Hydraulic design of leaves: insights from rehydration kinetics · resistance of a single pathway for rehydration versus the existence of distinct pools, compare the spatial patterns

Hydraulic design of leaves: insights fromrehydration kinetics

MACIEJ A. ZWIENIECKI1, TIMOTHY J. BRODRIBB3 & N. MICHELE HOLBROOK2

1Arnold Arboretum, 2Organismic and Evolutionary Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138,USA and 3School of Plant Science, University of Tasmania, Hobart Campus, Life Sciences Building, 111, Hobart, Australia

ABSTRACT

We examined the leaf hydraulic design in 10 species based ontheir rehydration kinetics. In all cases, a biphasic responsedescribed the temporal pattern of water uptake, with timeconstants of ~30 to 800 s and ~800 to 8000 s. The time con-stants of the fast phase were significantly shorter in the sixangiosperms (30 to 110 s) compared with the two single-veined conifer species (>400 s) examined, while the twomulti-veined gymnosperm species, Gnetum gnemon andGinkgo biloba, had time constants for the fast phase of~150 s. Among angiosperm species, the fast phase consti-tuted 50–90% of the total water absorbed, whereas in gym-nosperms 70–90% of the water uptake could be assigned tothe slow phase. In the four gymnosperms, the relative wateruptake corresponding to the fast phase matched to a gooddegree the relative volume of the venation and bundlesheath extension; whereas in the angiosperm species, therelatively larger water influx during the fast phase wassimilar in relative volume to the combined venation, bundlesheath extension, epidermis and (in four species) the spongymesophyll. This suggests a general trend from a design inwhich the epidermis is weakly connected to the veins (allfour gymnosperms), to a design with good hydraulic connec-tion between epidermis and veins that largely bypasses themesophyll (four of six angiosperms), to a design in whichalmost the entire leaf appears to function as a single pool.

Key-words: venation pattern; bundle sheath extension; tran-spiration stream; hydraulic compartmentalization.

INTRODUCTION

Leaves of terrestrial plants are topologically complex struc-tures charged with the task of creating an internal envi-ronment that permits a positive return on the plant’s invest-ments in photosynthetic tissues. Optimization of photosyn-thetic activity requires that leaves be thin, to facilitate lightpenetration, and porous, to allow CO2 diffusion within theleaf, making leaf structure at odds with the simultaneousdemand for water conservation. The inevitability of evapo-ration during CO2 uptake places tremendous pressure on

leaf hydraulic design as seen in the good correlation betweenleaf hydraulic conductance and assimilation rates (Brodribbet al. 2005). However, in addition to being able to supportsteady-state levels of transpiration, leaves must also copewith transient changes in water loss rates. The driving forcefor evaporative loss can change rapidly because of radiativeeffects on leaf temperature (Singsaas et al. 1999), as well asvariation in the thickness of the boundary layer as a result ofturbulent mixing of the surrounding air (Schuepp 1993).Although the valve-like action of guard cells provides leaveswith the ability to control rates of water loss to the environ-ment, stomatal closure involves the net movement of waterout of cells and thus proceeds at a finite rate (Buckley, Mott& Farquhar 2003). Differences in the timescales that charac-terize short-term variation in water loss rates and the corre-sponding response in stomatal aperture mean that leaveswill frequently experience imbalances in supply anddemand.In this paper,we explore how the internal hydraulicdesign of leaves contributes to their ability to maintainphysiological function in the face of rapid fluctuations intranspiration rate.

Transient imbalances between hydraulic supply anddemand can negatively impact leaf function in two ways.Loss of xylem conductivity due to cavitation can forcestomata to close leading to a decrease in photosyntheticcarbon gain, while desiccation can impair the metabolicactivities of living cells.Although a direct effect of leaf watercontent (or leaf water potential) on photosynthetic capacityremains a matter of debate, declines in water content belowthe turgor loss point have the potential to impair physiologi-cal processes (Matthews & Boyer 1984; Berkowitz & Kroll1988; Tang et al. 2002; Tezara et al. 2002). The challenge forleaf hydraulic design is that ameliorating one risk comes atthe price of accentuating the other. For example, the hydrau-lic capacitance of leaf cells means that the mesophyll can actto buffer the propagation of transient decreases in apoplasticpressure, thus protecting the xylem – but only by exposingthese living cells to significant changes in water content.Hydraulically separating the photosynthetic tissues from thetranspiration stream will minimize the degree to whichshort-term variation in transpiration rate affects the meso-phyll, but will speed up the rate at which fluctuations inleaf water potential are propagated to the xylem. Despiteincreasing interest in the movement of water through leaves(Tyree & Cheung 1977; Canny 1993, 1995; Fricke 2000; Sack

Correspondence: M. A. Zwieniecki. Fax: 617 496 5458; e-mail:[email protected]

Plant, Cell and Environment (2007) 30, 910–921 doi: 10.1111/j.1365-3040.2007.001681.x

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd910

mailto:[email protected]

et al. 2003; Nardini, Gortan & Salleo 2005; Sack, Tyree &Holbrook 2005), our understanding of leaf micro-hydrologyremains limited.Here,we use rehydration kinetics to explorethe hydraulic design of leaves, focusing on the degree towhich leaves function hydraulically as a single pool versusthe possibility that they exhibit substantial hydraulic com-partmentalization.

Leaf rehydration has long been used to study the move-ment of water in leaves (Weatherley 1963; Milburn 1966;Boyer 1974, 1977, 1985). Analysis of rehydration kineticssuggests that leaves should not be treated as uniformcapacitors because the rehydration of partially dried leavescould not be described using a single exponential (Cruiziatet al. 1980; Tyree et al. 1981). Although the idea that anumber of parallel pathways with different resistancesand/or spatially distinct water pools must exist to accountfor the observed kinetics was proposed (Tyree et al. 1981),further discussion of the physiological implications of thesepatterns has been limited. Leaves are composed of manycell types (e.g. epidermal cells, spongy and palisade meso-phyll, vascular parenchyma) which may differ in their indi-vidual hydraulic properties as well as the degree to whichthey are hydraulically connected to neighbouring cells.Thus, it is not surprising that leaves do not behave hydrau-lically as a single, well-mixed pool. What is unexpected,however, is that the rehydration of these diverse cell types,located within spatially defined regions of the leaf, produceskinetics that can be described by a small number of expo-nential processes.

