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This study investigated the role of quercitol in osmotic adjustment in field-grown Eucalyptus astringens Maiden subject to seasonal drought stress over the course of 1 year. The trees grew in a native woodland and a farm plantation in the semi-arid wheatbelt region of south Western Australia. Plantation trees allocated relatively more biomass to leaves than woodland trees, but they suffered greater drought stress over summer, as indicated by lower water potentials, CO2 assimilation rates and stomatal conductances. In contrast, woodland trees had relatively fewer leaves and suffered less drought stress. Plantation trees under drought stress engaged in osmotic adjustment, but woodland trees did not. Quercitol made a significant contribution to osmotic adjustment in drought-stressed trees (25% of total solutes), and substantially more quercitol was measured in the leaves of plantation trees (5% dry matter) than in the leaves of woodland trees (2% dry matter). We found no evidence that quercitol was used as a carbon storage compound while starch reserves were depleted under drought stress. Differences in stomatal conductance, biomass allocation and quercitol production clearly indicate that E. astringens is both morphologically and physiologically ‘plastic’ in response to growth environment, and that osmotic adjustment is only one part of a complex strategy employed by this species to tolerate drought.
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Most climate change scenarios suggest an increase in aridity in many areas of the globe (IPCC 2007), which makes investigations of plant responses to drought stress increasingly important. Higher plants have developed a range of mechanisms to adapt to soil moisture deficits, with escape, avoidance and tolerance strategies being the most important (Levitt 1972). Annual species such as grasses can escape drought completely by dying off during the driest times of the year. Perennial species such as trees and shrubs can avoid drought by maximizing water uptake (e.g. tapping permanent water sources via deep roots) or minimizing tissue water loss while maintaining high tissue water potentials (e.g. stomatal closure, sclerophyllous leaf morphology, leaf abscission). Other perennial plants have developed mechanisms for tolerating drought based on tolerating low tissue water potentials by having smaller cells, more rigid or more flexible cell walls or through osmotic adjustment (Morgan 1984).
Osmotic adjustment occurs when plants accumulate solutes for the purpose of maintaining positive cell turgor. Osmotic adjustment can occur either as an active process [i.e. the net accumulation of solutes on a dry weight (DW) basis] or as a passive process (i.e. a decrease in tissue water content that leads to an increase of solutes on a plant water basis, but not to a net increase on a DW basis) (Turner & Jones 1980). Adjustment is usually slow, but it is a potentially powerful adaptation to drought because it allows plants to maintain water absorption, cell turgor and metabolic activity during periods of drought stress, and also enables quick resumption of growth when water becomes available again (Chaves, Maroco & Pereira 2003).
Osmotic adjustment is a widely studied phenomenon, but the specific compounds responsible for maintaining cellular osmolarity remain unidentified and unstudied in most species. Previous work has shown that plants may regulate ion concentrations and/or draw on a variety of solutes, including organic acids, amino acids, quaternary ammonium compounds, carbohydrates and cyclitols (Hasegawa et al. 2000). Different solutes almost certainly vary in their metabolic cost, and it is reasonable to assume that plants have evolved to use solutes that provide the most benefits for investment. This assumption underpins recent work focusing on the role of ‘compatible’ solutes in osmotic adjustment, that is, solutes that do not inhibit cytosolic enzymes (as do high concentrations of ions), but rather protect cell membranes and metabolic processes under dehydration (Popp & Smirnoff 1995; Jaindl & Popp 2006).
Cyclitols are a group of compatible solutes that occur in appreciable concentrations in a wide range of plant species (Popp et al. 1997). Cyclitols such as pinitol, quebrachitol and quercitol can accumulate in relatively large amounts, up to several percent of dry matter on a whole-plant basis (Dittrich, Gietl & Kandeler 1971; Popp et al. 1997; Merchant et al. 2006a), and they have been shown to accumulate in response to drought (Nguyen & Lamont 1988) or salinity (Richter, Thonke & Popp 1990), which also suggests a role in stress tolerance. In the case of Eucalyptus, glasshouse studies have shown that concentrations of quercitol increase in the leaves of some species in response to salinity and drought stress (Adams et al. 2005; Merchant & Adams 2005). Species from low rainfall environments tend to produce significantly greater amounts of quercitol in leaf tissues than species from mesic environments (Merchant et al. 2006a), suggesting that quercitol accumulation may be correlated with species distribution as determined by water availability. However, quercitol is not always utilized as a dominant osmolyte, and its production varies greatly among different Eucalyptus species. Under some experimental conditions, quercitol concentrations (on a water content basis) may increase in leaf tissue only as a consequence of decreased leaf water content rather than active accumulation (Merchant & Adams 2005; Merchant et al. 2006b), and recent work has demonstrated that cyclitols can be a minor component of total solute concentration in species that maintain high leaf water contents and display strong stomatal sensitivity to water deficits (Warren, Bleby & Adams 2007).
