Interpretation of biphasic kinetics
Nine of the 10 species studied exhibited biphasic leaf uptake kinetics, suggesting that this is a widespread phenomenon among vascular plants (Fig. 4). More detailed studies on a subset of these species (Figs 6 & 7) support the hypothesis that this reflects underlying hydraulic compartmentalization, consistent with earlier studies using stable isotopes demonstrating that leaves are not well-mixed pools (Yakir, DeNiro & Rundel 1989; Yakir, DeNiro & Gat 1990; Wang & Yakir 1995). Our findings agree with pioneering studies by Tyree and colleagues in terms of the existence of complex (bi- or multiphasic) rehydration kinetics (Cruiziat et al. 1980; Tyree et al. 1981), but differ in the values of the reported time constants. Comparisons between our data and the earlier works are complicated by the fact that in these earlier studies, leaves were measured in a pressure bomb prior to their uptake rates being recorded. Pressurization results in marked decreases in the subsequent ability of leaves to absorb water (Brodribb & Holbrook 2003) and likely contributed to the differences in uptake kinetics reported in earlier studies and those presented here.
The uptake of water by partially dehydrated leaves occurs along a pathway that includes both the vasculature of the petiole and leaf veins, as well as the living tissues separating the xylem from each rehydrating cell. Because the actual amount of water uptake attributed to the fast phase is too large to be accounted for by refilling of embolized conduits, differences in the uptake kinetics between gymnosperms and angiosperms (Fig. 4) are likely to reflect structural aspects of the xylem that influence its hydraulic conductivity (Pittermann et al. 2005; Hacke et al. 2006). Careful comparative work demonstrates that estimates of leaf hydraulic conductance (Kleaf, mmol m−2 s−1 MPa−1) calculated from the fast uptake phase agree well with estimates of Kleaf determined using other approaches (Rockwell, unpublished data). This suggests that the fast phase corresponds with the pathway used by the transpiration stream. Estimates of Kleaf calculated from the data in this paper range from 0.17 to 10.6 [mmol(H2O) MPa−1 m−2 s−1], in good agreement with values reported for temperate angiosperm and gymnosperm leaves (Brodribb et al. 2005; Sack & Holbrook 2006).
The tentative association of the fast phase or compartment with the pathway involved in the transpiration stream highlights the question of the identity and underlying mechanisms responsible for the existence of the slow rehydration phase. The fact that a substantial component of the leaf hydraulic system is significantly decoupled from a faster phase suggests that an entirely new way of thinking about the internal hydraulic design of leaves is needed (Boyer 1985). In particular, the marked difference in time constants between the two phases raises the possibility that leaves may function internally as low pass filters, effectively buffering (protecting) critical tissues from transient changes imposed by variation in water loss rates.
Mechanisms of hydraulic compartmentalization
The idea that cells in close proximity might rehydrate at substantially different rates raises the question of ‘what aspects of leaf structure and/or physiology might allow such compartmentalization?’ The extent to which cells are hydraulically coupled will depend in part upon their physical contact. Thus, some degree of hydraulic compartmentalization could be achieved solely on the basis of the internal architecture of leaves. The fact that Larix laricina had the longest time constant for the slow compartment may, in part, reflect the relatively small degree of physical contact between the stele and the mesophyll (Fig. 8). It is unlikely, however, that geometry can account for the full range of hydraulic patterns exhibited here. Thus, differences in the hydraulic conductance governing the movement of water between cells and different tissue types are also likely to play an important role.
Figure 8. Typical cross section of each analysed species used to calculate relative distribution of volumes between different leaf tissue types. Sections are 10 µm thick and stained with alcian blue and safranin-O. Scale bars are 200 µm.
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A number of mechanisms that might contribute to hydraulic compartmentalization in leaves are known. For example, variation in the number and/or activity of plasmodesmata (Sowinski, Rudzinska-Langwald & Kobus 2003) or plasma membrane aquaporins (Chrispeels & Maurel 1994; Chrispeels et al. 2001), may underlie differences in the hydraulic linkages between tissues. The fact that the symplasm plays an important role in the post-xylary movement of water through leaves (Canny 1993, 1995; Sack, Streeter & Holbrook 2004) is consistent with the potential contribution of membrane level processes to leaf hydraulic design. Further studies characterizing the hydraulic linkages within leaves, as well as the contribution of both aquaporins and plasmodesmata, are needed to advance our understanding of the internal hydraulic properties of leaves.