The diversity of extant leaf structures and morphologiesprovides a powerful tool for the comparative analysis ofrehydration kinetics. Beginning with the simple designsoffered by coniferous leaves characterized by a single veinthrough to the reticulate, hierarchical venation of mostangiosperms, we can use variation in leaf anatomy and vas-cular supply to help pinpoint structural features associatedwith the principal characteristics of leaf hydraulic design.We can also examine the role of hydraulic inventions andmodifications in leaf anatomy such as the presence ofvessels, vein reticulation or bundle sheath extensions. In thispaper, we present data on the rehydration of leaves from 10species (six angiosperms and four gymnosperms), examine

the rehydration of leaves dried to various water potentialsto test whether the observed patterns reflect a change in theresistance of a single pathway for rehydration versus theexistence of distinct pools, compare the spatial patterns ofwater loss from leaves subject to rapid over-pressurizationusing a pressure chamber to test the idea that leaves exhibitsubstantial hydraulic compartmentalization, and use therelative sizes of the two phases in relation to the relativevolumes of the various tissue within each leaf to inform ourdiscussion of leaf hydraulic design.

METHODS

Plant material

A diverse group of 10 gymnosperm and angiosperm speciesrepresenting a range of venation types was studied(Table 1). In almost all cases, leaves were collected fromindividuals growing in either the greenhouse or experimen-tal garden on the campus of Harvard University, Cam-bridge, MA, USA. Leaves of Eucalyptus globulus werecollected from trees growing on the California Institute ofTechnology campus (Pasadena, CA, USA), shipped over-night in double plastic bags filled with moist paper towels,and measured the next day. All plants sampled appearedhealthy, without any symptoms of viral or bacterial infec-tion, insect activities or water stress.

Leaf rehydration

Rehydration kinetics was measured using a ‘reverse Polishguillotine’ (Fig. 1) that allows a leaf to be severed underwa-ter and attached to a water-filled tube in a single motion.The name of this device derives from the fact that it is theleaf, rather than the cutting blade that moves, thus avoidingperturbation of the hydraulic connection to the analyticbalance used to measure water flow into the excised leaf.The procedure involved cutting a 1- to 2-m-long branchduring the morning hours between 0800 and 1000 h (exceptfor E. globulus which was collected at midday). Brancheswere carried to the lab in a plastic bag with moist towelto limit transpiration. Prior to beginning the rehydration

Table 1. List of species examined with the major morphological and anatomical characteristics of their leaves

Species Venation type Xylem typeBundle sheathextension Mesophyll Stomata

Gnetum gnemon Multi-veined reticulate Vessels Present Palisade and spongy HypostomatousGingko biloba Multi-veined paralleled Tracheids Present Unstructured HypostomatousMetasequoia

glyptostroboidesSingle veined Tracheids Absent Unstructured Hypostomatous

Larix laricina Single veined Tracheids Absent Unstructured HypostomatousQuercus rubra Multi-veined reticulate Vessels Present Palisade and spongy HypostomatousAcer rubrum Multi-veined reticulate Vessels Present Palisade and spongy HypostomatousLiriodendron tulipifera Multi-veined reticulate Vessels Present Palisade and spongy HypostomatousPopulus nigra Multi-veined reticulate Vessels Present Palisade and spongy HypostomatousPhyllostachys aureosulcata Multi-veined paralleled Vessels Present Palisade AmphistomatousEucalyptus globulus Multi-veined reticulate Vessels Present Palisade Amphistomatous

Hydraulic design of leaves 911

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 910–921

experiment, the water potential of the leaf was estimated bymeasuring the water potential of two leaves, one locatedabove and one below the selected leaf. The target waterpotential was between -0.6 and -1.0 MPa so as to avoidmajor losses in conductivity because of cavitation, but toprovide sufficient water deficit so that the patterns of wateruptake would be discernable. However, leaves desiccated toa greater range of initial water potentials were measured intwo species.

The petiole of the target leaf was wrapped in parafilmand secured in the guillotine holder using Blu-tack (Bostik,Pty. Ltd., Notting Hill, VIC, Australia). A layer of highvacuum silicon grease (Dow Corning, Midland, MI, USA)was added to the petiole to help form a seal with the o-ring.The entire system (target leaf + guillotine) was then sub-merged in water to prevent evaporation from leaf surfacesduring the measurement period. All species were tested forcuticular uptake by submerging dehydrated leaves in waterwith petiole in the air. In no case was leaf water potentialincreased by this treatment, precluding the possibility ofwater uptake via the leaf surface. Any cut-ends of thebranch were covered with high vacuum silicon grease toprevent water entry. After the entire setup was prepared,the guillotine was deployed such that the leaf was severedfrom the branch and simultaneously connected to a tubelinked to a water reservoir seated on an analytic balance(Sartorius �0.01 mg, Goettingen, Germany). In the caseof Metasequoia glyptostroboides and Larix laricina, we

measured branchlets instead of individual leaves and thusthe measurements include the rehydration of a small pieceof stem. Water uptake was recorded at 1 s intervals for~9000 s, followed by ~500 s with the leaf (or branchlet) dis-connected so as to account for any evaporative losses thatmight occur from either the tube or the water reservoir. Wemeasured 5 to 10 leaves (or branchlets) for each species.The temperature of the water bath was maintained at 20 °Cand leaves were illuminated with 30 mmol m-2 s-1 through-out the measurement period, a fluence rate sufficient tosaturate any light-induced changes in leaf hydraulic conduc-tance using this measurement technique (Rockwell et al.,unpublished data). After accounting for the evaporationfrom the measurement system itself, a small linear uptakewas still observed in several species. This residual uptake,which we believe was due to capillary infiltration into leafintercellular spaces, was also accounted for before furtheranalysis.

