Isotopic inhomogeneity of leaf water: Evidence and implications for the use of isotopic signals transduced by plants

0916-7037/89i%3.w

+ .w

LETTER

Isotopic inhomogeneity of leaf water: Evidence and implications for the use of isotopic signals transduced by plants DAN YAKIR,*.*MICHAEL3. DENIRO,* ~0and PHILIP W. RUNDEL’ Laboratory of Biomedical and Env~onmen~ Sciences, University of C&fort&, Los Angeles, CA 90024, U.S.A. (Received May 9, 1989; accepted in revisedform August I, 1989)

Ahshract-Variations as large as 11%Oin 6 ‘*O values and 50% in BD values were observed among different fractions of water in leaves of ivy (Hederu helix) and sunflower (Heiianthus annuus). This observation contradicts previous experimental approaches to leaf water as an isotopically uniform pool. Using ion analysis of the water fractions to identify sources within the leaf, we conclude that the isotopic ~rn~tion of the water within ceils, which is involved in biosynthesis and therefore recotded in the plant organic matter, differs substantially from that of total leaf water. This conclusion must be taken into account in studies in which isotope ratios of fossil plant cellulose are interpreted in paleoclimatic terms. In addition, our results have implications for attempts to explain the Dole effect and to account for the variations of ‘sOf’60 ratios in atmospheric carbon dioxide, since the isotopic composition of cell water, not of total leaf water, influences the d I80 values of O2 and CO1 released from plants into the atmosphere. ation in the raQ/ I60 ratios of atmospheric CO, f FRANCEY and TANS,1987) may thus depend, in part, on interpretations of the factors that cause variability in leaf water 6 ‘*O values. Water in submerged leaves of aquatic plants has the same isotopic composition as the water in which the plants are growing ( BRIMUT, 1978 ) . This is not the case for emergent leaves of aquatic plants or for leaves of terrestrial plants. Isotopic fractionation during evaporation of water from leaf surfaces causes the water remaining in the leaf to become enriched in I80 and D relative to the source water taken up by a plant. Since the original discoveries of these enrichments-by ~N~A~INI et al, ( I965 ) for oxygen and WERSHAW et al. ( 1970) for hydrogen-numerous workers have measured isotope ratios of leaf waters. With one exception, in all previous studies leaf water was treated experimentally as an isotopically uniform water pool (e.g., GONRANTINI et al., 1965; WERSHAW et al., 1970; DCINGMAN et al., 1974; FERHIand LETOLLE, 1977; FARRIS and STRAIN, 1978; FOR~TEL,1978; ZUNDEL et al., 1978; ALLISONet al., 1985; LEANEY et al., 1985). In these studies, water from leaves was collected either by vacuum distillation or by azeotropic distillation. Both methods recover all the water in a leaf as a single sample. Based on analysis of total leaf water, a few workers inferred the possibility of isotopic inhomogeneity within leaves (ZIEGLER et al., 1976; FARRIS and STRAIN, 1978; LEANEYet al., 1985). WHITE ( 1983, 1988) collected water from white pine (Pinus strobus) leaves by vacuum distillation and by pressing. Based on differences between the 6D values of the two types of water samples, he concluded that bound water not released by pressing differs from total leaf water in D/H ratios. The physiolo~cal signi~~ce .of this observation is difficult to assess because WHITE f 1983, 1988) did not attempt to determine the physical or chemical nature of the reservoir holding the water not released by pressing the leaves.

INTRODUCEON

VARIATIONSIN THE isotopic composition of leaf water impart signals that are useful in studying a variety of biogeochemical cycles. First, the hydrogen and oxygen isotopic compositions of plant organic matter reflect those of leaf water (E-IN et al., 1976, 1977; DENIRO and EPSTEIN, 1979). Isotopic studies of cellulose, the most abundant organic constituent of plants, thus offer the possibility of reconstructing climatic and hydrological conditions if the factors that cause leafwater and ground water isotopic compositions to differ can be quantified (EPSTEIN et al., 1976, 1977; BURK and STIJIVER, 1981; LAWRENCEand WHITE, 1984; WHITE et al., 1985; EDWARDSand FRITZ, 1986 ) . Second, most atmospheric oxygen is derived from leaf water during photosynthesis. It is, therefore, the isotopic composition of leaf water that determines one of the biological influences on the isotopic composition of atmospheric oxygen. Understanding this component is essential to the study of the biogeochemical cycle of oxygen and its relation to the hydrological cycle (DOLE et al., 1954; BENDER et al., 1985). Third, a substantial portion of atmospheric carbon dioxide has been in contact with leaf water. FRANCEYand TANS ( 1987) have proposed that, because of the action of the enzyme carbonic anhydrase, this CO* is fully ~uilibmt~ with leaf water prior to its release from leaves into the atmosphere. Understanding Ia~tudin~ vari-

