Developmental Effects on Leaf Water Relations of two Evergreen Shrubs (Prunus laurocerasus L. and Ilex aquifolium L.)

Flora (1983) 173: 143-150

Developmental Effects on Leaf Water Relations of two Evergreen Shrubs (Prunuslaurocerasus L. and flex aquifolium L.) HEIDRUN KARLIC and HANNO RICHTER Botanisches Institut der Universitat fUr Bodenkultur, Wien, Osterreich

Summary Leaf water characteristics of two evergreen shrubs, Prunus laurocerasus L. and Ilew aquifolium L., were followed for one year by means of the pressure· volume curve technique. Osmotic potentials of newly emerged leaves were about 1 MPa higher (less negative) than those of mature, over-wintered leaves. This difference disappeared during the first months of leaf development, when the vacuolar solution in newly emerged leaves gradually became more concentrated. Daycourses of total water potential show the most negative values for fully mature leaves. Pressurevolume curves were used to convert these field data into corresponding values for the turgor potential, which were byfar higher for old than for young leaves. Recently emerged leaves in spring may therefore come under severe stress during periods of only moderately lowered total water potentials.

Introduction In the past few years, pressure-volume curves (PV curves; TYREE & HAMMEL 1972) have been frequently used to characterize the seasonal changes in tissue water relations (e.g. TYREE et al. 1978; ROBERTS et al. 1980; GROSS et al. 1980). Most of these studies (except the recent one by SYVERTSEN et al. 1981) have been carried out on deciduous trees or on conifers where only current foliage was examined. Changes in water relations parameters caused by developmental effects were therefore not easily separated from those due to environment and season. The purpose of our study was to show the interaction between state of leaf development and climatic factors in causing changes in such water relations parameters as total water potential (lJIt ), osmotic potential (lJIo) and turgor potential (lJIp ). In order to accomplish this goal we investigated seasonal and diurnal courses of water relations in two evergreen shrubs.

Material and Methods Investigations were carried out in 1978 and 1979 on Ilew aquifoliu.m L. and Prunuslaurocerasus L. growing in the Botanical Garden of the Agricultural University (Universitat fUr Bodenkultur) in Vienna. Both plants, shrubs of about 3 m height, became intermittently shaded by taller trees in the arboretum during the course of a day. The soil was kept well watered throughout the year so that pronounced drought adaptation (HINCKLEY et a!. 1978) could be excluded.

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Ilex was investigated from spring 1978 to spring 1979. Our observations on Prunu8 were started at the same time but came to a sudden end in .January 1979 because of severe frost injuries killing most of the lea ves. Young leaves of both species emerged in a single flush in mid-May. After about three weeks they were large enough to investigate them in a pressure chamber. At that time the previous year's leaves were dark green, and the two year old leaves had just started to turn yellow. Total water potentials for daycourses and pressure-volume curves were measured with a commercial pressure chamber (Soil-moisture Equipment Corp., Santa Barbara, Ca., USA). Pressurized air was used to increase the chamber pressure at a rate of about 0.03 MPa S-l. The petiole of a single leaf protruded through the rubber compression gland, and the cut surface was observed with a magnifying lens. The short petioles of Ilex aquifolium were elongated by means of two sharp cuts severing part of the leaf blade from both sides of the midrib (cf. BAUGHN & TANNER 1970; KARLIC & RICHTER 1979). During each daycourse documented, at least three leaves of each developmental stage were sampled at different times and resaturated by immersing their petioles in shallow water in a humid chamber for at least 12 h. PV curves coUld then be established on the day after the daycourse measurement. A leaf was rapidly weighed and inserted in the chamber; a balance pressure of less than 0.1 MPa was assumed to indicate full saturation. The chamber was then depressurized slowly, the leaf was removed from the chamber and allowed to transpire freely. Weights and corresponding balance pressures were repeatedly determined. At the end of a measurement cycle the leaves were dried to constant weight at 100°C. Values for relative water content (R) were calcUlated from the equation F-D R=-S-D where F is the fresh weight of the leaf corresponding to a given 'ljJt value, S is the saturated weight, and D is the dry weight. Data were then plotted as "type I" ('IjJ vs R-1) as well as "type II" ('IjJ-1 vs (1 - R)) diagrams (TYREE & RICHTER 1981). Linear regression analysis gave the lines of best fit and allowed for extrapolation. The main parameters evaluated were the osmotic potential at full saturation, 'ljJo(sat), and the osmotic potential at the turgor loss point, 'ljJo(TLP)' Seasonal and ontogenetic changes in these parameters could be compiled. Furthermore, type I diagrams were used to convert field data for 'ljJt into estimates for turgor potentials, 'ljJP' during daycourses measured in the garden.