The weight–time function of water flow from the balanceprovides a continuous record of leaf rehydration. Becausethere is a limit for water absorption by the dehydrated leaf(i.e. to zero water potential), one can use an exponentialgrowth to maximum function to describe these data. Weused both a single,

f t a e b t( ) = ∗ −( )− ∗1 1 ,

and a double exponential model:

f t a e c eb t d t( ) = ∗ −( ) + ∗ −( )− ∗ − ∗1 11 1

to describe water uptake by dehydrated leaves. In bothmodels, parameters a and c describe the volume of therespective compartments, and b, d their corresponding timeconstant.

Anatomy

The relative volumes of different tissue types were deter-mined from analysis of paraffin embedded leaf cross sec-tions. Fresh leaves were vacuumed infiltrated in FAA,dehydrated and embedded in paraffin (Ruzin 1999), sec-tioned at 10 mm using a microtome, and doubled stainedusing alcian blue (Sigma-Aldrich, St. Louis, MO, USA) andsafranin-O (Sigma-Aldrich). Images from five randomlyselected sections from the central lamina of five leaves weremade at 100¥ or 200¥ magnifications for a total of 25 picturesper species. Image analysis software (ImageJ, a publicdomain, Java-based image processing program developed atthe National Institutes of Health, Bethesda, MD, USA) wasused to measure the area within each cross section of thefollowing tissue types: vein (xylem, phloem and parenchymacells surrounding the vascular bundle), bundle sheath exten-sion (if present, defined as a parenchymatous extensionconnecting the vein to the epidermis),mesophyll (separatingspongy and palisade when morphologically distinct), upperand lower epidermis (measured together),and other types ofcells if present. Gnetum gnemon leaves contain largenumbers of non-lignified fibres, which were placed into their

Figure 1. Schematic and picture of the ‘reverse Polishguillotine’ used to connect a partially dehydrated leaf to awater-filled tube such that the entire period of water uptake bythe leaf can be monitored. The ‘reverse’ action of the guillotinederives from the fact that the blade is fixed in the lower position,while the specimen to be cut slides downwards across the blade,thus allowing the water-filled tube (located immediately belowthe blade) to remain fixed in place. The latter is critical forobtaining accurate measurements of the initial rates of waterflow. The seal between the severed petiole and the water filledtube was made using an o-ring. A split block allowed the leaf tobe inserted into the guillotine while still attached to the branch.The entire system was submerged under water to avoid air frombeing drawn into the severed xylem of the petiole, as well as toprevent water loss from the leaf surface. Because the water levelon the balance was approximately 5 cm lower than the waterlevel in the basin containing the leaf, any failure of the sealresulted in a backflow of water to the balance. In the few cases inwhich this occurred (primarily with Phyllostachys aureosulcatawhich has very thin petioles), the measurements were discarded.

912 M. A. Zwieniecki et al.


own category (Tomlinson & Fisher 2005). Using these areasand assuming that the thickness of the sample was uniform inthe field of view, we calculated the % contribution of eachtissue type to the cellular volume of the leaf.

Temporal and spatial patterns of waterextraction from over-pressurized leaves

Leaves of Populus nigra were cut off under water andallowed to rehydrate for several hours while supplied with a0.01% aqueous solution of sulforhodamine (Sigma). Leaveswere placed in a pressure chamber and pressurized to2.0 MPa. Pressurization took place over 5–10 s to avoidexcessive (>35 °C) increases in leaf temperature. Exudationfrom the petiole was collected into pre-weighed Eppendorftubes (0.5 mL) at intervals of 30 s during the initial phase,and subsequently at 60 s intervals. Each Eppendorf tubewas then weighed and the expressed mass per unit timeinterval calculated.

Leaves that were frozen in liquid nitrogen (LN) wereprepared in the same way as described above, before theywere placed in the pressure chamber containing a 0.25 Lpaper cup filled with LN at the bottom of the chamber(Fig. 2). Two thin wires attached to the rim of the cup pro-truded through the rubber fitting at the top of the chamber.The petiole was enclosed in a plastic tube to avoid crushingbefore being sealed in the chamber lid. A thermocoupleplaced on the leaf was used to avoid either overheating orovercooling the leaf, as well as to later confirm that the leaf

was successfully submerged in LN. Once the leaf and all thewires were fixed in the chamber top, the chamber was sealedand a pressure of 2.0 MPa applied for 30 s, after which theLN-filled cup was pulled up until the thermocouple regis-tered that the leaf had frozen (Fig. 2b). The cup containingthe frozen leaf was then carefully removed from the pressurechamber and the leaf transferred to a LN storage dewer.During the pressurization, special attention was neededbecause pressurization of the chamber causes the tempera-ture to rise, thus increasing evaporation of LN with conse-quent effects on chamber pressure (as well as temperature).With practice, we were able to maintain leaf temperaturewithin the range of 17–28 °C, while still achieving pressur-ization of 0.1 to 2.0 MPa within ~10 s. Fully hydrated leavesthat were not pressurized were frozen as a control.

The frozen leaves were then prepared for observation ina fluorescent microscope using previously described tech-niques (Cochard et al. 2004; Brodribb & Holbrook 2005).Pictures of leaf cross sections were taken using a long-distance (7 mm) objective (magnification 50¥; Zeiss). Thepresence of sulforhodamine in the bundle sheath allowed usto analyse the shapes of vein parenchyma cells, while chlo-rophyll autofluorescence from photosynthetic mesophyllcells was sufficient to allow analysis of their shape. Both thecross-sectional surface area of cells and their perimeterwere measured using Image J software. A unitless circular-ity index, [(cell circumference)2/(cell cross-sectional area)]/4p, was used to quantify deformation. A total of 42 bundlesheath cells and 28 palisade mesophyll cells were measuredfrom three pressurized and three control leaves.