Also, Department of Earth and Space Sciences, UCLA, Los Angeles, CA 90024, U,S.A. t Also, Biology Department, UCLA, Los Angeles, CA 90024, U.S.A. *Present address: Botany Department, Duke University, Durham, NC27706,U.S.A. - #Present address: DeDartment of Earth Sciences. University of Califomia,Santa Barbara, CA 93106,U.S.A. l

2769

2770

D. Yakir. M. J. DeNiro, and P. W. Rundel

In this study we show that different fractions of water in leaves of ivy (Hedera helix) and sunflower (Helianthus annuus) display large variations in oxygen and hydrogen stable

,~_

r- _....- ..--.I _..I----~-.-.‘

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isotopic composition. We identify the sources of the various water fractions within the leaf and conclude that the isotopic composition of the water within the cells, where all of the processes that relate leaf water isotopic compositions to those of organic matter and atmospheric O2 and CO2 occur, can be considerably different from that of total leaf water-the only value considered in the past. This observation will undoubtedly complicate attempts to interpret 6D and/or 6 “0 values of plant organic matter and atmospheric gases. On the other hand, the very existence of an isolated water pool within the cells suggests that this pool may be buffered against short-term isotopic variations that occur in the total leafwater, for example, in response to diurnal variations in temperature and humidity ( FORSTEL, 1978) or to changes in water availability ( FARRIS and STRAIN, 1978). The isotopic signals transduced by plants may therefore prove to be more reliable than would have been expected based on isotopic studies of total leafwater in reflecting long-term environmental changes.

-+60

METHODS Two adjacent young fully mature leaves detached at ‘7AM from the same specimen of either an ivy plant (Hedera helix) grown outside the laboratory or a month-old sunflower (Helianthus annuus) plant grown in the glasshouse were enclosed in a multiple compartment pressure chamber with their petioles (stems) protruding out through rubber sealing, with precautions taken to avoid evaporative water loss. The pmssute extraction method was essentially as described by JACHEITA et al. ( 1986). For the purposes of this study, water expressed through the petiole of the first leaf as the pressure applied to the leaves was increased was alternatively collected in volumetrically calibrated capillaries, which were immediately sealed for later oxygen and hydrogen isotopic analysis ( KISHIMA and SAKAI, 1980), or onto small preweighed filter paper disks, which were then weighed hefore being dropped into 2 ml of 1% nitric acid. This solution was used for ion analysis (ALEXANDERand MCANULTY, 198 I ) Water from the second leaf was collected at each pressure step on pmweighed filter papers that were reweighed on a microbalance to obtain a quantitative estimate of the volume of water expressed from the leaves. Water from a third leaf from each plant, collected simultaneously with the other two leaves, was distilled under vacuum, and the isotopic composition of total leaf water was then determined. This type of analysis of total leafwater represents the conventional approach taken in previous studies. isotopic compositions are reported as 6*X values, where 6*X = {[(*X/x~&J(*x/X)~&&l

- 1}‘10’%

*X/X is IsO/ I60 or D/H. The standard is Standard Mean Ocean Water (SMOW). The precision of the determinations of 6’*0 and 6D values were +0.5%0 and f I k, respectively. RESULTS

AND DISCUSSION

The results of the isotopic analysis reported in Figs. I a and 2a indicate that for both ivy and sunflower plants large variations in ‘*O and deuterium concentration exist among different water fractions expressed from the same leaf. This observation contradicts previous experimental approaches to leaf water as an isotopically uniform pool. Determining the amount of water expressed in each fmction analyzed enabled us to reconstruct, by simple mass balance calculations, the isotopic composition of total leaf water. The good agreement between these reconstructed isotopic

