Divergent Gas-exchange, Physiological, Isotopic and Compositional Responses of Two Wood-crop Species to Water Deficit: Ziziphus nummularia and Corymbia citriodora.
We assessed the response to drought in Corymbia citriodora, a fast growing wood-crop with high levels of water-loss, and Ziziphus nummularia, a tree species that occurs in arid areas of Asia and a potential alternative wood-crop plantation species in drought-prone regions. Z. nummularia was able to sustain higher levels of stomatal conductance at lower levels of soil water availability than C. citriodora. The leaves of Z. nummularia also contained higher levels of the antioxidant peroxidase, offering enhanced protection from drought induced oxidative stress. The carbon isotopic and nitrogen concentration of C. citriodora foliage was strongly affected by decreasing soil water availability, but a compositional effect was only apparent in Z. nummularia leaves at the lowest level of soil water.
The higher leaf levels of stomatal conductance and nitrogen are indicative of relatively high assimilation rates in Z. nummularia, suggesting that this species is capable of fully exploiting brief periods where conditions are limited for growth. These attributes, in combination with its inherent tolerance to drought, may make Z. nummularia a suitable wood crop species for rain-fed plantations in drought-prone areas.
Keywords: Carbon isotope composition; Drought; Lemon-scented eucalypts; Peroxidase; Stomatal conductance; Wood crop
Water availability is a major constraint to vegetation growth in many parts of the Earth, frequently limiting production of both food and biofuel crops (Chaves et al., 2003; Centritto et al., 2011b; Pinheiro and Chaves, 2011). Global population growth will necessitate improvements in agricultural practises, irrigation techniques and the development/identification of drought tolerant species to exploit previously unproductive lands (Grierson et al., 2011). Furthermore, future climatic changes are likely to result in more frequent drought events of greater duration in many regions. Prolonged water shortages are not only expected to occur more frequently in the future, but the extent of land affected by drought is expected to rise by 50% (IPCC, 2007). This is particularly relevant for fuel and hard-wood tree plantations that are often grown on marginal lands without supplementary irrigation (Thomas, 2008; Searchinger, 2010; Sedjo et al., 2013).
The identification of plant species likely to be vulnerable to water stress, and/or the traits that confer drought tolerance, are vital to the mitigation of the impacts of climate change to ensure the future security of food and fuel crops (Ren et al., 2007; Centritto et al., 2009; Chaves et al., 2009). Analysis of the photosynthetic and water-use responses of plants to drought may permit the elucidation of mechanisms that underpin plant responses and adaptation to water-deficits (Centritto et al., 2011b).
Water-deficit induces a number of responses in plants depending upon the species, duration and severity of drought, occurrence of co-existing biotic stresses and environmental factors such as temperature and light intensity. The primary negative effect of drought is diminished rates of photosynthesis caused by reductions in the diffusive uptake of CO2 and metabolic limitations (Loreto and Centritto, 2008; Pinheiro and Chaves, 2011). As water availability becomes limited plant stress hydraulic (Centritto et al., 2011b) and chemical signals, such as abscisic acid (ABA) (Wilkinson and Davies, 2002) and pH (Wilkinson et al., 1998; Tahi et al., 2007), cause stomata to close and stomatal conductance (Gs) to decrease (Haworth et al., 2013). This reduced Gs is often accompanied by reduced mesophyll conductance to CO2, resulting in lower availability of CO2 at the sites of carboxylation within the chloroplast envelope (Centritto et al., 2003; Centritto et al., 2011a; Flexas et al., 2013).
These reductions in Gs also result in changes to the carbon isotopic composition of leaves, as the discrimination of the lighter 12C in favour of the heavier 13C isotope becomes less pronounced (Farquhar et al., 1989). Metabolic limitations to CO2-uptake such as diminished regeneration of RuBP caused by impaired ATP production (Lawlor and Tezara, 2009) tend to occur under conditions of severe water-deficit following an extended duration of drought (Centritto et al., 2003; Centritto et al., 2009; Centritto et al., 2011a). To protect the photosynthetic physiology from the effects of reduced photochemical energy use, a corresponding increase in the non- photochemical dissipation of light energy as heat occurs (Harbinson et al., 1990). During episodes of extreme or prolonged water-deficit these protective mechanisms may become impaired, resulting in inhibition of the photosynthetic physiology (Pinheiro and Chaves, 2011).
As photosynthesis becomes increasingly diminished by the reduced uptake of CO2 during drought stress, this results in increased generation rates of harmful reactive oxygen species (ROS) within the leaf (Reddy et al., 2004). These ROS are mopped-up' by anti-oxidants such as peroxidase (POX) as part of the cell's protective metabolism. The capacity to mop-up' these harmful ROS via anti-oxidant cycling may influence the resistance to drought of a particular plant species or variety (Fu and Huang, 2001; TA1/4rkan et al., 2005). The sustainable production of biofuel crops is of increasing importance in the developed world as a carbon neutral energy source and in the developing world as a source of fuel for domestic uses such as cooking and heating.
The establishment of commercial stands of fast growing tree species as a hard and fuel-wood biomass crop is increasing in many countries, providing economic benefits and reducing the clearance of slower growing mature mixed species forests that provide important ecosystem services and enhance biodiversity (Searchinger, 2010). Many of these tree crops have rapid growth; however, this accumulation of biomass is often accompanied by high water consumption. Wood-fuel crops are often grown under rain-fed conditions the relative high water requirements frequently restrict their cultivation in marginal land types. This, in turn, may lead to either higher pressure and eventually loss of native forests to provide fuel wood, or the conversion of productive agricultural land from food to biofuel crop production, that would result in exacerbating food security concerns (Grierson et al., 2011).