Relation between uptake kinetics and leaf anatomy
There were large differences between species in the relative contributions of each tissue type to overall leaf volume (Figs 8 & 9). We explored the idea that leaves consist of spatially distinct hydraulic pools by comparing the relative volumes of water absorbed during the two phases with the relative volumes of different tissue types. This analysis assumes that the relative water deficit is the same for all tissues, such that the amount of water absorbed during rehydration scales linearly with their relative volumes. In the case of the gymnosperm species examined, the relative size of the fast phase compares well with the total volume of the veins and bundle sheath extension. In all but two of the angiosperm species examined, the relative size of the fast phase best matched the relative volume of veins, bundle sheath extensions, both lower and upper epidermides, and spongy mesophyll. For the two angiosperm species that lack a spongy mesophyll layer (Phyllostachys aureosulcata and E. globulus), the relative volume of the fast phase was so large that it had to be assigned to nearly the entire leaf. In contrast, the relative size of the slow phase corresponds to the relative size of the epidermis and mesophyll tissues in gymnosperms, and with only the palisade mesophyll in four of the six angiosperm species (i.e. excluding Phyllostachys and Eucalyptus).
Figure 9. Relative contribution of each tissue type for each species. Thick lines with vertical error bars indicate the per cent water corresponding to the fast phase (below the line) and the per cent water corresponding to the slow phase (above the line). Error bars denotes SE of the mean.
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Significance of hydraulic compartmentalization
Leaf hydraulic design is likely to play a significant role in leaf photosynthetic performance, in part because species with high photosynthetic rates typically operate with small hydraulic margins. For example, the volume of water passing through a transpiring Quercus rubra leaf in 30 min can exceed the total amount of water in the leaf when it is fully hydrated. Stated another way, a rapidly transpiring Q. rubra leaf will use up its entire water content above the turgor loss point in only 75 s (Zwieniecki, Boyce & Holbrook 2004). The extent to which this substantial and potentially time-varying flux of water through the leaf might affect the performance of the photosynthetic cells depends both on the efficiency with which the xylem is hydraulically coupled to the epidermis, as well as on the degree to which the mesophyll is isolated from the transpiration stream. Although further measurements are needed to quantify the degree of hydraulic isolation between specific tissues, we explore here the potential consequences of such compartmentalization.
We focus on two aspects of leaf hydraulic design: the hydraulic linkage between the xylem and epidermis, and the degree to which the photosynthetic tissues are hydraulically uncoupled from the transpiration stream. The former has implications for stomatal control of xylem tensions, while the latter bears on the effects of transient imbalances in supply and demand on the water status of the mesophyll. Three scenarios for the connection between xylem, epidermal layers and photosynthetic cells are presented in Fig. 10. In the first scenario, the vein is hydraulically separated from the rest of the leaf; the second presents a mixed design in which the epidermis is hydraulically linked to the veins, but the mesophyll remains separated; while the third scenario describes leaves in which all tissues are equally well coupled. Each of the hydraulic designs proposed here has distinct physiological implications and leads to predictions for leaf behaviour in response to water management (Pickard 1982; Canny 1993, 1995; Zwieniecki et al. 2002; Sack et al. 2003).
Figure 10. Schematic of three scenarios for leaf hydraulic design describing the hydraulic linkages between different tissues. Solid lines depict water flow, dashed lines describe diffusion of water vapour, and Ø denotes high resistance between tissue types.
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The 10 species examined here support the existence of all three designs. Based on the relative volumes of the two phases, we propose that the four gymnosperm species examined have a relatively weak hydraulic connection between the vein and the rest of the leaf, corresponding to the first scenario. In these species, we suggest that the fast phase is limited to the vein (including the transfusion tissue) and bundle sheath extension (if present) and that the slow phase includes the mesophyll and epidermis. This hypothesis is supported by the lack of specialized non-photosynthetic tissue connecting the vein to the epidermis in Larix laricina and M. glyptostroboides. In the other two gymnosperms, Ginkgo biloba and Gnetum gnemon, anatomical separation of the vein from the mesophyll and epidermis is not obvious, although in both cases there is an endodermal-like cell layer surrounding the vein that might provide the hydraulic separation of vein from the rest of the leaf (Esau 1977; Fahn 1990).