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Waterexpressed&of total) ’ FIG. 1. The relationships between the relative volume of water expressed from a leaf of an ivy plant, given as a percentage of total leaf water volume, and (A) the stable hydrogen or oxygen isotopic composition of the expremed water, and (B) the pressure applied on the leaf or the lithium concentration in the expressed water. Ion

analysis included 18additional ions, all of which gave similar results. The pressure at zero turgor point ($:) and the corresponding water volume expressed( WD.) are aho indicated in ( B) . The solid points in (A) indicate the isotopic composition of the water that remained in the leaf at the end of the experiment, which was collected by vacuum distillation.

values and those of total leaf water distilled from control leaves (Table 1) indicates that no detectable evaporational water loss or other adverse effects resulted from the pressure extraction. In the second part of this study we used the results of the ion analysis (Figs. lb and 2b) to incorporate the isotopic data reported above into a physiological framework. Following the lead of JACHETTA et al. ( 1986), the results for the ion analysis are interpreted as follows. As pressure on a leaf is increased, water from various parts of the leaf is expressed through the petiole as an inverse function of the resistance to flow. When pressure is first applied to the leaves, water flows out from the low-resistance petiole and veins, which effectively form a system of tubes. Next, water flows from the continuum of cell walls within the leaf which offer some resistance to flow. Finally, as the pressure reaches values high enough to counter the resistance offered by cytoplasmic membranes, the major resistance barrier rn the water flow pathway (BOYER, 1974), water within cells, referred to as the symplast, begins to flow. Hence, water expressed from the petiole of the leaf over the time-course of

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Isotopic inhomogeneity of leaf water

-1 A

-50

I

I

Wier expressed (sbof%+ol) FIG. 2. The relationships between the relative volume of water expressed from a leaf of a sunflower plant, given as a percentage of total leaf water volume, and (A) the stable hydrogen or oxygen isotopic composition of the expressed water, and (B) the pressure applied on the leaf or the iron concentration in the expressed water. Ion analysis included 18 additional ions, all of which gave similar results. The pressure at zero turgor point ($i) and the corresponding water volume expressed ( WD,) are also indicated in ( B) . The solid points in

(A) indicate the isotopic composition of the water that remained in the leaf at the end of the experiment, which was collected by vacuum distillation.

our experiments is assumed to represent the sequence petiole + veins - cell walls - symplast, with each fraction replacing its predecessor on its way out through the petiole. Ion concentration is initially relatively high as water is expressed from the petiole and veins through which ions are transported from the roots (Figs. 1b and 2b). Water stored

Table 1. Isotopic composition of total leaf The reconstructed values were water. calculated hy mass balance fran the isotopic values of each water fraction expressed from the leaf and its corresponding relative volume (as reported in Figs. 1, 2) together with the isotopic value and relative volume of the water that remained in the leaf at the end of the pressure expression. The observed values were obtained by analyzing water vacuum-distilled from a separate adjacent leaf of the same plant.

