophloea. Generally, hydraulic conductance is expected to decline with increasing water stress as a result of increasing resistances along the conducting pathway (Blizzard and Boyer 1980). The decline in K sL observed in the water-stressed plants was, however, insignificant: 0.2 and 0.3 mmol MPa –1 m –2 s –1 for A. tortilis and A. xanthophloea, respectively. Two possible reasons for this are that (1) a more competent transport system developed following the imposition of water stress and (2) there was an improved balance between the absorbing root and transpiring leaf surface area in the water-stressed plants. Slower cell expansion during periods of limited water availability, associated with a high carbohydrate concentration, permits rapid primary and secondary wall growth, leading to formation of cells with smaller pit-membrane pores, which are less vulnerable to cavitation (Tyree and Sperry 1989). Thus, the water-stress treatment may have led to the development of better-adapted water-conducting vessels. Water-stressed plants continually shed leaves between Days 4 and 6 following each watering event. This may have improved or maintained the balance between absorbing root and transpiring leaf surface area (shown as Rdm:LA ratio), and is likely to have improved root-to-shoot hydraulic conductance (see Meinzer and Grantz 1990). There was an absolute increase in root growth in waterstressed plants. The results, therefore, show a strong link between water supply from the soil by roots and the transpiring

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leaf surface area. Furthermore, they show that carbon allocation is one way by which plants determine the effectiveness of water supply from roots to shoots. Increased resource allocation to the roots compared to leaves leads to high hydraulic conductance, and this represents a vital adaptation to drought stress (Kramer 1980). This means that, for a given rate of transpiration, a species like A. tortilis, which has a high hydraulic conductance, will undergo less reduction in ΨL during the day. The capacity to dampen the decline in ΨL during water stress ensures prolongation of photosynthesis during drought periods and has implications for plant survival and productivity in dry environments (Cochard et al. 2002). Acacia xanthophloea, however, exhibited an exaggerated dehydration avoidance mechanism associated with stomatal closure that must be linked to a low K sL, and with the disadvantage that declining ΨL may have approached the threshold for xylem cavitation (Tyree and Sperry 1989). That water-stressed plants of both species were able to restore ΨL when they were re-watered during the moderate water-stress treatment, however, indicates that severe xylem cavitation did not occur during drought and that effective water transport to the shoots was restored when drought was relieved. However, cavitation occurred in control plants of A. xanthophloea during severe water stress, inhibiting resumption of water transport and restoration of ΨL, and causing the death of aerial shoots in the control plants. Water-stressed A. tortilis showed a significant (P < 0.05) reduction in Π100 compared with controls, which may have been largely due to solute accumulation in the cells, as determination of cell osmotic potential at full turgor eliminates increased solute concentration as a result of decreased cell volume (Jones 1996). The observed solute increase, therefore, constitutes osmotic adjustment (Hsiao 1973, Osonubi and Davies 1978). As a result of the 6-day water stress cycle, A. tortilis adjusted osmotically by 0.48 MPa, whereas A. xanthophloea adjusted an insignificant 0.16 MPa. Osmotic adjustment may constitute an adaptation to drought stress when it causes a decrease in Ψtlp (Morgan 1984), i.e., maintenance of turgor down to lower values of ΨL. Osmotic adjustment significantly improves soil water uptake under dry conditions (Tyree and Jarvis 1982), and allows maintenance of open stomata with larger apertures and a higher stomatal conductance and net photosynthesis rate down to lower values of ΨL (Myers and Landsberg 1989). In the current study, A. tortilis maintained higher ΨL and stomatal conductances when the soil was drying. Although these observations could be explained by osmotic adjustment, an alternative explanation is that they were the result of reduced LA. Water-stressed A. tortilis had limited LA, resulting in less water loss from the pots. This would mean delayed pot dehydration, hence the plants experienced favorable soil water status for longer. Another explanation might relate to root system characteristics: A. tortilis had a higher R:S ratio, had more roots distributed over the entire soil mass and may have had a higher water permeability across the root surfaces (as shown by high K sL). Thus, repeatedly water-stressed plants were able to draw water from a large soil mass, but were required to supply only a relatively small transpiring leaf surface.



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In A. xanthophloea, repeated water stress resulted in a reduction in ε from 18.7 to 9.9 MPa. Elastic modulus reflects the mechanical properties of the cell wall such that fully turgid tissue is expected to possess the greatest apparent ε (Blake et al. 1991). Bulk modulus of elasticity will, therefore, decline with decreasing cell water content (Tyree and Jarvis 1982). A more elastic tissue, as denoted by a lower modulus of elasticity, indicates that turgor potential declines less rapidly per unit loss of water (Blake et al. 1991). For a given Π, increased elasticity facilitates turgor maintenance over a greater range of water contents, hence improving water uptake from a drying soil (Tyree and Jarvis 1982). Ability to improve cell wall elasticity during water stress as observed in A. xanthophloea may account for its increased water-stress tolerance during severe stress and might also have contributed to its quick recovery when water stress was alleviated, by allowing greater carbon utilization in cell repair processes and more rapid growth after the relief of water stress (Blake et al. 1991). Active solute accumulation, however, shifts photosynthates away from growth towards cell turgor regulation (Dale and Sutcliffe 1986, Munns 1988), and could have accounted for the reduced growth rate and slow recovery observed in water-stressed A. tortilis after re-watering. Response patterns shown by greenhouse plants of the two Acacia species when subjected to different degrees of water stress reflected those observed in mature trees growing under natural conditions (Otieno et al. unpublished). Although the water-stress treatment was limited to watering every seventh day, the root mass of the experimental plants was confined to a limited soil volume, compared with the volume that may be explored by plants in the field, hence their tissue water status dropped to an extent that might not be experienced by fieldgrown trees until several months without rainfall. Acacia xanthophloea trees are associated with marshes and swamps (Noad and Birnie 1987), where water availability is high. They grow rapidly during rainy seasons but lose a large number of aboveground shoots during drought (Otieno et al. unpublished observations). Similar observations were made for seedlings of the same species subjected to different watering regimes in the greenhouse. The results provide detailed information on the physiological adaptations possessed by the two Acacia species and help to explain the pattern of distribution exhibited by mature trees growing naturally in the Kenyan savanna. Conclusions This study showed that behavior patterns exhibited by mature trees growing under natural conditions are reflected closely in young greenhouse-grown plants subjected to experimental manipulation. The results therefore help to explain the distribution pattern observed in these species in their natural environment. Plants previously subjected to water stress maintained a favorable ΨL and survived longer than well-watered control plants when subjected to severe water stress, suggesting that preconditioning improves the ability of seedlings to survive water stress. However, the capacity of a species to overcome drought was an overriding factor in water-stress resistance, as

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