Furthermore, the high water requirements of these fast growing biofuel species frequently makes them vulnerable to drough t, thus constraining their potential cultivation in the provision of fuel-wood (Thomas, 2008; Orikiriza et al., 2009; Agaba et al., 2010).
The Australian Eucalypt, Corymbia citriodora, exhibits rapid growth and has been planted extensively as a hard and fuel-wood species in Australia, Central and South East Asia, Africa and South America. To be economically productive, plantations of C. citriodora require consistent water availability to maintain growth and prevent loss of plants (Thomas, 2008; Agaba et al., 2010). Drought results in a pronounced decline in rates of photosynthesis and Gs in C. citriodora. Additionally, under severe water-deficit the emission of the biogenic volatile organic compound isoprene declines, indicating that the efficacy of any protective role against thermal and oxidative damage is diminished (Brilli et al., 2013). To fully exploit marginal land types for wood production it may be necessary to identify alternative tree species that may be more adapted to growth and survival under water-deficit conditions (Allen et al., 2010).
The genus Ziziphus grows in arid areas of Africa and Asia and is composed of shrubs and trees that possess structural and physiological adaptations to prevent water- loss (Clifford et al., 1998; Arndt et al., 2001). Z. nummularia has been suggested as possible fruit and wood- crop species for growth in water-limited areas (Pandey et al., 2010). The ability of Z. nummularia to tolerate water- stress may permit its growth in commercial hard and fuel- wood plantations in marginal land-types more prone to drought than those required by C. citriodora. Furthermore, the afforestation of low-grade upland areas and marginal lands by a drought tolerant species may assist in the prevention of desertification and soil degradation (Zuazo and Pleguezuelo, 2009).
The gas-exchange and physiological responses of C. citriodora and Z. nummularia to water-deficit were investigated to assess the suitability of both trees as hard and fuel-wood species in areas likely to be affected by drought events. This study aimed to: (i) assess the Gs response of C. citriodora and Z. nummularia relative to the content of soil water available for transpiration, to characterise the ability of the respective species to sustain gas-exchange and hence CO2 uptake during water-deficit; (ii) investigate changes in leaf water potential and leaf area as possible adaptations to drought; (iii) analyse shifts in the carbon isotopic and nitrogen composition of leaves as potential indicators of drought stress, and; (iv) study alterations in peroxidase activity as an indicator of the efficiency of physiological protective mechanisms in mopping-up harmful ROS generated during drought stress.
Materials and Methods
Plant Material and Experimental Design
Two-year-old saplings of C. citriodora and Z. nummularia were grown in 5 dm3 pots containing a mixture of commercial soil, sand and manure (1:1:1) in the glasshouse at Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi. The seedlings of both species were collected from areas adjacent to the Chenab River in Muzaffargarh District, south-western Punjab, Pakistan. All the saplings were regularly watered and fertilized with Hoagland solution once a week to supply mineral nutrients at free access rates. On the afternoon prior to the beginning of the experiment, all of the plants were fully irrigated and the excess water was allowed to drain overnight. After draining, the pots were weighed to 1-g precision on a digital balance (model ACS Electronic Scale, Meezan Ltd., Rawalpindi, Pakistan) to determine the weight at pot water capacity (Initialpot weight). Each pot was then enclosed in a plastic bag that was tied around the stem of the sapling to prevent evaporation from the soil.
Twelve (C. citriodora) and (Z. nummularia) plants were then water-stressed by withholding water, while an equal number of control plants were watered to pot capacity each day. The development of water-deficit stress was characterised by recording the daily pot weight relative to Gs values at the start and end of the drought treatment 20 days later, and then expressed as the fraction of transpirable soil water (FTSW). The mean daily weight of twelve pots of each species and treatment was used to calculate FTSW (Sinclair and Ludlow, 1986; Brilli et al., 2013). The physiological lower limit of available soil water was defined as the FTSW at which stomatal conductance approached zero (i.e., soil water decreased to a level where there was no longer water available to support transpiration) (Sinclair and Ludlow, 1986; Centritto et al., 2011a). Once this level was achieved, the water-stressed pots were weighed to determine the final pot weight (Finalpot weight).
Thereafter, every morning the plastic bags were unwrapped to weigh the water-stressed saplings (Dailypot weight) and to water the control plants. Then, the FTSW was calculated for individual pots as follows:
Estimation of Physiological and Chemical Parameters
Stomatal conductance (Gs) was measured on sunny days between 08:00 a.m. and 10:00 a.m. with a steady-state porometer (AP4-UM-3, Delta-T Devices Ltd., Cambridge, England) by enclosing a portion of a single fully expanded leaf in the porometer head whilst avoiding the midrib. The youngest fully expanded leaf was selected from the uppermost canopy of each plant, this leaf was then marked and its levels of Gs recorded throughout the experiment. Three measurements of Gs were recorded for each leaf per day; these were then averaged to produce a mean value for each plant, and the average of 12 plants taken for each point in Figure 1. Leaf area was measured by using planimetric techniques on leaves developed after the onset of the water- stress treatment. Paper replicas of the planar leaf surface were made and then measured using a leaf area meter (LI 3100, LI-COR Inc., Lincoln, NE, USA). The pre-dawn leaf water potential of C. citriodora was measured using a pressure bomb/pressure chamber.
Unfortunately due to th e relatively small leaf size of Z. nummularia it was not possible to measure leaf water potential without collecting a large number of leaves and causing significant injury to the plant that would likely have biased other physiological measurements. Non-destructive measurements such as Gs and recording of pot weight for the calculation of FTSW was undertaken each day on all twelve plants. Destructive samples of leaves were collected at FTSW levels of 100, 45, 15 and 5% on three replicates.