in cell walls is most likely depleted in ions as a result of active uptake by the cytoplasm ( JACHETTAet al., 1986). The decrease in ion concentration after the initial output (Figs. 1b and 2b) therefore indicates that water from the cell walls is now being expressed. As pressure is increased, water is being forced out of the cells through cytoplasmic membranes, which are impermeable to passive ion transport. This water is thus effectively filtered with regard to ions, and the observed further decrease in ion concentration, down to the level of distilled water, indicates, as previously interpreted ( JACHETTAet al., 1986)) that membrane-filtered symplastic water is now being collected. Increasing the pressure on the leaf even further apparently causes a sharp increase in permeability (or the disruption) of the cell membranes and a release of ions into the water flow expressed through the petiole. This explains the large increase in ion concentrations at the end of the runs (Figs. lb and 2b). The ion data provide an indication of the likely source of each water fraction expressed from the leaf. Using this information, we assigned isotopic values (Table 2) to each of the three main water compartments in the leaf, i.e., water in the veins, cell walls and symplast, as follows. Ideally one would, expect a change in isotopic composition in a stepwise fashion as water is expressed from one compartment, then another. It is clear, however, that some degree of mixing occurs as water is slowly forced from its original location across the leaf into the conductive system and out through the petiole. This mixing causes gradual changes from one isotopic value to another as one or another compartment is expressed (Figs. 1a and 2a). The isotopic compositions measured in the first water fractions expressed from the leaves represent the isotopic composition of vein water. Vein water normally contains unfractionated soil water (WERSHAW et al., 1970). Taking into account the fact that soil water may be somewhat more enriched than the irrigation water due to evaporative water loss from the soil ( FONTES, 1980)) there is good agreement between the isotopic composition assigned to the vein water (Table 2) and that of the irrigation water, which had a 6’*0 value of -14X%0 and a 6D value of -lOO?&. As the pressure was increased, the expressed water contained less vein water, which is gradually being replaced by water from cell walls. The first short plateau apparent in both isotope ratios in both plants, after about 5% of total leaf water is expressed, probably reflects the complete shift from vein to cell wall water. If this is the case, the isotopic value of the plateau may be taken to represent the isotopic composition

Table

2.

Isotopic composition of the three main compartments. Isotopic values for the three compartments were obtained frcm the isotope data presented in Figs. lA, 2A based on the interpretation of the ion analysis (Figs. lR, 28). as discussed in the text.

lea,f water

Sunflower

Ivy Sunflower

IVY

Reconstructed Observed

6’*0

60

aI80

6D

-4.R -5.2

-51 -47

-6.0 -6.2

-60 -54

Water in veins Water in cell walls Symplastic water

6'80

60

(;'a0

60

-14.0 -1l.l-l - 2.6

-83 -Rl -3n

-12.8 - 9.5 - 4.7

-95 -82 -46

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Yakir. M. J. DeNiro, and P. W. Rundei

of the water retained in cell walls (Table 2). As discussed above, increasing the pressure further causes symplastic water to be forced out of the cells. Thus, sympfastic water gradually replaces cell wall water in the main veins leading out of the leaf through the petiole. Previous studies have indicated that cell membranes have no isotopic fractionation effects (ALI.IsON et al., 1985), and therefore there is no isotopic filtering effect before the increase in permeability for disruption) of the membrane occurs at a high pressure, as was inferred from the ion analysis. Symplastic water was considerably more enriched in “0 and deuterium, at the time of sample collection, than water in either the veins or cell walls (Table 2). The isotopic value of the expressed water therefore increased as the contribution of symplastic water became predominant (Figs. la and Za). A second plateau is expected when symplastic water is the only water being expressed. However. this point could not be reached under the conditions of the present study because, under extreme pressure, the petiole fractured and its distal portion was expelled from the pressure chamber. Nevertheless, for a first approximation, we assumed that the expected final symplastic plateau is only slightly higher than the last isotopic value obtained (Table 2). This ~sumption is supported by the observation that the oxygen isotopic composition of the water that remained in the leaf aFter it was removed from the pressure chamber, which was collected by vacuum distillation, showed the same isotopic composition as the last water fraction expressed (Figs. la and 2a). We note, however, that the bD values of the water rem~ning in the leaves were lower than those of the last fractions expressed. This suggests that some water with 6D value lower than that of the symplastic water fraction could not be expressed by the pressure sufficient to express symplastic water. This observation could be accounted for if the so-called bound water, strongly attached to cellulose or other hydrophilic macromolecules, is depleted in D relative to the symplastic water fraction. Based on the differences between the bD values of water in white pine (Pinus strobus) leaves obtained by vacuum distillation and by pressing on the leaves, WHITE ( 1983. 1988) concluded that the D/H ratios of bound water and total leaf water differed. This conclusion is not inconsistent with our explanation ofthe discrepancy between the 6D values of the last fraction expressed from the leaves and the water that remained in the leaves at the end of the experiment. The isotopic values of the three main leaf water compartments, based on the above interpretations, are summarized in Table 2. It is important to note that environmen~l effects, which can produce dramatic changes in the isotopic composition of total Ieaf water ( FARRIS and STRAIN. 1978; FoRSTEL, 1978), most likely do not affect all three compartments equally. It is therefore possible that the relationship among the isotopic values of the three leaf water compartments, as presented in Table 2, may vary considembly under different conditions. For example, we expect that the observations reported here will prove to represent the minimum isotopic variations among different fractions of leaf water over the course of a day. The reasoning behind this expectation, which is presented in expanded form elsewhere ( YAKIR et al., 1990). may be summarized as follows. The existence of an isotopic& distinct sympIastic water pool requires its relative isolation from environmental factors, such as humidity, whose