Results Each PV curve results from three leaves harvested at different times during the day; each data point in Figs. 1 and 3 corresponds to one 'Pt measurement. The three leaves in a given age group behaved very similarly; there was no detectable effect of the harvest time. Changes in the vacuolar solute content during the course of a day are therefore either absent or of a very transient nature and easily reversed during resaturation of a detached leaf. Type I and type II plots of the same data set yield always slightly different values for the osmotic potential at full saturation, 'Po(sat). The reasons for these discrepancies are quite complex (RICHTER et al. 1980; TYREE & RICHTER 1981). Overall, the numerical values derived from type II transformations prove to be somewhat more reliable, especially where large amounts of apoplastic water are present. We found the mean difference between transformations to be 0.045 (± 0.045)

Developmental Effects on Leaf Water Relations

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MPa with Prunus and 0,06 (± 0,045) MPa with Ilex. Type I values were always the less negative onel-;, The maximum difference between the two estimates for lJIo(sat) was 0,2 MPa in newly emerged leaves of Ilex aquifolium, This may be due to the more scattered data points derived from three young leaves at a time of rapid development, In mid-.June four different typeH of leaves could be distinguished on the twigs of Prunus (Fig, 1): small. supple ones towards the apex, two to three weeks old; larger leaves already partly in the shade of the youngest ones, about four weeks old; fully mature, dark-green organs from the previous year's growth; and finally two year old senescent leaves just turning yello,Y, The most negative osmotic potentials could be measured on the mature leaves (lJIo(sat) = - 1.93 MPa); senescent leaves contained somewhat less solutes (lJIo(sat) = - 1.63 MPa), The two samples of the new leaf generation demonstrate that the osmotic potential becomes more negative in the 9a

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course of leaf expansion: lesl' than two weeks difference in age mean nearly 0.5MPa difference in Po(sat) ( - 0.86 MPa in the smallleavel' V8. - 1.34 in the larger oneR). The osmotic potentials of three weeks old leaves of Ilex aquifolium differed subRtantially from those of one year old leaves (Fig. 3). po(sat) was - 0,9:{ in new vs. - 1.76 MPa in mature leaves. These momentary pictures are corroborated by the Heasonal pattern for l[fo(sat). Thi" parameter declined in both tlpecies during thc cxpanISive growth of leaves by about 1 MPa. :From August ommrd, the younger leave I' looked like laRt years generation in color and size; there were no longer significant differences in PY and daycourse data between the generation,;, and their water relations were probably identical during the rest of the year until the onset of leaf senescence in the older leaves in next spring. Fig. 5 illustrates this behavior for Ilex aquifolium.

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P\' curves were then used to convert values for total water potential, as measured during a daycourse, to values for the turgor potential, 'Pp. PV curves and day courses are necessarily obtained from different sets of leaves. Any ov-erinterpretation of 'Pp curves derived in this way should be avoided. Likewise, the small differences between parameters derived from type I and type II transformations become quite unimportant in comparison with the uncertainties introduced by the natural variability of leaves. Our curves for 'Pp drop to negative values at some points during periods of stress; we must however emphasize that this does not imply the existence of a state of tension ("negative turgor") in the cell. Negative figures are derived as the difference ,,'henever a field value for 'Pt becomes lower than the osmotic potential at the turgor lOSt-; point in the P\- curve. Such negative values provide therefore an estimate for the severity of leaf wilting. From mid-May to late July the young leaves retained higher total water potentials during periods of high atmospheric demand than the mature leaves. In flex aquljolium (Fig. 4) the most negative 'Pt values of mature leaves drop to -1.6 MPa, while the minima of the young leaveR reach just - 1.0 MPa, even though they are more exposed to radiation and wind due to their position on the end of twigs. The calculated daycourse for lpp in the same figure shows clearly that the 'Pt values of the young leaves, although being remarkably more positive than those of mature leaves. nevertheless approached 'Po(TLP) during the noon hours or dropped even below this point. The mature leaves had positive turgor potentials of more than 0.2 MPa during the whole day. The daycourRes for Prunus laurocerasus (Fig. 2) show again that mature, one year old leaves have lower total water potentials but higher turgor than newly emerged leaves. Yellow. senescent organs behave differently. Their extremely low

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total water potentials are the result of severe wilting. A slight resaturation of the old leaves could be observed in .June (Fig. 2), while on July 3 the wilting process continued throughout the night. Soon afterwards most of the yellow leaves were shed.