RESULTS

Rehydration kinetics

For the 10 species examined, a double exponential functionbetter fit the measured rehydration kinetics than did thesingle exponential function (Fig. 3), although the discrep-ancy between the two models varied between species. Theadjusted R2 of the single exponential model ranged from0.75 to 0.96, while for the double exponential model R2

values ranged from 0.92 to 0.99. The only species for whicha significant improvement of fit between the single anddouble exponential model was not observed was E. globu-lus, with the R2 for single and double exponential modelsbeing respectively 0.96 and 0.99.

The double exponential model describes leaf rehydrationas consisting of two phases: the fast phase has time con-stants ranging from 30 to 800 s, while the slow phase hastime constants ranging from 800 to 8000 s. Time constantsfor the fast phase were shorter (i.e. rehydration occurredmore quickly) in all of the angiosperm species examinedcompared to all of the gymnosperms (Fig. 4). The time con-stants for this phase were significantly longer in the twoconifers, than in all other species. The two multi-veinedgymnosperms (Gnetom gnemon and Ginkgo biloba) hadtime constants of the fast phase that were longer than in allof the angiosperms (>150 versus <110 s), but the difference

Wires to

pull LN

container

(a) (b)

Termocouple

Pressure

Figure 2. Diagram of apparatus used to freeze-pressurizeleaves. After pressurization for the specified time interval, aplastic, non-compressible cup filled with liquid nitrogen (LN)initially placed at the chamber bottom (a) is pulled up to the topof the chamber using fine wires inserted through the pressurechamber gasket surrounding the leaf petiole (b). A thermocoupleattached to the leaf allowed the pressurization rate to beregulated so as to avoid either overheating or cooling, as well asto confirm that the leaf had successfully been immersed in LNbefore the pressure was released.



was not significant. In contrast, there was no obvious differ-ence in the time constants of the slow phase between gym-nosperms and angiosperms. Most species had timeconstants between 3000 to 5500 s. Eucalyptus globulus hada significantly shorter slow phase time constant, consistentwith the fact that this species was equally well fit by a singleexponential model. The slow phase time constant of Larixlaricina was significantly longer than for all other species,although it included rehydration/resistance of small portionof the stem because we could not measure single leaves.

The relative volume of water absorbed during the fastand slow phases showed consistent differences between

angiosperms and gymnosperms. The dominant pool ofwater in the gymnosperms was associated with the slowphase, with 70 to 90% of the total water flowing into the leafbeing attributed to this slower pool. In contrast, in theangiosperm species the relative volume associated with theslow phase was much smaller, ranging from approximately10 to 50% of the total amount of water absorbed duringrehydration (Fig. 5).

Underlying basis for biphasic kinetics

Having established that biphasic rehydration kineticsoccurs broadly across vascular plants, an important ques-tion is to interpret these patterns in terms of the underlyingphysical and biological phenomena. One potential explana-tion is that the biphasic response reflects an increase in theresistance of a single pathway for rehydration during theperiod of water uptake because of generation of intercellu-lar pressure differentials (Fricke 2000). If this is true, thenthe relative size of the fast and slow phases should dependupon the water deficit. To test this idea, we measured leafrehydration for two species (Acer rubrum and Lirioden-dron tulipifera) across a broad range of initial leaf waterpotentials (0.2 to 1.4 MPa). Both species exhibited biphasicrehydration kinetics across the full range of initial leafwater potential (see also Tyree et al. 1981), and the relativesizes of the two phases were independent of the degree ofdesiccation experienced by the leaf prior to rehydration(Fig. 6).

This led us to explore an alternative scenario in which thebiphasic kinetics reflects the rehydration of two spatiallydistinct regions within the leaf, by examining the spatialdistribution of water loss from within leaves of Populusnigra. The analysis started with the measurement of water

Figure 3. Representative rehydration kinetic curve for an Acerrubrum leaf dehydrated to ~ -1 MPa. The experimental data(solid dashed line being the raw data from the balance) havebeen fit with both a single and double exponential rise tomaximum. Inset shows water uptake partitioned between the fastand slow phase, calculated from the double exponential model.

Figure 4. Calculated time constantsfrom two exponential rise to maximummodel of rehydration kinetics for (a) fastphase and (b) slow phase. The grey areadenotes gymnosperm species. Error barsdenote SE of the mean. Small lettersabove the bars denote significancedifferences (P < 0.05) resulting fromone-way analysis of variance. G. gnemon,Gnetum gnemon; G. biloba, Gingkobiloba; M. glyptostroboides, Metasequoiaglyptostroboides; L. laricina, Larixlaricina; Q. rubra, Quercus rubra; A.rubrum, Acer rubrum; L. tulipifera,Liriodendron tulipifera; P. nigra, Populusnigra; P. aureosulcata, Phyllostachysaureosulcata; E. globulus, Eucalyptusglobulus.

(a) (b)



efflux from hydrated leaves pressurized to 2.0 MPa using apressure chamber. There was significant efflux over the first40 s (accounting for ~40% of water forced from the leaf),followed by slower efflux extending for approximately10 min (Fig. 7a).The temporal dynamics of water efflux wasconsistent with prior observation of rehydration kinetics ofpoplar leaves (Fig. 4). The time constant of the fast com-partment was calculated to be ~30 s, a value slightly lowerthen calculated from the rehydration curves.The slow phasetime constant, however, was in the range of ~300 s – muchlower than in the case of the rehydration kinetics. Thisdifference may result from the fact that over-pressurizationforces very dry air through the petiole, accelerating evapo-ration from the collection vials. Underestimation of waterexudation due to evaporation will have a disproportionateeffect on the calculation of the time constant of theslow phase because of the increasingly smaller exudationvolumes as the leaf approaches equilibrium with theapplied pressure.

To determine the source of water of the fast phase, wefroze leaves in LN after 30 s of exposure to 2.0 MPa; as acontrol we froze fully hydrated leaves. All leaves had beenpreviously infiltrated with sulforhodamine to allow us toquantify changes in cell shape in sections of frozen tissuesusing fluorescence microscopy (Fig. 7b,c). We used a simple‘circularity index’ to describe changes in cell shape in termsof their deviation from a circular cross section: [(cellcircumference)2/(cell cross-sectional area)]/4p. A smallervalue of this parameter indicates greater deviation from

a circular shape. There was no difference in the shapeof palisade mesophyll cells measured from control andpressure-dehydrated leaves (Fig. 7d–f). However, there wasa significant decrease in the circularity index of parenchymacells surrounding minor leaf veins (Fig. 7g–j), suggestingpreferential loss of water from these cells. Cross-sectionalarea of the bundle sheath cells was smaller, but due tovariation in cell size, the difference was not significant.