What are the physiological consequences of design 1? The most obvious prediction is that in transpiring leaves, the water potential of the epidermis, and thus the stomata, will be significantly lower than that of the xylem. One consequence of this is that stomata could be forced to shut before the xylem experiences a significant drop in pressure. Stomatal closure that significantly preceded the threshold for cavitation has been reported in ferns (Brodribb & Holbrook 2004). This pattern of early stomatal closure has been described as providing leaves with a ‘safety margin’, but might simply reflect the presence of a significant hydraulic discontinuity between xylem and epidermis. Thus, leaves in which the epidermis is hydraulically disconnected from the xylem are not efficient in utilizing their hydraulic system to maximize photosynthetic activity. In addition, under fluctuating atmospheric conditions, their stomata may be forced to limit gas exchange despite abundant soil water availability.
In design 2, we propose that the epidermis and bundle sheath extensions form part of the fast phase, while the palisade and possibly some of the spongy mesophyll belong to the slow phase. Based on the relative volumes of the fast and slow phases, we suggest that this design corresponds to four of the six angiosperm species examined (i.e. excluding Eucalyptus and Phyllostachys). In this design, the easiest route for the water is to flow out of the vein, through the well-connected cells of the bundle sheath extension, to the epidermis where water evaporates from the internal surfaces of stomata or nearby cells. This results in the mesophyll being largely bypassed by the transpiration stream. Whether such hydraulic separation of the mesophyll could result solely from leaf geometry, or requires some degree of restricted water movement between specific cell types (e.g. between the adaxial epidermis and palisade parenchyma cells) is not known.
The physiological consequence of design 2 derives from the epidermis and stomata being hydraulically well linked to the xylem. This allows the water potential of the epidermis to closely follow that of the xylem and thus the threshold for stomatal closure can be much closer to the cavitation threshold than in design 1. Consistent with this, a small ‘safety margin’ between xylem cavitation and stomatal closure has been observed in a number of angiosperm species (Brodribb & Holbrook 2004). Hydraulic separation of the mesophyll from the transpiration stream allows these cells to be buffered from rapid changes in the transpiration rate. One could argue that disconnecting mesophyll cells from the transpiration stream reduces the capacity of the leaf to deal with sudden increases in water loss rates given that the mesophyll cells themselves could act as water capacitors. However, the amount of water in these cells could support transpiration for no more than a few tens of the seconds before their turgor loss point is reached. Moreover, minimizing short-term changes in the water status of mesophyll cells might be advantageous given their photosynthetic role.
In design 3, all major tissue types belong to the fast phase. Based on results from the rehydration kinetics, we believe that such a design is highly probable for E. globulus and Phyllostachys aureosulcata where only a small per cent of the total leaf volume corresponds to the slow phase. This suggests that both the mesophyll and epidermis in these two species are well linked hydraulically to the vasculature. This interpretation is consistent with the very high density (packing) of the mesophyll, the presence of stomata on both surfaces, and the lack of morphological separation between spongy and palisade mesophyll. From a physiological perspective, design 3 has similar properties as design 2, in that leaves would be expected to operate near the cavitation threshold. However, the mesophyll would not be protected from sudden changes in transpiration and thus the photosynthetic cells could be exposed to higher temporal variation in water potential.
The three hydraulic design scenarios discussed here result from a consideration of rehydration kinetics in conjunction with anatomical studies. Although assignment of the two phases to spatially distinct tissues within the leaf remains a hypothesis, our goal is to stimulate discussion concerning the hydraulic designs of leaves and to motivate research that will provide a better understanding of both the biological and physical principles governing water flow in leaves. We have shown that rehydration kinetics provides a powerful tool for describing the movement of water within leaves at negative water potentials. The data presented here represent a significant step in our understanding of leaf hydraulic design. In essence, they motivate a conceptual shift from the idea that leaves function hydraulically as a single pool of water that simply evaporates from the internal surfaces of mesophyll and/or epidermis, to one in which leaves are understood to consist of well-organized water pools separated by hydraulic resistance of sufficient magnitude to maintain water potential disequilibria for sufficient time to have a significant impact on leaf performance.