Quercitol functions as a stable osmoticum in Eucalyptus species and contributes significantly to the cellular osmotic potential in leaves, but its role in osmotic adjustment is not yet fully understood. Most studies investigating quercitol have been performed on juvenile trees in glasshouse studies, and most have only investigated its accumulation in leaves. It is currently unknown if quercitol concentrations show similar responses in mature trees in the field, where the environmental conditions are different and complex, and the onset of drought stress is less abrupt as compared to glasshouse experiments. Previous studies of Quercus robur indicate that quercitol can be translocated between leaves and woody above-ground tissues during the year (Popp et al. 1997), and appreciable amounts of quercitol have been measured in xylem sap of a range of Eucalyptus species (Merchant, unpublished results). It appears possible that quercitol may be translocated between different organs during the course of the year, and that it may therefore play a role in carbon transport or carbon storage.
The primary aim of this study was to investigate whether the concentration of quercitol in different plant tissues of a semi-arid Eucalyptus species changes in response to drought stress on a whole-plant level in the field. The objectives of the study were to investigate: (1) the concentration of quercitol in different tissues (leaves, branches, stems, roots) at different times of the year; (2) the contribution of quercitol to total osmolytes and possible osmotic adjustment in the different tissues; and (3) the translocation of quercitol between the different tree organs during the season. The species chosen for this study was Eucalyptus astringens (brown mallet), which grows naturally in the semi-arid wheatbelt region of southern Western Australia. This region has a typical Mediterranean-type climate, and E. astringens experiences significant drought stress during the hot, dry summer months and little or no drought stress during the cooler, wet winter months. We compared two neighbouring stands, a native woodland and a farm plantation, where preliminary work showed that plantation trees were more drought stressed than woodland trees, particularly in summer.
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The Dryandra woodland region experienced typical Mediterranean-type climatic conditions over the course of this study (Fig. 1). The hot and dry summer period lasted from December 2001 to April 2002, and the first substantial rainfalls occurred in May 2002. Total precipitation for 2002 was 350 mm, and most (70%) of that rainfall occurred in the winter months between June and September. There were a few frosty days in late July and early August, where minimum temperatures were below zero.
Figure 1. Climate data for Narrogin in 2002, 10 km from the field sites in Dryandra Woodland, Western Australia. Daily maximum temperature (solid line), daily minimum temperature (dashed line), daily precipitation events (bars).
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Stands of E. astringens were relatively homogenous in stand structure and even aged at both sites, but there were clear differences between the sites with respect to tree age, stand structure and biomass allocation. Plantation trees were 4 years old compared to around 10 years old in the woodland, had been planted at a lower stem density than the natural regeneration in the woodland and clearly had greater access to light and nutrients (NO3- and NH4+) compared to the woodland trees. Plantation trees allocated approximately 35% of their biomass to the stem, 20% to branches, 20% to leaves and the remaining 20% to roots (Table 1). In comparison, woodland trees allocated relatively less biomass to branches and leaves and more to stems, approximately 50% to stems, 15% to branches, 15% to leaves and 20% to roots (Table 1). Patterns of biomass allocation did not change significantly during the year at either site (Table 1).
Table 1. Allocation of biomass and quercitol among different plant tissues (leaves, branches, stems and roots) of Eucalyptus astringens grown in a native woodland or a plantation in Dryandra, Western Australia
| || ||Leaves||Branches||Stems||Roots|
| Woodland||March||12.7 a||15.8 a||49.5 a||22.0 a|
|May||15.2 a||12.7 a||52.7 a||19.4 a|
|July||16.1 a||11.8 a||52.1 a||20.0 a|
|September||16.8 a||13.0 a||51.4 a||18.8 a|
|December||15.1 a||15.2 a||50.0 a||19.7 a|
| Plantation||March||32.6 a||21.8 ab||23.9 a||21.7 a|
|May||37.4 a||22.5 ab||17.8 a||22.3 a|
|July||36.6 a||21.4 ab||17.8 a||24.3 a|
|September||34.9 a||20.3 a||21.5 a||23.3 a|
|December||30.5 a||25.4 b||23.3 a||20.8 a|
| Woodland||March||27.3 a||18.5 a||33.8 ab||20.4 a|
|May||33.5 a||13.2 a||38.1 ab||15.1 a|
|July||26.4 a||12.4 a||45.4 b||15.8 a|
|September||33.8 a||20.3 a||31.0 a||14.9 a|
| Plantation||March||62.0 a||16.9 a||10.7 a||10.3 a|
|May||71.9 a||10.9 a||9.7 a||7.5 a|
|July||49.4 a||17.5 a||17.3 a||15.8 b|
|September||50.9 a||21.3 a||13.4 a||14.4 ab|
|December||42.5 a||26.1 a||12.0 a||19.4 b|
The long dry period over the summer months had a profound effect on the water relations and gas exchange of all E. astringens trees measured in this study (Fig. 2). Trees at both sites had very negative pre-dawn leaf water potentials in the summer months and high leaf water potentials in winter (Fig. 2a), and this trend was mirrored in our gas exchange data in that photosynthesis and stomatal conductance were lowest at the height of summer and highest in winter (Fig. 2b,c). Plantation trees were considerably more stressed in summer compared to woodland trees, as evidenced by the very negative pre-dawn leaf water potentials (down to −6 MPa), very low CO2 assimilation rates (<2 µmol m−2 s−1) and extremely low stomatal conductance (<10 mmol m−2 s−1) of the plantation trees in February and April 2002. In contrast, the woodland trees had less negative pre-dawn water potentials in summer (down to −4 MPa) as well as appreciable CO2 assimilation (5–10 µmol m−2 s−1) and at least fivefold greater stomatal conductance (50–70 mmol m−2 s−1). In winter, trees at both sites recovered from drought stress and had similar water potentials (−1 to −2 MPa), CO2 assimilation rates (12–23 µmol m−2 s−1) and stomatal conductance (120–240 mmol m−2 s−1).