DISCUSSION

Interpretation of biphasic kinetics

Nine of the 10 species studied exhibited biphasic leafuptake kinetics, suggesting that this is a widespread phe-nomenon among vascular plants (Fig. 4). More detailedstudies on a subset of these species (Figs 6 & 7) supportthe hypothesis that this reflects underlying hydraulic com-partmentalization, consistent with earlier studies usingstable isotopes demonstrating that leaves are not well-mixed pools (Yakir, DeNiro & Rundel 1989; Yakir,DeNiro & Gat 1990; Wang & Yakir 1995). Our findingsagree with pioneering studies by Tyree and colleagues interms 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 arecomplicated by the fact that in these earlier studies, leaveswere measured in a pressure bomb prior to their uptakerates being recorded. Pressurization results in markeddecreases in the subsequent ability of leaves to absorbwater (Brodribb & Holbrook 2003) and likely contributedto the differences in uptake kinetics reported in earlierstudies and those presented here.

The uptake of water by partially dehydrated leavesoccurs along a pathway that includes both the vasculatureof the petiole and leaf veins, as well as the living tissuesseparating the xylem from each rehydrating cell. Because

Fast phaseSlow phase

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Figure 5. Relative water uptake calculated from the doubleexponential rise to maximum model of rehydration kinetics forfast phase and slow phase. Error bars denote SE of the mean.

Liriodendron tulipiferaRegression (R2=0.054)Acer rubrumRegression (R2=0.001)

Figure 6. Relative volume of the fast phase (rehydrating in lessthan 100 s) for two species (Acer rubrum and Liriodendrontulipifera) dehydrated to different initial water potentials.



the actual amount of water uptake attributed to the fastphase is too large to be accounted for by refilling of embo-lized conduits, differences in the uptake kinetics betweengymnosperms and angiosperms (Fig. 4) are likely to reflectstructural aspects of the xylem that influence its hydraulicconductivity (Pittermann et al. 2005; Hacke et al. 2006).Careful comparative work demonstrates that estimatesof leaf hydraulic conductance (Kleaf, mmol m-2 s-1 MPa-1)calculated from the fast uptake phase agree well withestimates of Kleaf determined using other approaches

(Rockwell, unpublished data). This suggests that the fastphase corresponds with the pathway used by the transpira-tion stream. Estimates of Kleaf calculated from the data inthis paper range from 0.17 to 10.6 [mmol(H2O)MPa-1 m-2 s-1], in good agreement with values reported fortemperate angiosperm and gymnosperm leaves (Brodribbet al. 2005; Sack & Holbrook 2006).

The tentative association of the fast phase or compart-ment with the pathway involved in the transpiration streamhighlights the question of the identity and underlying

(a) (b)

(c)

(d) (e) (g) (h)

(j)(f)

Figure 7. (a) Cumulative water lost from leaves pressurized to 2.0 MPa. Values are scaled relative to the total leaf water content to takeinto account variation in leaf size. (b) View of frozen vein cross section from pressurized leaf (arrows indicate typical cell deformation;image is 150 mm on a side). (c) View of vein cross section of fully hydrated leaf. (d,e,f) Shape and calculated circularity index for palisademesophyll cells, while (g,h,j) show the same vein parenchyma cells. *** indicates a significant difference (P < 0.001) between hydrated(control) and pressurized leaves.



mechanisms responsible for the existence of the slow rehy-dration phase. The fact that a substantial component of theleaf hydraulic system is significantly decoupled from afaster phase suggests that an entirely new way of thinkingabout the internal hydraulic design of leaves is needed(Boyer 1985). In particular, the marked difference in timeconstants between the two phases raises the possibility thatleaves may function internally as low pass filters, effectivelybuffering (protecting) critical tissues from transient changesimposed by variation in water loss rates.

Mechanisms of hydraulic compartmentalization

The idea that cells in close proximity might rehydrate atsubstantially different rates raises the question of ‘whataspects of leaf structure and/or physiology might allowsuch compartmentalization?’ The extent to which cells arehydraulically coupled will depend in part upon their physi-cal contact. Thus, some degree of hydraulic compartmental-ization could be achieved solely on the basis of the internalarchitecture of leaves. The fact that Larix laricina had thelongest time constant for the slow compartment may, inpart, reflect the relatively small degree of physical contactbetween the stele and the mesophyll (Fig. 8). It is unlikely,however, that geometry can account for the full range ofhydraulic patterns exhibited here. Thus, differences in thehydraulic conductance governing the movement of waterbetween cells and different tissue types are also likely toplay an important role.

A number of mechanisms that might contribute tohydraulic compartmentalization in leaves are known. Forexample, variation in the number and/or activity ofplasmodesmata (Sowinski, Rudzinska-Langwald & Kobus2003) or plasma membrane aquaporins (Chrispeels &Maurel 1994; Chrispeels et al. 2001), may underlie differ-ences in the hydraulic linkages between tissues. The factthat 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 thepotential contribution of membrane level processes to leafhydraulic design. Further studies characterizing the hydrau-lic linkages within leaves, as well as the contribution of bothaquaporins and plasmodesmata, are needed to advance ourunderstanding of the internal hydraulic properties of leaves.