Discussion WALTER (1931) shows how changes in osmotic potentials may be brought about in two ways. "Active" changes are due to alterations in the solute content of cells during leaf development or during adaptation to climatic factors like drought or cold. They lead to a change in osmotic potential at a given value for relative water content, e.g. at full saturation (R = 1). "Passive" changes are caused by dehydration and rehydration of the vacuole and are thus a direct consequence of changes in relative water content, with the number of molecules in the vacuolar solution remaining constant. Early studies on seasonal and ontogenetic changes in Po did not care much for information on the relative water content of the leaves sampled. This information is however a prerequisite for discerning between the two types of Po changes. TYREE et al. (1978) recognized the advantage of PV curves for the demonstration of "active" osmotic changes: comparisons can be made between potentials at full saturation or at any other fixed level of hydration. Our data (Fig. 1 and 3) show again the characteristic decline in the osmotic potentials of developing leaveR already observed by TYREE et al. (1978); HIKCKLEY et al. (1980); GROSS et al. (1980) and SYVERTSE~ et al. (1981), among others. Significant changes in fully mature leaveR do not occur over a long period of the year. Senescent leaves show less negative values for Po(sat) than the mature generation, indicating some depletion of vacuolar solutes in the process of aging. Pressure-volume curves may however also be used to reach a better understanding of water relations during a daycourse. In the field, mature leaves reach constantly more negative values for Pt during periods of high evaporative demand than young leaves. The reasons for this behavior certainly do not become clear ,,"hen only the daycourse of total water potential is considered. At first sight a number of different explanations seem to be plausible. We could for instance assume less frictional resistance in the transition zone between twig and young leaf as compared to the old growth. As an alternative one could postulate for young leaves more sensitive stomatal behavior leading to closure at higher turgor potentials; RASCHKE & ZEEVAART (1976) preferred this explanation for their observation that the youngest leaves of Xanthium strumarium transpire the least amount of water per unit leaf area. A plot of the turgor potential Pp as derived from the comparison of field data and the corresponding PV curve shows however clearly that a third explanation is more to the point, at least in our case. The values for total water potential in young leaves hover around the turgor loss point for much of the day. Although the minimum values for P t stay far higher in expanding leaves, there is nevertheless far more leeway for fully mature organs with low osmotic potentials before reaching

Devplopmpntal Effeets on Leaf \Vater' Relations

149

PO(TLP), which if; a critical thre~hold value: some preliminary observations of stomatal behavior in our objects as well as more detailed results from a number of other shrubs (HIKCKLEY et al. 1980) make it very likely that a decline of potentials to a value near the turgor loss point triggers stomatal closure, thus stopping a further potential decline very effectively. The twigs of our evergreen shrubs present therefore, during the flush period from May to July, an unexpected distribution of potentials: young and small leaves inserted towards the apex have frequently less negative total water potentials than the mature generation further down the same twig. Such a behavior is not fully understood unless we take the high resistances towards water transport into account which are found in angiospermous plants at the transition from stem to leaf (ZIMMERMANN 1978; RUCKENBAUER & RICHTER 1980). These resistances ensure a degree of autonomy for every leaf; each can then adjust its transpirational water loss and its stomatal activity according to the osmotic counterbalance available in the leaf cells. P t in the twig xylem will thus always remain above the least negative of the separate leaf potentials. Transpirational water losses of different age groups of leaves may be considerably different due to varying patterns of stomatal closure. SYVERTSEN et al. (1981) presented recently a detailed study of water relations in Citru8. The response of the evergreen leaves was not completely equivalent with what we found in flex and Prunu8. Osmotic potentials in spring leaves are higher, but total water potentials drop nevertheless to lower values than in old organs. This result could be due to a particularly high cuticular conductance in this early flush. Leaves emerging during a second flush in summer behave rather like our objects, which indicates a remarkable variability in a single tree. Overall, there is again proof for the pronounced stres,; imposed on expanding leaves; this makes water relations during periods of leaf growth a matter of particular importance in the life of field plants.