Figure 2. Pre-dawn leaf water potential (a), CO2 assimilation (b) and stomatal conductance (c) in Eucalyptus astringens grown in a native woodland (closed symbols) and a plantation (open symbols) in Dryandra, Western Australia. Error bars are standard deviation of n = 6 samples; different letters indicate statistically significant differences between different sampling events at one location, analysis of variance (anova), Tukey honestly significantly different (HSD) P < 0.01.
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The distinct seasonal cycle observed in leaf water relations and gas exchange was not generally reflected in the osmolality of tree tissues (Fig. 3). In woodland trees, the concentration of osmotically active substances in leaves, stems and roots did not change during the course of the year when calculated on a dry matter basis. However, when calculated on a plant water basis, the concentration of total osmolytes in woodland trees tended to be greater in the summer months compared to the winter months. The seasonal differences in osmolyte concentrations in the woodland trees indicated there was a change in tissue water content during the year, with lower tissue water content in the summer months resulting in greater tissue osmolality. In plantation trees, on the other hand, concentrations of osmotically active substances did not vary seasonally and showed the same pattern on a DW or plant water basis, except in leaves, which had significantly greater concentrations of osmotically active substances in summer compared to winter.
Figure 3. Osmotically active substances in different tree tissues of Eucalyptus astringens trees grown in a native woodland (left-hand panels) and a plantation (right-hand panels) in Dryandra, Western Australia. Concentration of osmotically active substances in leaves (open circles), branches (closed triangles), stems (closed squares) and structural roots (grey diamonds) is expressed in mmol kg−1 dry weight (upper panels) or in mmol L−1 plant water (lower panels). Error bars are standard deviation of n = 5 trees. Different letters in the panels indicate statistically significant differences between different sampling events in L = leaves, S = stems, B = branches and R = roots [analysis of variance (anova), Tukey honestly significantly different (HSD), P < 0.01].
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Substantial quercitol concentrations were measured in all plant tissues, with leaves showing the greatest concentrations (between 150 and 270 mmol kg−1 DW), and branches, stems and roots containing around 70 mmol kg−1 DW (Fig. 4). On average, quercitol contributed about 15% to the total osmotically active substances in leaves at both sites. Quercitol concentrations did not change substantially between seasons in tissues of woodland or plantation trees, except in the leaves of plantation trees, where the summer concentration of quercitol (270 mmol kg−1 DW) was double that in winter (140 mmol kg−1 DW). For these leaves, the contribution of quercitol to osmotic adjustment in response to drought was approximately 25% (calculated from the difference in the foliar concentration of total osmolytes between winter and summer).
Figure 4. Quercitol concentration (upper panels), sum of soluble carbohydrate concentration (middle panels) and starch concentration (lower panels) in different tree tissues of Eucalyptus astringens trees grown in a native woodland (left-hand panels) and a plantation (right-hand panels) in Dryandra, Western Australia. Concentration of quercitol; carbohydrates (glucose, fructose, sucrose); and starch in leaves (open circles), branches (closed triangles), stems (closed squares) and structural roots (grey diamonds). Error bars are standard deviation of n = 5 trees; different letters indicate statistically significant differences between different sampling events in leaves (abc) and roots (xyz) [analysis of variance (anova), Tukey honestly significantly different (HSD), P < 0.01]; statistically significant differences of concentrations in the other organs are not displayed.
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Woodland trees and plantation trees showed marked differences in the distribution of the total amount of quercitol per tree (Table 1). Woodland trees stored around one-third of total quercitol in their leaves and one-third in the stem, while the remaining third was equally divided between branches and roots. In contrast, plantation trees stored half or more of their quercitol in their leaves, while the remaining half was roughly equally divided between the stem, branches and roots. There was no significant change in quercitol allocation among plant tissues during the year in trees at either site.
In the neutral fraction of solutes, the concentration of total soluble carbohydrates was much less than the concentration of quercitol in trees from both sites (Fig. 4). Total soluble carbohydrates did vary according to the time of measurement, but there was no clear seasonality or relationship with water deficit or gas exchange parameters (Fig. 2). Starch concentrations, on the other hand, showed a clear seasonality in trees at both sites. Starch concentrations in all tissues were low in the summer months, increased in autumn to a maximum in winter and decreased again in spring to return to a minimum in summer (Fig. 4). This pattern was most pronounced in the roots and leaves, but also evident in other tree tissues. It was noticeable that starch reserves in plantation trees were almost completely depleted in February 2002, when these trees were most drought stressed.