Relation between uptake kinetics andleaf anatomy

There were large differences between species in the relativecontributions of each tissue type to overall leaf volume(Figs 8 & 9). We explored the idea that leaves consist ofspatially distinct hydraulic pools by comparing the relativevolumes of water absorbed during the two phases with therelative volumes of different tissue types. This analysisassumes that the relative water deficit is the same for alltissues, such that the amount of water absorbed duringrehydration scales linearly with their relative volumes. In

the case of the gymnosperm species examined, the relativesize of the fast phase compares well with the total volume ofthe veins and bundle sheath extension. In all but two of theangiosperm species examined, the relative size of the fastphase best matched the relative volume of veins, bundlesheath extensions, both lower and upper epidermides, andspongy mesophyll. For the two angiosperm species that lacka spongy mesophyll layer (Phyllostachys aureosulcata andE. globulus), the relative volume of the fast phase was solarge that it had to be assigned to nearly the entire leaf. Incontrast, the relative size of the slow phase corresponds tothe relative size of the epidermis and mesophyll tissues ingymnosperms, and with only the palisade mesophyll in fourof the six angiosperm species (i.e. excluding Phyllostachysand Eucalyptus).

Significance of hydraulic compartmentalization

Leaf hydraulic design is likely to play a significant role inleaf photosynthetic performance, in part because specieswith high photosynthetic rates typically operate with smallhydraulic margins. For example, the volume of waterpassing through a transpiring Quercus rubra leaf in 30 mincan exceed the total amount of water in the leaf when it isfully hydrated. Stated another way, a rapidly transpiringQ. rubra leaf will use up its entire water content abovethe turgor loss point in only 75 s (Zwieniecki, Boyce &Holbrook 2004). The extent to which this substantial andpotentially time-varying flux of water through the leafmight affect the performance of the photosynthetic cellsdepends both on the efficiency with which the xylem ishydraulically coupled to the epidermis, as well as on thedegree to which the mesophyll is isolated from the transpi-ration stream. Although further measurements are neededto quantify the degree of hydraulic isolation between spe-cific tissues, we explore here the potential consequences ofsuch compartmentalization.

We focus on two aspects of leaf hydraulic design: thehydraulic linkage between the xylem and epidermis, and thedegree to which the photosynthetic tissues are hydraulicallyuncoupled from the transpiration stream. The former hasimplications for stomatal control of xylem tensions, whilethe latter bears on the effects of transient imbalances insupply and demand on the water status of the mesophyll.Three scenarios for the connection between xylem, epider-mal layers and photosynthetic cells are presented in Fig. 10.In the first scenario, the vein is hydraulically separated fromthe rest of the leaf; the second presents a mixed design inwhich the epidermis is hydraulically linked to the veins, butthe mesophyll remains separated; while the third scenariodescribes leaves in which all tissues are equally wellcoupled. Each of the hydraulic designs proposed here hasdistinct physiological implications and leads to predictionsfor leaf behaviour in response to water management(Pickard 1982; Canny 1993, 1995; Zwieniecki et al. 2002;Sack et al. 2003).

The 10 species examined here support the existence of allthree designs. Based on the relative volumes of the two



phases, we propose that the four gymnosperm speciesexamined have a relatively weak hydraulic connectionbetween the vein and the rest of the leaf, corresponding tothe first scenario. In these species, we suggest that the fastphase is limited to the vein (including the transfusiontissue) and bundle sheath extension (if present) and thatthe slow phase includes the mesophyll and epidermis. Thishypothesis is supported by the lack of specialized non-photosynthetic tissue connecting the vein to the epidermisin Larix laricina and M. glyptostroboides. In the other twogymnosperms, Ginkgo biloba and Gnetum gnemon, ana-tomical separation of the vein from the mesophyll and

epidermis is not obvious, although in both cases there is anendodermal-like cell layer surrounding the vein that mightprovide the hydraulic separation of vein from the rest of theleaf (Esau 1977; Fahn 1990).

What are the physiological consequences of design 1? Themost obvious prediction is that in transpiring leaves, thewater potential of the epidermis, and thus the stomata, willbe significantly lower than that of the xylem. One conse-quence of this is that stomata could be forced to shut beforethe xylem experiences a significant drop in pressure. Sto-matal closure that significantly preceded the threshold forcavitation has been reported in ferns (Brodribb & Holbrook

Figure 8. Typical cross section of eachanalysed species used to calculate relativedistribution of volumes between differentleaf tissue types. Sections are 10 mm thickand stained with alcian blue andsafranin-O. Scale bars are 200 mm.

Gnetum gnemon

Gingko biloba

Metasequoia glyptostroboides

Larix laricina

Quercus rubra

Acer rubrum

Liriodendron tulipifera

Populus nigra

Phyllostachys aureosulcata Eucalyptus gobulus



2004). This pattern of early stomatal closure has beendescribed as providing leaves with a ‘safety margin’, butmight simply reflect the presence of a significant hydraulicdiscontinuity between xylem and epidermis. Thus, leaves inwhich the epidermis is hydraulically disconnected from thexylem are not efficient in utilizing their hydraulic system tomaximize photosynthetic activity. In addition, under fluctu-ating atmospheric conditions, their stomata may be forced tolimit gas exchange despite abundant soil water availability.

In design 2, we propose that the epidermis and bundlesheath extensions form part of the fast phase, while thepalisade and possibly some of the spongy mesophyll belongto the slow phase. Based on the relative volumes of the fastand slow phases, we suggest that this design corresponds tofour of the six angiosperm species examined (i.e. excludingEucalyptus and Phyllostachys). In this design, the easiestroute for the water is to flow out of the vein, through thewell-connected cells of the bundle sheath extension, to theepidermis where water evaporates from the internal sur-faces of stomata or nearby cells. This results in the meso-phyll being largely bypassed by the transpiration stream.Whether such hydraulic separation of the mesophyll could

result solely from leaf geometry, or requires some degree ofrestricted water movement between specific cell types (e.g.between the adaxial epidermis and palisade parenchymacells) is not known.