Acknowledgements We thank Prof. T. M. HINCKLEY, Seattle, for his continous interest III this work and for stimulating discussions. ERNST SCHARFETTER drew the figures. Financial support came from Projects 1465 and 371l5, Fonds zur Forderung der Wissenschaftlichen Forschung, Vienna.

References BAUGHN, .J. W., & TANNER, C. B. (1971l:) Excision effects on leaf water potential of five herbaceous species. Crop Sci. 16: 184~190. GROSS, K., l'HA:\I-NG1JYEN, T., & UNGER, H. (1980): Tagliche und saisonale Anderungen des Wasserpotentials und seiner Komponenten in den Kronen von Fichten unterschiedlichen Alters. Allg. For'st- u ..Jagd-Ztg. 151: 69~79. HINCKLEY, T. M., DUHME, F., HINCKLEY, A. R., & RICHTER, H. (1980): Water relations of drought hardy shrubs: osmotic potential and stomatal reactivity. Plant, Cell and Environment 3: 131~ 140. LASSOIE, J. P., & RUNNING, S. W. (1978): Temporal and spatial variations in the water status of forest trees. Forest Sci. Monographs 20: 1 ~ 72. 10 Flora, lld. 173

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KARLIC, H., & RICHTER, H. (1979): Storage of detached leaves and twigs without changes in water potential. New Phytol. 83: 379-3~4. RASCHKE, K., & ZEEVAART, .J. A. D. (197H): Abseisic acid content, transpiration, and stomatal conduetanef' as related to leaf age in plants of Xunthium strumrlrium L. Plant Physiol. 58: 1H9-174. HICHTER, H., DeH~lE, F., GLATZEL, G., HINCKLEY, T. M., & KARLIC, H. (19~O): Some limitations and applications of the pressure· volume curve technique in ecophysiological research. In: GRACE, .J., FORD, E. D., & .JARVIS, P. G. (Eds.): Plants and their atmospheric envimnment, 2!l3 - 272. BhH"kwell, Oxfol'd. ROBERTS, S. \Y., STRAIX, B. R., & KXOERR, K. R. (1980): Seasonal patterns of leaf water relations in four eo·oecurring forest tl'ee speeies: parameter's from pressurf'·volume eurves. Oecologia (Bed.) 46: 330-337. RUcKExBArER, P., & RICHTER, H. (19~()): Frictional resistances to water transport in watercultured wheat plants. Phyton (Austr'ia) 20: 37 -45. SYVERTSEN, ,J. P., SMITH, c\L L., and ALLEX, .J. C. (19fH): Growth rate and water relations of

Citrus leaf flushes. Ann Bot. 47: 97 -105. TYREE, c\L T., CHElTXG, Y. N. S., McGREfWR, M. E., & TALBOT, A ..J. B. (1978): The charaderistics of seasonal and ontogenetic changes in the tissue-water r'elations of Acer, PopulUS, Tsuga, and Picea. Can ..J. Bot. 56: (j35-!i47. & HA:\lMEL, H. T. (1972): The measurement of the turgor p,'eSSlue and the water' relations of plants by the pressme-bomb teehnique .. r. Exp. Bot. 23: 2(;7 - 2~2. & RICHTER, H. (1981): Alternative methods of analysing water potf'ntial isoterms: Some e>lutions and elarifications. 1. The impact of non-ideality and of some experimental errors . .J. Exp. Bot. 32: 643-653. WALTER, H. (1931): Die Hydratur der Pflanze und ihre physiologiseh-okologische Bedeutung. 174 p. Fischer, .Tena. ZUIMERMAXX, 1\1. H. (197~): Hydmulic architecture of some diffuse-porous trees. Can ..J. Bot. 56:

22~6-2295.

Reeeived C\larc'h 19, 1982

Authors' address: H. KARLIC and H. RICHTER, Botanisches Institut der Universitat fiir Boclenkultur, Grf'gor-Mendel-Stra13e 33, A - 1180 'Vien.

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