To determine total leaf nitrogen, three leaf samples of three replicates were oven dried at 65C and then ground into powder. Nitrogen concentration of the dried leaf powder (0.2 g) was determined calorimetrically, measuring absorbance at 655 nm, as described by Anderson and Ingram (1993). The carbon isotopic composition (d13C) of 1 mg of ground freeze-dried ground leaf tissue was measured using an elemental analyser (NA 1500, Carlo Erba, Milan, Italy) as described by (Farquhar and Richards, 1984).
To assess peroxidase (POX) activity, newly developed fully expanded leaves (three plants per water treatment) were immediately frozen in liquid nitrogen, lyophilized (Cryodos-50, Telstar, Spain), and then stored at -80C until analysed. The frozen leaves were weighed and then 1 mg ground with 1.5 ml of 0.1 M sodium phosphate (pH 6.5) buffer containing 5% (w/v) polyvinylpyrrolidone (PVP). The Peroxidase activity was assayed using the Guaiacol test (Chance and Maehley, 1955).
Data were analysed using a factorial ANOVA (two-way maximum interactions) to determine the main effects of water treatment and species on all dependent variables measured using Sigma Plot 10.0. A chi-square goodness of fit was used to test whether all variables were normally distributed. No interaction was observed between variables.
C. citriodora and Z. nummularia exhibited pronounced reductions in Gs as water stress increased. However, these Gs reductions occurred at different stages of the drought treatment, possibly reflecting differential responses to reductions in soil water availability between C. citriodora and Z. nummularia (Fig. 1a). Z. nummularia (0.5 mol m-2 s-1 at 100% FTSW) exhibited significantly higher rates of Gs than C. citriodora (0.2 mol m-2 s-1 at 100% FTSW) throughout the experiment as the soils dried (Table 1).
Nonetheless, differences in the kinetics of drought were apparent between the two species as Gs of Z. nummularia began to reduce as the fraction of transpirable soil water (FTSW) declined below 80%, while C. citriodora maintained full rates of Gs until the FTSW reached 50%. The reduction in Gs at higher FTSW values in Z. nummularia was accompanied by a more gradual decline in Gs at lower FTSW values than was apparent in C. citriodora. As drought progressed the leaf-water potential of drought-treated C. citriodora plants declined (Fig. 2) following a similar pattern to that observed in Gs (Fig. 1a). The leaf-water potential of control plants remained constant throughout the experiment, while the drought stressed plants
Table 1: ANOVA of stomatal conductance (gs) values of C. citriodora and Z. nummularia following drought"
Source of Variation###SS###df###MS###F###P-value###F crit
Table 2: ANOVA of leaf area values of C. citriodora and Z. nummularia following drought"
Source of Variation###SS###df###MS###F###P-value###F crit
exhibited a 20% decline at 45% FTSW (co-incident with the level of soil water availability at which Gs values fell), and pronounced reductions of 60% and 125% at FTSW levels of 15 and 5%, respectively (Fig. 2).
C. citriodora possessed significantly greater leaf area than Z. nummularia (Table 2), accounting for the observation that while leaf-level Gs was higher in Z. nummularia, whole plant transpiration was greater in C. citriodora. The leaf area of Z. nummularia under both control and water-deficit conditions was constant throughout the experimental period. In contrast, well- watered control C. citriodora plants exhibited a significant ~100% increase in leaf area over the 20-day experimental period, reflecting the rapid growth of the species under optimal conditions. Leaf area in drought-stressed C. citriodora, despite exhibiting a 21.8% increase by day-10 (45% FTSW) was not significantly altered over the course of the study (Fig. 3). This suggests that neither C. citriodora or Z. nummularia reduced available leaf area as an acclimation response for coping with severe drought over a 20-day period.
The carbon isotopic composition (d13C) of the leaves of C. citriodora and Z. nummularia became enriched in the heavier 13C isotope as drought progressed (Fig. 4). The d13C of C. citriodora leaves was more sensitive to the early stages of drought as a significant difference between the carbon isotope values of control and drought treatment leaves was apparent at 45%, and this upturn in d13C was increasingly evident as FTSW declined further; whereas, the leaves of Z. nummularia only showed a significant increase in d13C values at the lowest 5% FTSW level. The divergence between the bulk carbon isotopic composition of leaves of C. citriodora and Z. nummularia (Table 3) exposed to drought may reflect differences in the growth rate of new foliage between the leaves (Fig. 3) and leaf- lifespan, in addition to compositional alterations of metabolites within the leaves. A similar pattern of shifts in composition was evident in the total nitrogen concentration of the leaves of the two species.
The leaves of Z. nummularia showed a progressive decline in total nitrogen as the drought progressed (Table 4). In contrast, the leaves of C. citriodora exhibited a 22% increase in total nitrogen as the FTSW fell to 45%, before total nitrogen fell by 38% relative to control 100% FTSW values (Fig. 5).
Levels of the protective antioxidant peroxidase (POX) within the leaves of the two species examined in this study showed divergent responses to drought stress. Z. nummularia exhibited a decline in POX activity as the FTSW reached 5%. In contrast, POX activity in C.
Table 3: ANOVA of carbon isotopes values of C. citriodora and Z. nummularia following drought"
Source of Variation###SS###df###MS###F###P-value###F crit
Table 4: ANOVA of total leaf nitrogen values of C. citriodora and Z. nummularia following drought"
Source of Variation###SS###df###MS###F###P-value###F crit
citriodora progressively increased with the severity of drought (Fig. 6). However, it is noteworthy that while the relative changes in POX levels during drought differ between the two species, absolute levels of POX are generally higher in drought stressed Z. nummularia than drought stressed C. citriodora, with the exception of those recorded at 5% FTSW (Table 5). This may be indicative of a greater antioxidant protective capacity in leaves of Z. nummularia than C. citriodora in the event of water deficit occurring, or the use of different antioxidant systems between the two species.
