Osmoregulation in leaves
Our data reveal that different compounds contribute to overall changes in concentrations of osmotically active substances among these eucalypt species. Significant concentrations (up to 150 mmol L−1) of quercitol were found in leaf water of E. polyanthemos, E. tricarpa, E. cladocalyx, E. astringens, E. viridis and E. oleosa (all species considered as ‘xeric’; Table 1). In contrast, most species regarded as more ‘mesic’– namely E. maculata, E. macrorhyncha, E. camaldulensis, E. ovata, E. globulus and E. rubida– contained no quercitol under WD conditions; however, they showed equally significant concentrations of sucrose (up to 200 mmol L−1) in leaf water.
In addition to the contribution of quercitol and sucrose to osmotic potential, the contrasting biochemistry of these compounds (and their accumulation) has important consequences for their function in plant tissues. While ‘osmotic adjustment’ (defined as the accumulation of solutes under WD; Turner 1986) can be achieved in both cases, sucrose is a vital primary metabolite, readily metabolized, and therefore not a stable osmoticum. In E. globulus, it was shown that during periods of drought stress, concentrations of sucrose may rapidly increase (Quick et al. 1992) presumably because of: (1) increased activity of sucrose–phosphate synthase (Quick et al. 1992) at the expense of starch biosynthesis; (2) decreased rate of export from the cell either by compartmentalization away from transport loading sites and/or a consequence of inhibition of phloem transport (Quick et al. 1992); or (3) starch hydrolysis. Importantly, sucrose can be metabolically recycled to facilitate growth during periods of relief from stress. Such characteristics may be advantageous under the temporary nature of osmotic stress experienced by trees from high-rainfall areas.
In contrast to sucrose, quercitol and other cyclitols are highly stable and metabolically inactive because of the absence of reactive aldehyde or ketone groups. We hypothesize that for low-rainfall Eucalyptus species, the continuation of physiological activity during prolonged dry conditions requires the presence of such stable osmotica. The stability of cyclitols – such as quercitol – improves their function as an osmolyte as they do not undergo short-term fluctuations (Paul & Cockburn 1989).
This pattern supports previous data (Adams et al. 2005; Merchant et al. 2006) that Eucalyptus species from contrasting environments may adopt contrasting responses to arid conditions. White et al. (2000) recently suggested that inherently low osmotic potentials may confer advantages in low-rainfall environments. A review of previous investigations reveals that inherently lower water potentials are commonly observed in eucalyptuses growing in low-rainfall environments, including Eucalyptus melliodora, Eucalyptus microcarpa (Clayton-Greene 1983), E. polyanthemos, Eucalyptus behriana, Eucalyptus macrocarpa (Myers & Neales 1986), Eucalyptus microtheca (Tuomela 1997) and Eucalyptus leucoxylon, (White et al. 2000). Our data suggest that such osmotic adaptations are specifically related to changes in quercitol and sucrose concentrations. Our tentative conclusion is that we have identified a solute (quercitol) responsible for significant species and edaphic condition-dependent variation in osmotic potential in eucalyptuses.
Subcellular compartmentalization of sucrose and quercitol further enhances osmolytic significance (see Paul & Cockburn 1989; Popp et al. 1997). Quantification of cytoplasmic osmotic potential during WD is difficult given the current inability to determine the extent of subcellular compartmentalization of solutes. To date, only Paul et al. (1989) has presented evidence of subcellular compartmentalization of cyclitols. He showed accumulations of up to 230 mol m−3 in the chloroplast and about 100 mol m−3 in the cytosol of the herbaceous halophyte Mesembryanthemum crystallinum. Concentrations of this magnitude make a highly significant contribution to osmoregulation (Popp et al. 1997). For trees more generally, including those investigated here, cyclitols commonly reach over 10 times these concentrations (Popp et al. 1997).
Measurement of total cellular osmolality (osmoles of solute per kilogram of solvent) is an approximation of the sum of individual solute concentrations. Nevertheless, significant changes in solute concentrations are reflected in cellular osmolality. We showed that in all but three species (E. globulus and the two most xeric species, E. viridis and E. oleosa), the pool of osmotically active substances in leaves increased in response to WD, as might be expected from previous studies on osmotic relations (see Morgan 1980; Turner & Jones 1980; Chaves et al. 2003). Increases in tissue concentrations of osmotically active substances can be attributed to either an accumulation of solutes and/or decreased water content. It is noteworthy that water deficit led to significantly decreased RWC in all but three species –E. maculata, and the two lowest rainfall species, E. oleosa and E. viridis. The lack of significant increases in cellular osmolality in E. oleosa and E. viridis are thus largely explained by the maintenance of high RWC. For these two species, changes in osmotic potential may arise as a result of changes in cell wall elasticity as drought-tolerant species tend to have lower bulk modulus of elasticity (BEM) (Clayton-Greene 1983) such as those found in Eucalyptus platypus (White et al. 2000). Mallees – a particular growth form of Eucalyptus– are generally regarded as particularly adapted to dry conditions. The two mallee species included in this experiment (E. viridis and E. oleosa) originate from low-rainfall environments (Table 1) and display structural mechanisms such as thick cuticles, high leaf surface to volume ratios and sunken stomata, congruent with water conservation.