The physiological consequence of design 2 derives fromthe epidermis and stomata being hydraulically well linkedto the xylem. This allows the water potential of the epider-mis to closely follow that of the xylem and thus the thresh-old for stomatal closure can be much closer to the cavitationthreshold than in design 1. Consistent with this, a small‘safety margin’ between xylem cavitation and stomatalclosure has been observed in a number of angiospermspecies (Brodribb & Holbrook 2004). Hydraulic separationof the mesophyll from the transpiration stream allows thesecells to be buffered from rapid changes in the transpirationrate. One could argue that disconnecting mesophyll cellsfrom the transpiration stream reduces the capacity of theleaf to deal with sudden increases in water loss rates giventhat the mesophyll cells themselves could act as watercapacitors. However, the amount of water in these cellscould support transpiration for no more than a few tensof the seconds before their turgor loss point is reached.

Figure 9. Relative contribution of eachtissue type for each species. Thick lineswith vertical error bars indicate the percent water corresponding to the fastphase (below the line) and the per centwater corresponding to the slow phase(above the line). Error bars denotes SEof the mean.

Vein

Extension

Fibes

Epidermis

Spongy

Palisade

Other

Split between

fast phase

(below the line)

and slow phase

(above the line)

Gnetu

m g

nem

on

Gingko

bilo

ba

Met

aseq

uoia

glypto

strob

oides

Larix

laric

ina

Querc

us ru

bra

Acer r

ubru

m

Liriod

endro

n tu

lipife

ra

Populu

s nig

ra

Phyllo

stach

ys a

ureo

sulca

ta

Eucaly

ptus g

obulu

s

Figure 10. Schematic of three scenariosfor leaf hydraulic design describing thehydraulic linkages between differenttissues. Solid lines depict water flow,dashed lines describe diffusion of watervapour, and Ø denotes high resistancebetween tissue types.

Design 1 Design 2 Design 3



Moreover, minimizing short-term changes in the waterstatus of mesophyll cells might be advantageous given theirphotosynthetic role.

In design 3, all major tissue types belong to the fast phase.Based on results from the rehydration kinetics, we believethat such a design is highly probable for E. globulus andPhyllostachys aureosulcata where only a small per cent ofthe total leaf volume corresponds to the slow phase. Thissuggests that both the mesophyll and epidermis in these twospecies are well linked hydraulically to the vasculature.Thisinterpretation is consistent with the very high density(packing) of the mesophyll, the presence of stomata on bothsurfaces, and the lack of morphological separation betweenspongy and palisade mesophyll. From a physiological per-spective, design 3 has similar properties as design 2, in thatleaves would be expected to operate near the cavitationthreshold. However, the mesophyll would not be protectedfrom sudden changes in transpiration and thus the photo-synthetic cells could be exposed to higher temporal varia-tion in water potential.

The three hydraulic design scenarios discussed here resultfrom a consideration of rehydration kinetics in conjunctionwith anatomical studies. Although assignment of the twophases to spatially distinct tissues within the leaf remains ahypothesis,our goal is to stimulate discussion concerning thehydraulic designs of leaves and to motivate research that willprovide a better understanding of both the biological andphysical principles governing water flow in leaves. We haveshown that rehydration kinetics provides a powerful tool fordescribing the movement of water within leaves at negativewater potentials.The data presented here represent a signifi-cant step in our understanding of leaf hydraulic design. Inessence, they motivate a conceptual shift from the idea thatleaves function hydraulically as a single pool of water thatsimply evaporates from the internal surfaces of mesophylland/or epidermis, to one in which leaves are understood toconsist of well-organized water pools separated by hydraulicresistance of sufficient magnitude to maintain water poten-tial disequilibria for sufficient time to have a significantimpact on leaf performance.

ACKNOWLEDGMENTS

We would like to thank Andrea Leigh and Anna Zwien-iecka for providing crucial assistance in collecting data fromthe reverse Polish guillotine, and Anna Zwieniecka for allaspects of microscope slide preparation and image analysis.This work was supported by NSF competitive grant numberIOB-0517071 and the Andrew W. Mellon Foundation.

REFERENCES

Berkowitz G.A. & Kroll K.S. (1988) Acclimation of photosynthesisin Zea mays to low water potentials involves alterations in pro-toplast volume reduction. Planta 175, 374–379.

Boyer J.S. (1974) Water transport in plants: mechanism ofapparent changes in resistance during absorption. Planta 117,187–207.

Boyer J.S. (1977) Regulation of water movement in whole plants.In Integration of Activity in the Higher Plant (ed. D.H. Jennings),pp. 455–470. Cambridge University Press, Cambridge, UK.

Boyer J.S. (1985) Water transport. Annual Review of Plant Physi-ology 36, 473–516.

Brodribb T.J. & Holbrook N.M. (2003) Stomatal closure during leafdehydration, correlation with other leaf physiological traits.Plant Physiology 132, 2166–2173.

Brodribb T.J. & Holbrook N.M. (2004) Stomatal protection againsthydraulic failure: a comparison of coexisting ferns and angio-sperms. New Phytologist 162, 663–670.

Brodribb T.J. & Holbrook N.M. (2005) Water stress deforms trac-heids peripheral to the leaf vein of a tropical conifer. PlantPhysiology 137, 1139–1146.

Brodribb T., Holbrook N., Zwieniecki M. & Palma B. (2005)Leaf hydraulic capacity in ferns, conifers and angiosperms:impacts on photosynthetic maxima. New Phytologist 165, 839–846.

Buckley T.N., Mott K.A. & Farquhar G.D. (2003) A hydromechani-cal and biochemical model of stomatal conductance. Plant, Cell& Environment 26, 1767–1785.

Canny M.J. (1993) The transpiration stream in the leaf apoplast –water and solutes. Philosophical Transactions, Series B-BiologicalSciences 341, 87–100.

Canny M. (1995) Apoplastic water and solute movement – newrules for an old space. Annual Review of Plant Physiology andPlant Molecular Biology 46, 215–236.

Chrispeels M.J. & Maurel C. (1994) Aquaporins: the molecularbasis of facilitated water movement through living plant cells?Plant Physiology 105, 9–13.

Chrispeels M., Morillon R., Maurel C., Gerbeau P., Kjellbom P.& Johansson I. (2001) Aquaporins of plants: structure, function,regulation, and role in plant water relations. Current Topics inMembranes 51, 277–334.