This study has shown contrasting physiological, morphological and compositional responses to drought stress between Z. nummularia and C. citriodora. C. citriodora is characterised by high rates of water-use to sustain photosynthesis and rapid growth that has led to its extensive use in fuel and hard-wood plantations. However, the results of this and previous investigations (eg. Thomas, 2008; Agaba et al., 2010; Brilli et al., 2013) indicate that seedlings of C. citriodora are relatively incapable of tolerating extended periods of water deficit. In contrast, Z. nummularia was able to maintain Gs at lower levels of soil water availability than C. citriodora (Fig. 1a). This suggests that the physiological and morphological adaptations of Z. nummularia that permit it to survive in arid regions (Clifford et al., 1998; Arndt et al., 2001) may also make it a suitable species for plantations in drought prone marginal lands (Pandey et al., 2010).
The ability of Z. nummularia to sustain higher rates of Gs at lower levels of soil water availability than C. citriodora may be related to differences in the water-uptake and transport systems between the two species (Fig. 1b). Z. nummularia possesses
Table 5: ANOVA of POX values of C. citriodora and Z. nummularia following drought"
###ANOVA among POX activity of both plant species
Source of Variation###SS###df###MS###F###P-value###F crit
a large deep and extensive root system that allows it to access water in rocky arid soils (Pandey et al., 2010). This effective water-uptake system permits Z. nummularia to sustain photosynthesis during episodes of water deficit. Furthermore, drought tolerance in tree species is often associated with resistance to xylem embolism (Brodribb et al., 2003). As a tree species occupying arid water-limited environments with a high vapour pressure deficit it may be expected that Z. nummularia exhibits a high level of resistance to xylem cavitation that allows it to maintain transport of water to the photosynthetic organs during episodes of low water availability and high transpirative demand (Sun et al., 2011).
Z. nummularia reduces from full Gs at higher levels of soil water availability (80% FTSW) than C. citriodora, which maintains full Gs until soil water levels are lower (45% FTSW). This may represent different water-use behaviours or acclimation responses between the two species, with the more drought tolerant Z. nummularia exhibiting more conservative water-use (isohydric species) and the earlier modification of leaf physiology than C. citriodora (Figs. 1 and 5) (anisohydric species) (see Maseda and Fernandez, 2006; Centritto et al., 2011b). The native habitat of C. citriodora is sub-tropical and tropical summer dry forests of north-eastern Australia that receive 500800 mm of precipitation annually, with most falling during the winter. During the dry period C. citriodora frequently loses a high proportion of older leaves and branches to reduce water loss (Pook, 1985; Prior et al., 1997).
Relatively few episodes of drought occur during the winter-wet period in the native habitat of C. citriodora. Z. nummularia occurs in arid drought prone areas that frequently experience a large range in temperatures and levels of annual precipitation as low as 100 mm (Gupta et al., 2002; Orwa et al., 2009; Pandey et al., 2010); potentially inducing selective pressures favouring the ability to tolerate persistent water-deficit stress (Kolb and Sperry, 1999). The adaptation of Z. nummularia to drought is apparent in early stages of soil drying (at 80% FTSW), the resultant plant water-uptake behaviour and the capacity of the leaf physiology obstruct the negative effects of potential drought that were employed by the plants in this study (Figs. 1 and 6).
Enhanced antioxidant levels are associated with increased tolerance to drought induced oxidative stress in Olea europea (Sofo et al., 2005; Aganchich et al., 2009). Levels of the protective antioxidant POX were 875% higher in unstressed (100% FTSW) leaves of Z. nummularia than C. citriodora (Fig. 6). Z. nummularia maintained these comparatively higher levels of POX activity under drought stress until the FTSW declined below 15%, whereupon POX activity fell by 65.8% as FTSW reached 5%. In contrast, levels of POX activity rose with increasing levels of drought stress in C. citriodora. This may suggest that leaves of Z. nummularia possess greater capacity against oxidative stress, or that C. citriodora utilises alternative antioxidant systems such as the glutathione and ascorbate systems (Noctor and Foyer, 1998).
This divergence in the activity of POX between the two species may reflect differing degrees of drought tolerance and investment in foliage. C. citriodora produces large numbers of leaves with high photosynthetic rates and relatively short leaf lifespans of less than one-year (Poorter and Bongers, 2006; Laclau et al., 2009). A common response to drought marked by the onset of the dry season in Eucalypts is to rapidly lose older leaves, thus reducing photosynthetic area from which transpirative water-loss can occur (Pook, 1985; Prior et al., 1997). The leaves of Z. nummularia are generally more robust with a longer leaf lifespan reflecting a greater investment in structural material such as lignin (Niinemets, 1999; Haworth and Raschi, 2014). In response to water deficit, Z. nummularia generally does not shed leaves to reduce total leaf area unless the drought event is particularly prolonged or severe (Pandey et al., 2010).
This would be indicative of a relatively lower level of investment in the foliage of C. citriodora (Niinemets, 2001; Poorter et al., 2009), and the selective pressures exerted by the environments of the two species may have shaped their respective responses to drought (Kane and Rieseberg, 2007). In this study the total leaf area of stressed C. citriodora and Z. nummularia remained constant throughout the drought period (Fig. 3). This may be due to the 20-day duration of the experiment, if water-deficit had been allowed to progress over a longer time period reductions in leaf area may have occurred.
