Abscisic acid and the after-effect of water stress on stomatal opening potential

Institut fiir Allgemeine Botanik, Universitat Hamburg

Abscisic Acid and the After-Effect of Water Stress on Stomatal Opening Potential K. DORFFLING,

J. STREICH, W. KRUSE and

BARBEL MUXFELDT

With 8 figures Received April 19, 1976 . Accepted June 26, 1976

Summary Isolated leaves of ecologically different plants (mesophytes: Pisum sativum L., H elianthus annuus L., Vieia faba L.; hygrophytes: Tradescantia X andersoniana LUDW. et ROHw., Menyanthes trifoliata L. and Mentha aquatica L.) were wilted for a short time, so that the fresh weight was reduced to 80-60 % of that of the original. Subsequently they were transferred to water for recovery. During the stress period and during the time of recovery, the stomatal pore size and the abscisic acid (ABA) content of the leaves were measured. A delay in the opening reaction of the stomata relative to the recovery of leaf turgor was observed with the mesophytes. The duration of this after-effect of stress on stomatal opening was directly correlated with the ABA level. The ABA level increased two-to five-fold during the stress period and decreased after the stressed leaves had been transferred to water. The application of (± )-ABA to stressed leaves for a duration of one hour after the transfer to water prolonged the duration of stomatal closure, the prolongation being dependent on the concentration applied. The applied ABA increased the ABA level of the leaf and this level decreased subsequently in the same manner as did the natural ABA level. In the hygrophytes investigated, the stomatal opening was not delayed relative to recovery of leaf turgor, and the ABA level did not increase in response to water stress but remained constant (Menyanthes) or decreased (Tradescantia, Mentha). It is concluded that the after-effect of wilting on stomatal opening is caused primarily by the increased level of ABA. The possible importance of the delay of stomatal opening in the stress physiology of mesophytic and hygrophytic plants is discussed. Key words: abscisic acid, water stress, stomata.

Introduction In many plant species a period of water stress has an «after-effect» on stomatal opening, whereby the opening movement of the stomata is delayed for several hours or even days after a period of water stress. This reaction occurs although leaf water-deficit is eliminated within a much shorter time. STALFELT (1955), who first described this phenomenon, supposed it to be a «safety mechanism» against serious water loss. Z. Pflanzenphysiol. Bd. 81. S. 43-56. 1977.

44

B. DORFFLlNG,

J. STREICH, W. KRUSE and

BARBEL MUXFELDT

Without direct experimental evidence, ALLAWAY and MANSFIELD (1970) postulated the after-effect to be based upon the accumulation of a stomatal inhibitor in the leaves. Direct evidence that abscisic acid (ABA) might be involved was presented by WRIGHT (1972) and HIRON and WRIGHT (1973). In dwarf bean seedlings subjected to a continuous stream of warm air, an increased ABA level and an increased leaf resistance was observed both during the wilting period and after full turgidity had been regained. No data, however, were presented by HIRON and WRIGHT regarding the duration of stomatal closure and its possible relation to the ABA level. However, from recent experiments concerning the relationship between leaf water status, ABA levels, and stomatal resistance, BEARDSELL and COHEN (1975) concluded that impaired stomatal functioning after water stress in corn and sorghum is not associated with increased levels of ABA. Nevertheless, some of their data (figs. 2 and 7) clearly indicate such a relationship. In a previous paper (DORFFLING et al., 1974) it was observed that the stomata of pea exhibit a delay in their opening reaction which is more or less correlated to the decrease of the ABA level after a period of wilting. The present study was undertaken in order to investigate more precisely the relationship between the after-effect of wilting on stomata and the ABA level. Plants of different ecological character were employed to examine whether the after-effect is related to ecological adaptation to water stress.