Cochard H., Froux F., Mayr F.F.S. & Coutand C. (2004) Xylem wallcollapse in water-stressed pine needles. Plant Physiology 134,401–408.

Cruiziat P., Tyree M., Bodet C. & Logullo M. (1980) Kinetics ofrehydration of detached sunflower leaves following substantialwater-loss. New Phytologist 84, 293–306.

Esau K. (1977) Anatomy of Seed Plants, 2nd edn. John Wiley &Sons, New York, NY, USA.

Fahn A. (1990) Plant Anatomy, 4th edn. Pergamon Press, NewYork, NY, USA.

Fricke W. (2000) Water movement between epidermal cells ofbarley leaves – a symplastic connection? Plant, Cell & Environ-ment 23, 991–997.

Hacke U.G., Sperry J.S., Wheeler J.K. & Castro L. (2006) Scaling ofangiosperm xylem structure with safety and efficiency. TreePhysiology 26, 689–701.

Matthews M.A. & Boyer J.S. (1984) Acclimation of photosynthesisto low leaf water potentials. Plant Physiology 74, 161–166.

Milburn J. (1966) The conduction of sap: 1. Water conduction andcavitation in water stressed leaves. Planta 96, 34–42.

Nardini A., Gortan E. & Salleo S. (2005) Hydraulic efficiency of theleaf venation system in sun- and shade-adapted species. Func-tional Plant Biology 32, 953–961.

Pickard W.F. (1982) Distribution of evaporation in the sub-stomatal chamber, the possibility of transpiration-linked porenarrowing, and the pathway of water near the site of evapora-tion. Annals of Botany 49, 545–548.

Pittermann J., Sperry J.S., Hacke U.G., Wheeler J.K. & SikkemaE.H. (2005) Torus-margo pits help conifers compete withangiosperms. Science 310, 1924–1924.

Ruzin S.E. (1999) Plant Microtechnique and Microscopy. OxfordUniversity Press, New York, NY, USA.



Sack L. & Holbrook N.M. (2006) Leaf hydraulics. Annual Reviewof Plant Biology 57, 361–381.

Sack L., Cowan P., Jaikumar N. & Holbrook N. (2003) The‘hydrology’ of leaves: coordination of structure and function intemperate woody species. Plant, Cell & Environment 26, 1343–1356.

Sack L., Streeter C.M. & Holbrook N.M. (2004) Hydraulic analysisof water flow through leaves of sugar maple and red oak. PlantPhysiology 134, 1824–1833.

Sack L., Tyree M.T. & Holbrook N.M. (2005) Leaf hydraulic archi-tecture correlates with regeneration irradiance in tropical rain-forest trees. New Phytologist 167, 403–413.

Schuepp P.H. (1993) Tansley Review No. 59: leaf boundary layers.New Phytologist 125, 477–507.

Singsaas E., Laporte M., Shi J., Monson R., Bowling D., Johnson K.,Lerdau M., Jasentuliytana A. & Sharkey T. (1999) Kinetics ofleaf temperature fluctuation affect isoprene emission from redoak (Quercus rubra) leaves. Tree Physiology 19, 917–924.

Sowinski P., Rudzinska-Langwald A. & Kobus P. (2003) Changes inplasmodesmata frequency in vascular bundles of maize seedlingleaf induced by growth at sub-optimal temperatures in relationto photosynthesis and assimilate export. Environmental andExperimental Botany 50, 183–196.

Tang A.C., Kawamitsu Y., Kanechi M. & Boyer J.S. (2002) Photo-synthetic oxygen evolution at low water potential in leaf discslacking an epidermis. Annals of Botany 89, 861–870.

Tezara W., Mitchell V., Driscoll S.P. & Lawlor D.W. (2002) Effectsof water deficit and its interaction with CO2 supply on the bio-chemistry and physiology of photosynthesis in sunflower. Journalof Experimental Botany 53, 1781–1791.

Tomlinson P.B. & Fisher J.B. (2005) Development of nonlignifiedfibers in leaves of Gnetum gnemon (Gnetales). American Journalof Botany 92, 383–389.

Tyree M.T. & Cheung Y.N.S. (1977) Resistance to water flow inFagus grandifolia leaves. Canadian Journal of Botany 55, 2591–2599.

Tyree M., Cruiziat P., Benis M., Logullo M. & Salleo S. (1981) Thekinetics of rehydration of detached sunflower leaves from differ-ent initial water deficits. Plant, Cell & Environment 4, 309–317.

Wang X. & Yakir D. (1995) Temporal and spatial variations in theoxygen-18 content of leaf water in different plant species. Plant,Cell & Environment 18, 1377–1385.

Weatherley P. (1963) The pathway of water movement across theroot cortex and leaf mesophyll in transpiring plants. In The WaterRelations of Plants (eds A. Rutter & F. Whitehead), pp. 85–100.Blackwell, London, UK.

Yakir D., DeNiro M. & Rundel P. (1989) Isotopic inhomogeneity ofleaf water – evidence and implications for the use of isotopicsignals transduced by plants. Geochemica at Cosmochimica Acta53, 2769–2773.

Yakir D., DeNiro M.J. & Gat J.R. (1990) Natural deuterium andoxygen-18 enrichment in leaf water of cotton plants grown underwet and dry conditions: evidence for water compartmentationand its dynamics. Plant, Cell & Environment 13, 49–56.

Zwieniecki M.A., Melcher P.J., Boyce C.K., Sack L. & HolbrookN.M. (2002) The hydraulic architecture of the leaf venation inLaurus nobilis L. Plant, Cell & Environment 25, 1445–1450.

Zwieniecki M.A., Boyce C.K. & Holbrook N.M. (2004) Hydrauliclimitations imposed by crown placement determine final size andshape of Quercus rubra L. leaves. Plant, Cell & Environment 27,357–365.

Received 7 January 2007; received in revised form 10 April 2007;accepted for publication 11 April 2007



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Hydraulic design of leaves: insights from rehydration kinetics · resistance of a single pathway for rehydration versus the existence of distinct pools, compare the spatial patterns