However, well-watered C. citriodora exhibited a rapid doubling of total leaf area during the experimental period, while the canopy size of Z. nummularia did not significantly increase. This corroborates the high growth rate in Eucalypts observed in other studies (Mooney et al., 1978; Poorter et al., 1990), and suggests that under growth conditions where the supply of water is unimpeded, a species such as C. citriodora is an effective hard and fuel-wood crop species due to the rapid accumulation of biomass. Nonetheless, such ideal conditions of uninterrupted water supply are unlikely to occur in real word situations, particularly in rain-fed forest plantations on marginal lands where the death of seedlings due to drought constitutes a significant economic loss (McDowell et al., 2008; Agaba et al., 2010).
The effect of the different leaf economic strategies of the species analysed in this study may also be evident in the compositional changes of the leaves following drought.
Carbon isotopic analysis of the leaves of C. citriodora indicated progressive increases in d13C as soil water availability declined (Fig. 4); indicative of diminished Gs resulting in increased uptake of 13C within the leaves (Farquhar et al., 1989; Feng et al., 2013). In contrast, Z. nummularia, despite showing a decline in Gs at higher FTSW values than C. citriodora, did not exhibit any significant increases in leaf carbon isotopic composition until the level of soil water available for transpiration reached its lowest (5% FTSW). As the carbon isotopic composition of the bulk leaf was analysed in both cases, the lack of a detectable isotopic shift in the leaves of Z. nummularia at FTSW values above 5% may reflect the greater structural content invested in the leaves.
The absence of alteration evident over the experimental period in the total leaf area of Z. nummularia (Fig. 3) is indicative that the leaves grew prior to the stress treatment and therefore the carbon isotopic composition of much of this structural material will likely reflect the more favourable growth conditions. The faster growth and lower structural content in the foliage of C. citriodora was more likely to show the carbon isotopic effect of drought in the analysis of bulk leaf material. The lack of any increase in the carbon isotopic ratio of the leaves of Z. nummularia may also be indicative of the importance of Gs in determining the degree of Z. nummularia carbon isotope discrimination. Leaf-level Gs of Z. nummularia was consistently higher than that of C. citriodora. An increase in d13C values of C. citriodora coincided with a reduction of Gs values from ~0.2 to 0.1 mol m-2 s-1 at 45% FTSW.
Stomatal conductance values of Z. nummularia only declined below 0.1 mol m-2 s-1 at the lowest level of available soil water recorded of 5% FTSW (Fig. 1); raising the possibility that the apparent lack of leaf carbon isotopic response to drought stress is due to the relatively high rates of Gs sustained by Z. nummularia. However, the carbon isotopic composition of a leaf is generally associated with the long-term WUE of a plant, and often determined by Gs (Farquhar and Richards, 1984; Farquhar et al., 1989). The d13C of C. citriodora would suggest a lower WUE than Z. nummularia, yet the Gs values of Z. nummularia were consistently greater than those of C. citriodora, possibly suggesting more efficient CO2-uptake in Z. nummularia; potentially associated with the observations of higher leaf nitrogen concentration in this study (Fig. 5).
Nonetheless, to constrain these potential sources of variation, future studies of the effect of drought-stress on the isotopic composition of wood-crop species should utilise the analysis of recently synthesised photosynthates (Scartazza et al., 1998) or compound specific analysis of wax compounds on newly developed leaves (Tipple et al., 2013).
Similar patterns of the effect of drought on foliage composition are evident in the nitrogen concentration of the leaves of the two species. The total nitrogen concentration of Z. nummularia leaves was unaffected as the available soil water declined from 100 to 15% FTSW. A significant reduction in leaf nitrogen was only apparent at the lowest level of water availability (5% FTSW). In contrast, the leaves of C. citriodora showed progressive reductions in nitrogen concentration as FTSW declined from 45 to 5% (Fig. 5). These divergent compositional responses may reflect different physiological adaptations to water-stress and/or the differential degrees of investment in the leaves between the two species. It is however noteworthy that the longer lived foliage of Z. nummularia possessed significantly higher levels of leaf nitrogen than the foliage of C. citriodora.
This pattern is not consistent with numerous observations of leaf economic strategies, where those leaves representing a greater proportional investment with longer leaf lifespans generally possess lower levels leaf nitrogen relative to leaf mass (Poorter et al., 1990; Poorter et al., 2009). Leaf-level photosynthetic rates are often positively associated with the concentration of nitrogen (Evans, 1989), and rates of stomatal conductance (Haworth et al., 2011). Therefore, the higher levels of leaf nitrogen in the foliage of Z. nummularia are consistent with observations of relatively higher rates of Gs than were found in C. citriodora. This may suggest that despite being adapted to relatively arid conditions, Z. nummularia is capable of high rates of leaf- level photosynthesis during periods of water availability to fully exploit favourable growth conditions (Attiwill and Clayton-Greene, 1984; Hetherington and Woodward, 2003).
During drought-stress Z. nummularia can then reduce photosynthetic rates and Gs to conserve water (isohydric behaviour), and in these periods the comparatively high levels of antioxidant activity (Fig. 6) protect the leaf from the harmful effects of oxidative stress.
The fast growth and rapid accumulation of biomass make C. citriodora a highly effective wood crop species in areas of high water availability. However, C. citriodora is relatively intolerant of drought stress, leading to impaired photosynthesis and the frequent loss of both seedlings and young trees (Fig. 1) (Agaba et al., 2010; Brilli et al., 2013). Z. nummularia possesses physiological (Figs. 1 and 6) and morphological adaptations (Clifford et al., 1998; Arndt et al., 2001; Haworth and McElwain, 2008) to the negative effects of drought stress. The apparent capacity of Z. nummularia leaves to fully exploit favourable growth conditions, combined with the ability to tolerate periods of water deficit, are highly desirable in a biomass crop species grown in drought prone rain-fed marginal lands.