In E. globulus, the absence of changes in cellular osmolality was surprising given that: (1) it is native to high-rainfall and cool environments; and (2) reductions in osmotic potential have been reported in previous studies (White, Beadle & Worledge 1996; Pita & Pardos 2001). Previous studies have also detected significant decreases in transpiration and stomatal conductance in E. globulus under drought conditions (Quick et al. 1992; Pita & Pardos 2001; Pita, Gasco & Pardos 2003), a response that may be common to related species such as Eucalyptus grandis (Whitehead & Beadle 2004). In our study, stomatal sensitivity to stress seems the likely explanation of the ability of E. globulus to maintain a relatively high RWC.
In every water deficit experiment, the imposition of a consistent level of physiological drought across species that differ in structure and physiology, is a difficult, if not impossible task. Species-specific variation in growth habits and water use frequently serves to confound experimental designs. For example, while ψpdwn is often used as a surrogate measure of physiological stress, there are numerous associated difficulties (Flexas & Medrano 2002). In isohydric species (such as grapevine), leaf water potential may remain high despite severe stress (Flexas & Medrano 2002) while in our experiment with 13 eucalyptuses, ψpdwn dropped significantly by between 2 and 4 MPa in all species. Reductions in ψpdwn of this magnitude encompass the range of osmotic pressures experienced by a number of Eucalyptus species under field conditions (eg, White et al. 1996, 2000). The applied treatments generally reduced (but did not stop) growth – a response often associated with the need to maintain turgor through a diversion of carbon to non-growth processes (Chaves et al. 2003). Hence, this experiment allowed study of acclimation processes critical to survival and growth under water-limited conditions.
Osmoregulation in roots
Increasing concentrations of osmotically active substances in response to water deficit is perhaps of even greater significance in roots, as it is a major facilitation mechanism for water uptake from dry(ing) soils (Chaves et al. 2003). The present study showed increases in concentrations of low-molecular-weight metabolites in root tissues of Eucalyptus species subjected to WD. The accumulation of quercitol and mannitol accounted for approximately 10–20% of observed solute accumulation (on a DW basis) in root tissues of xeric species. Previous detections of reductions in osmotic potential in root tissues of Pinus radiata (Zou, Sands & Sun 2000), Pinus pinaster (Nguyen & Lamant 1989), Pinus banksiana, Picea glauca (Koppenaal, Tschaplinski & Colombo 1991) and Prunus avium × pseudacerasus and Prunus cerasus (Ranney, Bassuk & Whitlow 1991) indicate that such a response mechanism may be common to a number of tree species.
The only comparative data for eucalyptuses are those of Morabito et al. (1996), who demonstrated increased K+ concentrations in root tissues of E. microtheca during osmotic stress. In that study, the influence of the rooting medium and potential confounding by nutrient status precluded clear conclusions. We note that expression of solute concentrations on a DW basis limits our ability to determine the overall contributions to cellular osmolarity. Nevertheless, quercitol represents approximately 10% of the DW of osmotically active substances in root tissues of low-rainfall species and quercitol and mannitol together accumulate in response to water deficit. These solutes therefore play significant roles in the amelioration of the effects of low external water potentials.
Potential role in chemotaxonomy
Interestingly, seedlings of all species in the present study contained at least some quercitol in leaf or root tissues at the beginning of the experiment. The major distinction was that ‘xeric’ species from low-rainfall environments maintained high quercitol concentrations during growth under drought stress. Assuming that quercitol accumulation is beneficial, it is the retention of quercitol in leaf tissues that provides a link with aridity tolerance. Quercitol has been isolated previously in drought/salt-tolerant Eucalyptus species (Adams et al. 2005), and initial results of a genus-wide screening of the occurrence of cyclitols among Eucalyptus species corroborates a link to drought adaptation within the genus (Merchant et al. 2006). The presence of quercitol in tissues of all 13 Eucalyptus species selected in this study, even those from mesic environments, indicates that while the physiological and genetic capacity to synthesize this compound is widely present in eucalyptuses, it is expressed differently in species from differing habitats.
Several authors have attempted to delineate Eucalyptus species based on initial growth rates (Davidson & Reid 1980; Duff, Reid & Jackson 1983), foliar nutrient concentrations (Lambert & Turner 1983), volatile leaf oils (Li, Madden & Potts 1995, 1996) and respiratory metabolism (Anekonda et al. 1999) and combinations of these parameters (Noble 1989). It is likely, however, that the geographical distribution of Eucalyptus species is dependent on a variety of these factors, but particularly related to the availability of water (Adams 1996). Our results suggest the inclusion of an additional taxonomic component of the responses of quercitol concentrations to WD.
In conclusion, we can answer the questions addressed in the present study as follows: (1) cyclitols respond to drought exposure, but responses are different in different groups of Eucalyptus species. In ‘xeric’ species, tissue concentrations of quercitol on a leaf water basis increased upon exposure to WD, and contributed to an overall increase in leaf osmolality. On a leaf DW basis, quercitol concentration in leaves decreased during the growth of WW plants and WD-treated plants of ‘mesic’ species, whereas it was maintained in water deficit treated ‘xeric’ species; (2) cyclitols (mainly quercitol) are present and accumulate in both leaves and roots of these ‘xeric’ species; and (3) seedlings of all 13 Eucalyptus species investigated contained at least low amounts of cyclitols. The constitutive presence and significant concentration of quercitol in eucalyptus leaf and root tissues demonstrated here point towards a biochemical link between taxonomy, physiology and acclimation to aridity.