diurnal variations affect the isotopic composition of total leaf water. The water in cell walls, on the other hand, comprises the active tmnspimtion flux and is likely to have its isotopic composition directly influenced by changes in environmental conditions. The isotope ratios of water in cell walls will thus fluctuate over the course of a day to a greater extent than those of water in the symplasm. It follows that differences ir! the isotopic composition of the two pools will reach a min” imum. This minimum wilt most likely occur in the early morning hours (when we collected our samples j before the large heavy isotope enrichment in total leaf water. relative to unfmctionated source water in the veins and branches, occurs in response to midday humidity minima ( WERSHA\S et al., 1970; ZUNDEL et al., I978 ) _ Using the isotopic compositions of the main water compartments given in Table 2, we estimated, based on simple mass balance equations, the relative volume of each compartment. The results of these calculations, presented in Table 3. are in agreement with previous estimates made in other plants based on indirect physiological (non-isotopic } ap proaches ( TYRREand JARVIS, 1982 ) . For example. a standard inte~re~tion of the PV curves (TURNER. f 98 1) reported in Figs. lb and 2b produced comparable estimates of cell wall water volumes of 15 and 25W of total leaf water for ivy ano sunflower leaves, respectively, CONCLtiSIONS This work represents the first attempt to provide a detailed picture of ieaf water compartmentation from isotopic perspectives. It is important to emphasize that the fact that leaf water is not isotopically homogenous, as demonstrated in this study, must be taken into account in any attempt to use stable isotopy of plant matter or of gases whose isotopic compositions are affected by interactions with plants in geochemical, paieoclimatic, or environmental studies. We have demonstrated that the last water fractions expressed from leaves under pressure, considerably enriched in D arid I80 relative to total leaf water, originated from within the parts of leaves bounded by the cytoplasmic membrane, the major resistance barrier in the water pathway. This is most si~ifi~nt because it is this water reservoir inside the cells that is involved in the biosynthesis of organic matter and is therefore the major. direct source for the hydrogen and oxygen isotopic signal in plant organic matter (EPSTEIN et al., 1976. 1977: DENIRO and EPSTEIN, 1979). The isotopic composition of the cell water, not that of total leaf water, must be considered in the future in studying biochemical fractionation effects of pro-

Tahie 3. The estimated volumes of the three md?n water compartments in leaves, given as d percentage of total Jeaf water volume. Volume "as estieated based on "ass balance CaIculatians using the isotopic cwnposrtion of each water crnpartnent and that of the total leaf water as reported in Tables 1 and 2,

Isotopicinhomogeneity of leaf water cesses that utilize leaf water, such as those occurring during photosynthesis and subsequent carbohydrate synthesis (e.g., STERNBERGet al., 1986). Similarly, it is the water within cells, not total leaf water, that is subject to the photolysis reaction of phot~~~~~, and therefore only its isotopic composition will influence that of molecuku oxygen released by plants to the atmosphere. Finally, because carbonic anhydrase is largely confined within cells in plants, the oxygen isotopic composition of the water within cells is the relevant quantity to be included in consideration of the biological factors influencing the 6 ‘*O values of atmospheric CO2 (FRANCJZY and TANS, 1987). Ac~owied~~-We

thank David Winterfor performingthe mass spectrometricdeterminations,Ellie Dzuro for preparingthe manuscriptand tables, and acknowledge support from NSF GrantsDMB 84-05003, DMB 88-96201, BNS 84-18280 and BNS 89-16273, DDE Grant DE-87ER60615 and BARD-United States-Israel Binationat Agricultural Research and Development Fund Grant S&0024-85.

Editorial handling: G. Faure

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