Nonetheless, further work is required to fully assess the relative impact of drought on rates of biomass accumulation in Z. nummularia (eg. Monclus et al., 2006), as this is the critical criterion in any successful hard or fuel-wood crop. Nonetheless, the results of this study suggest that the ability to tolerate drought through effective stomatal control, maintenance of Gs at low levels of soil water availability and enhanced protective mechanisms against oxidative stress in Z. nummularia may permit its potential exploitation as a highly effective wood plantation species in rain-fed marginal lands in drought prone areas.
In conclusion, this study has shown a clear distinction between the respective anisohydric and isohydric drought responses of C. citriodora and Z. nummularia. Z. nummularia reduced Gs at higher FTSW levels than C. citriodora, but was able to maintain higher rates of Gs at lower levels of soil water availability. Foliage of Z. nummularia exhibited less compositional change in terms of the ratio of carbon isotopes and nitrogen concentration in response to water deficit than C. citriodora. Leaves of Z. nummularia also possessed higher levels of the antioxidant POX than C. citriodora, possibly as a pre-emptive protective mechanism against the harmful effects of the frequent drought events that occur in the native habitat of Z. nummularia.
This apparent ability to sustain Gs during episodes of reduced water availability, combined with elevated antioxidant mechanisms that confer tolerance to the harmful physiological effects of drought, may permit the use of Z. nummularia as an effective hard and fuel-wood plantation species in drought prone areas. While C. citriodora displays rapid growth, ideal for use as a wood crop species in areas where drought events are relatively infrequent, the results of this and other studies suggest that it is relatively intolerant of water-deficit conditions, exhibiting reduced gas-exchange and growth. The observations of previous studies may suggest that the attributes required for high rates of photosynthesis and accumulation of biomass under optimal growth conditions may be mutually exclusive to the attributes that confer drought tolerance. However, the high rates of Gs and foliar nitrogen concentration would suggest that Z. nummularia is capable of sustaining high rates of leaf-level photosynthesis.
This adaptation may permit Z. nummularia to fully exploit brief intervals where conditions are favourable to growth.
This work was supported by the Ministero dell'Istruzione dell'UniversitA e della Ricerca of Italy: PRIN 2010-2011 PRO-ROOT and Progetto Premiale 2012 Aqua. MH acknowledges funding from a Marie Curie IEF (2010- 275626).
Agaba, H., L.J.B. Orikiriza, J.F.O. Esegu, J. Obua, J.D. Kabasa and A. Huettermann, 2010. Effects of hydrogel amendment to different soils on plant available water and survival of trees under drought conditions. Clean-Soil Air Water, 38: 328335
Aganchich, B., S. Wahbi, F. Loreto and M. Centritto, 2009. Partial root zone drying: regulation of photosynthetic limitations and antioxidant enzymatic activities in young olive (Olea europaea) saplings. Tree Physiol., 29: 685696
Allen, C.D., A.K. Macalady, H. Chenchouni, D. Bachelet, N. McDowell, M, Vennetier, T. Kitzberger, A. Rigling, D.D. Breshears and E. Hogg, 2010. A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests. For. Ecol. Manage., 259: 660684
Anderson, J.M. and J.S.I. Ingram, 1993. Tropical Soil Biology and Fertility: A Handbook of Methods. Wallingford, U.K: C.A.B. International Arndt, S., S. Clifford, W. Wanek, H. Jones and M. Popp, 2001. Physiological and morphological adaptations of the fruit tree Ziziphus rotundifolia in response to progressive drought stress. Tree Physiol., 21: 705715
Attiwill, P.M. and K.A. Clayton-Greene, 1984. Studies of gas exchange and development in a subhumid woodland. J. Ecol., 72: 285294
Brilli, F., T. Tsonev, T. Mahmood, V. Velikova, F. Loreto and M. Centritto, 2013. Ultradian variation of isoprene emission, photosynthesis, mesophyll conductance, and optimum temperature sensitivity for isoprene emission in water-stressed Eucalyptus citriodora saplings. J. Exp. Bot., 64: 519528
Brodribb, T.J., N.M. Holbrook, E.J. Edwards and M.V. Gutierrez, 2003. Relations between stomatal closure, leaf turgor and xylem vulnerability in eight tropical dry forest trees. Plant Cell Environ., 26: 443450
Centritto, M., F. Brilli, R. Fodale and F. Loreto, 2011a. Different sensitivity of isoprene emission, respiration and photosynthesis to high growth temperature coupled with drought stress in black poplar (Populus nigra) saplings. Tree Physiol., 31: 275286
Centritto, M., M. Lauteri, M.C. Monteverdi and R. Serraj, 2009. Leaf gas exchange, carbon isotope discrimination, and grain yield in contrasting rice genotypes subjected to water deficits during the reproductive stage. J. Exp. Bot., 60: 23252339
Centritto, M., F. Loreto and K. Chartzoulakis, 2003. The use of low [CO2] to estimate diffusional and non-diffusional limitations of photosynthetic capacity of salt-stressed olive saplings. Plant Cell Environ., 26: 585594
Centritto, M., R. Tognetti, E. Leitgeb, K. Strelcova and S. Cohen, 2011b. Above ground processes - anticipating climate change influences, In: M. Bredemeier, S. Cohen, D.L. Godbold, E. Lode, V. Pichler and P. Schleppi (eds.). Forest Management and the Water Cycle: An Ecosystem-Based Approach, pp: 3164. London: Springer, The Netherlands
Chance, B. and A.C. Maehley, 1955. Assay of catalases and peroxidases, In: S.P. Colowick and N.O. Kaplan (eds.). Methods in Enzymology. Vol. II, pp: 764775. New York: Academic Press, New York, USA
Chaves, M., J. Flexas and C. Pinheiro, 2009. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann. Bot., 103: 551560
Chaves, M.M., J.P. Maroco and J.S. Pereira, 2003. Understanding plant responses to drought-from genes to the whole plant. Funct. Plant Biol., 30: 239264
Clifford, S.C., S.K. Arndt, J.E. Corlett, S. Joshi, N. Sankhla, M. Popp and H.G. Jones, 1998. The role of solute accumulation, osmotic adjustment and changes in cell wall elasticity in drought tolerance in Ziziphus mauritiana (Lamk.). J. Exp. Bot., 49: 967977
Evans, J., 1989. Photosynthesis and nitrogen relationships in leaves of C3 plants. Oecologia, 78: 919
Farquhar, G. and R. Richards, 1984. Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes. Funct. Plant Biol., 11: 539552
Farquhar, G.D., J.R. Ehleringer and K.T. Hubick, 1989. Carbon isotope discrimination and photosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol., 40: 503537
Feng, Q., M. Centritto, R. Cheng, S. Liu and Z. Shi, 2013. Leaf functional trait responses of Quercus aquifolioides to high elevations. Int. J. Agric. Biol., 15: 6975
Flexas, J., U. Niinemets, A. GallACopyright, M. Barbour, M. Centritto, A. Diaz- Espejo, C. Douthe, J. GalmACopyrights, M. Ribas-Carbo, P. Rodriguez, F. Rossello, R. Soolanayakanahally, M. Tomas, I. Wright, G. Farquhar and H. Medrano, 2013. Diffusional conductances to CO2 as a target for increasing photosynthesis and photosynthetic water-use efficiency. Photosynth. Res., 117: 4559
Fu, J. and B. Huang, 2001. Involvement of antioxidants and lipid peroxidation in the adaptation of two cool-season grasses to localized drought stress. Environ. Exp. Bot., 45: 105114
Grierson, C.S., S.R. Barnes, M.W. Chase, M. Clarke, D. Grierson, K.J. Edwards, G.J., Jellis, J.D. Jones, S. Knapp, G. Oldroyd, G. Poppy, P. Temple, R. Williams and R. Bastow, 2011. One hundred important questions facing plant science research. New Phytol., 192: 612
Gupta, N., S. Meena, S. Gupta and S. Khandelwal, 2002. Gas exchange, membrane permeability, and ion uptake in two species of Indian jujube differing in salt tolerance. Photosynthetica, 40: 535539
Harbinson, J., B. Genty and N.R. Baker, 1990. The relationship between CO2 assimilation and electron transport in leaves. Photosynth. Res., 25: 213224
Haworth, M., C. Elliott-Kingston and J. McElwain, 2013. Co-ordination of physiological and morphological responses of stomata to elevated [CO2] in vascular plants. Oecologia, 171: 7182
Haworth, M., C. Elliott-Kingston and J.C. McElwain, 2011. Stomatal control as a driver of plant evolution. J. Exp. Bot., 62: 24192423
Haworth, M. and J. McElwain, 2008. Hot, dry, wet, cold or toxic Revisiting the ecological significance of leaf and cuticular micromorphology. Palaeogeography, Palaeoclimatology, Palaeoecology, 262: 7990
Haworth, M. and A. Raschi, 2014. An assessment of the use of epidermal micro-morphological features to estimate leaf economics of Late Triassic-Early Jurassic fossil Ginkgoales. Rev. Palaeobot. Palynol., 205: 18
Hetherington, A.M. and F.I. Woodward, 2003. The role of stomata in sensing and driving environmental change. Nature, 424: 901908
IPCC, 2007. Climate Change 2007: Impacts, Adaptation and Vulnerability. Contribution of Working Group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge Cambridge University Press, Cambridge, UK
Kane, N.C. and L.H. Rieseberg, 2007. Selective sweeps reveal candidate genes for adaptation to drought and salt tolerance in common sunflower, Helianthus annuus. Genetics, 175: 18231834
Kolb, K.J. and J.S. Sperry, 1999. Differences in drought adaptation between subspecies of sagebrush (Artemisia tridentata). Ecology, 80: 23732384
Laclau, J.P., J.C. Almeida, J.L.M. Goncalves, L. Saint-AndrACopyright, M. Ventura, J. Ranger, R.M. Moreira and Y. Nouvellon, 2009. Influence of nitrogen and potassium fertilization on leaf lifespan and allocation of above-ground growth in Eucalyptus plantations. Tree Physiol., 29: 111124
Lawlor, D.W. and W. Tezara, 2009. Causes of decreased photosynthetic rate and metabolic capacity in water-deficient leaf cells: a critical evaluation of mechanisms and integration of processes. Ann. Bot., 103: 561579
Loreto, F. and M. Centritto, 2008. Leaf carbon assimilation in a water- limited world. Plant Biosyst., 142: 154161
Maseda, P.H. and R.J. Fernandez, 2006. Stay wet or else: three ways in which plants can adjust hydraulically to their environment. J. Exp. Bot., 57: 39633977
McDowell, N., W.T. Pockman, C.D. Allen, D.D. Breshears, N. Cobb, T. Kolb, J. Plaut, J. Sperry, A. West and D.G. Williams, 2008. Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought New Phytol., 178: 719739
Monclus, R., E. Dreyer, M. Villar, F.M. Delmotte, D. Delay, J.M. Petit, C. Barbaroux, D. Le Thiec, C. BrACopyrightchet and F. Brignolas, 2006. Impact of drought on productivity and water use efficiency in 29 genotypes of Populus deltoidesA- Populus nigra. New Phytol., 169: 765777
Mooney, H., P.J. Ferrar and R. Slatyer, 1978. Photosynthetic capacity and carbon allocation patterns in diverse growth forms of Eucalyptus. Oecologia, 36: 103111
Niinemets, A., 1999. Research review. Components of leaf dry mass per area thickness and density alter leaf photosynthetic capacity in reverse directions in woody plants. New Phytol., 144: 3547
Niinemets, A., 2001. Global-scale climatic controls of leaf dry mass per area, denisty and thickness in trees and shrubs. Ecology, 82: 453469
Noctor, G. and C.H. Foyer, 1998. Ascorbate and glutathione: Keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol.Biol., 49: 249279
Orikiriza, L.J.B., H. Agaba, M. Tweheyo, G. Eilu, J.D. Kabasa and A. Huettermann, 2009. Amending soils with hydrogels increases the biomass of nine tree species under non-water stress conditions. Clean-Soil Air Water, 37: 615620
Orwa, C., A. Mutua, R. Kindt, R. Jamnadass and A. Simons, 2009. Zizyphus nummularia Rhamnaceae (Burm. F.) Wight and Arn. Agroforestree Database:a tree reference and selection guide version 4.0
Pandey, A., R. Singh, J. Radhamani and D.C. Bhandari, 2010. Exploring the potential of Ziziphus nummularia (Burm. f.) Wight et Arn. from drier regions of India. Genet. Resour. Crop Evol., 57: 929936
Pinheiro, C. and M.M. Chaves, 2011. Photosynthesis and drought: can we make metabolic connections from available data J. Exp. Bot., 62: 869882
Pook, E., 1985. Canopy dynamics of Eucalyptus maculata Hook. III Effects of drought. Aust. J. Bot., 33: 6579
Poorter, H., A. Niinemets, I. Poorter, I.J. Wright and R. Villar, 2009. Causes and consequences of variation in leaf mass per area (LMA): a meta- analysis. New Phytol., 182: 565588
Poorter, H., C. Remkes and H. Lambers, 1990. Carbon and nitrogen economy of 24 wild species differing in relative growth rate. Plant Physiol., 94: 621627
Poorter, L. and F. Bongers, 2006. Leaf traits are good predictors of plant performance across 53 rain forest species. Ecology, 87: 17331743
Prior, L., D. Eamus and G. Duff, 1997. Seasonal and diurnal patterns of carbon assimilation, stomatal conductance and leaf water potential in Eucalyptus tetrodonta saplings in a wetdry savanna in northern Australia. Aust. J. Bot., 45: 241258
Reddy, A.R., K.V. Chaitanya, M. Vivekanandan, 2004. Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. J. Plant Physiol., 161: 11891202
Ren, J., W. Dai, Z. Xuan, Y. Yao, H. Korpelainen and C. Li, 2007. The effect of drought and enhanced UV-B radiation on the growth and physiological traits of two contrasting poplar species. For. Ecol. Manage., 239: 112119
Scartazza, A., M. Lauteri, M.C. Guido and E. Brugnoli, 1998. Carbon isotope discrimination in leaf and stem sugars, water-use efficiency and mesophyll conductance during different developmental stages in rice subjected to drought. Aust. J. Plant Physiol., 25: 489498
Searchinger, T.D., 2010. Biofuels and the need for additional carbon. Environ. Res. Lett., 5: 024007
Sedjo, R.A., B. Sohngen, A. Riddle, 2013. Wood bioenergy and land use: a challenge to the Searchinger hypothesis. Indust. Biotechnol., 9: 319327
Sinclair, T. and M. Ludlow, 1986. Influence of soil water supply on the plant water balance of four tropical grain legumes. Funct. Plant Biol., 13: 329341
Sofo, A., B. Dichio, C. Xiloyannis and A. Masia, 2005. Antioxidant defence in olive trees during drought stress: changes in activity of some antioxidant enzymes. Funct. Plant Biol., 32: 4553
Sun, S.J., M.P. Zhang and X. Wan, 2011. Seasonal variation in water use of Ziziphus jujuba in the South aspect of Taihang Mountains with deuterium isotope signature. Sci. Silvae Sin., 5: 008
Tahi, H., S. Wahbi, R. Wakrim, B. Aganchich, R. Serraj and M. Centritto, 2007. Water relations, growth, photosynthesis and water use efficiency in tomato plant subjected to partial rootzone drying (PRD) and regulated deficit irrigation (RDI). Plant Biosyst., 141: 265274
Thomas, D.S., 2008. Hydrogel applied to the root plug of subtropical eucalypt seedlings halves transplant death following planting. For. Ecol. Manage., 255: 13051314
Tipple, B.J., M.A. Berke, C.E. Doman, S. Khachaturyan and J.R. Ehleringer, 2013. Leaf-wax n-alkanes record the plantwater environment at leaf flush. Proc. Natl. Acad. Sci., 110: 26592664
TA1/4rkan, I., M. Bor, F. Ozdemir and H. Koca, 2005. Differential responses of lipid peroxidation and antioxidants in the leaves of drought- tolerant P. acutifolius Gray and drought-sensitive P. vulgaris L. subjected to polyethylene glycol mediated water stress. Plant Sci., 168: 223231
Wilkinson, S., J.E. Corlett, L. Oger and W.J. Davies, 1998. Effects of xylem pH on transpiration from wild-type and flacca tomato leaves - A vital role for abscisic acid in preventing excessive water loss even from well-watered plants. Plant Physiol., 117: 703709
Wilkinson, S. and W.J. Davies, 2002. ABA-based chemical signalling: the co-ordination of responses to stress in plants. Plant Cell Environ., 25: 195210
Zuazo, V.H.D. and C.R.R. Pleguezuelo, 2009. Soil-erosion and runoff prevention by plant covers: a review, In: E. Lichtfouse, M. Navarrete, M., P.Debaeke, S. VACopyrightronique, C. Alberola (eds.). Sustainable Agriculture, pp 785811. Springer Netherlands