Materials and Methods Plant Material

Two different types of plants were used: 1. plants normally growing under conditions in which short periods of water stress often occur (mesophytic character). These plants were Pisum sativum L. var. Kleine Rheinlanderin, Helianthus annuus L. var. bismarckianus and Vicia faba L. var. Hangdown Weil\; 2. plants normally growing in moist soil and in a humid atmosphere (hygrophytic character). These were Tradescantia X andersoniana LUDw. et ROHW., Meny anthes trifoliata L. and Mentha aquatica L. Seedlings of peas, sunflowers and broad beans were grown in pots filled with garden soil or sand (pea) in climate chambers; pea seedlings were also grown in water culture. The seedlings were irradiated daily with fluorescent lamps (Philips (TLF 65 W/34 De Luxe, 8000 lux) from 6 am to 10 pm. The temperature was 25°C during the day and 20°C during the dark period; relative humidity was 50 to 600/0. Experiments were carried out on fully expanded leaves of plants, which were between 15 and 30 days old. In a greenhouse, cuttings from Tradescantia were grown under humid conditions until they had developed roots. Six days before the newly developed leaves were taken for experiments, the cuttings were transferred to the climate chambers. Leaves from Menyanthes were taken in August from a batch of plants growing in a pond in the Botanical Garden. Mentha aquatica grew in water basins in a greenhouse. Treatment of the leaves

To induce a water deficit, leaves were cut with a razor blade early in the morning and placed on dry filter paper with their lower surfaces resting against the paper. The pre-existing temperature and conditions of light and humidity were not altered. This treatment lasted one Z. Pflanzenphysiol. Bd. 81. S. 43-56. 1977.

ABA and the after-effect of water stress

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half to five hours, in one experiment as long as 23 h. During this period the fresh weight was reduced to 80-60 Ofo of that of the original. After the treatment (time zero), the leaves were transferred to plexiglas boxes (lOX lOX 5.5 ern) half-filled with tap water for recovery. Care was taken that the petioles were in good contact with the water. Control leaves were not wilted and placed on water immediately after cutting. In some experiments with peas, the whole plants were wilted by withholding water. The leaves were then cut from the wilted plants and transferred to water. The results obtained did not differ from those results obtained with leaves wilted after isolation. At different intervals during the entire treatment the weight and the stomatal opening of individual leaves were measured. To determine the weight, leaves were taken out of the plexiglas box, dried quickly between layers of filter paper and weighed. The whole procedure did not take more than 15 sec. Afterwards the leaves were transferred again to the plexiglas boxes. The stomatal diameter was measured microscopically. Leaves were fastened onto glass slides with their lower surfaces upwards and total of 50 stomata from five different areas were measured within two minutes. These leaves were subsequently discarded. Determination of ABA At different time intervals during the wilting and recorvery periods batches of 20 to 50 leaves were immediately frozen and afterwards extracted three times with 96 % ethanol. For the calculation of the loss of natural ABA during the purification procedure 3-5 ,ug trans-ABA (Schuchardt, Munich) were added as an internal standard to the ethanol used for the first extraction (LENTON et al., 1971). The ethanol was removed under vacuum and the residue was suspended in water. This suspension was adjusted to pH 9 with 0.1 M NaOH and extracted three times with ether. The ether fractions were discarded. The aqueous phase was acidified to pH 3 with 0.1 M HCL and again extracted with ether. The ether was removed and the residue dissolved in methanol. This solution was purified by thin-layer chromatography on Silica Gel HF 254 (Merck) with isopropanol-20 N ammonia-water 80 : 5 : 15, vlv, and afterwards with bcnzcne-ethylacetate-aceric acid 100: 100 : 1, v/v. Synthetic ABA (Hoffmann La-Roche, Basel, Switzerland) and trans-ABA (Schuchardt, Munich) were used as markers. The zones corresponding to ABA and trans-ABA were scraped off together and subjected to gas-liquid chromatography. The samples were methylated with diazomethane and chromatographed on 1 I 8" steel columns 150 ern in length and coated with 3 Ofo QF1 or 2 Ofo Epon on 80/100 mesh Chromosorb W. N 2 was used as the carrier gas. The column temperature was 195°C, the detector (electron capture) temperature was 240 °C and the injector temperature was 235°C. Calculation of the amount of ABA was performed according to LENTON et al. (1971) or by means of standard concentrations of ABA. Application of ABA to leaves To investigate the possibility of modifying the after-effect duration, ABA was added to the tap water on which the wilted leaves had been placed for recovery. After one hour, the leaves were transferred to tap water again. The concentration was 10-4 M to 10- 6 M racemic ABA. [There is evidence, however that only the (+ )-enantiomer induces stomatal closure (KRIEDEMANN et al., 1972).] The stomatal diameter of these leaves was compared with that of leaves set on ABA-free water for recovery. During the treatment the ABA level was measured as described above.

Results Effect of water stress on ABA content and stomatal behaviour

When isolated leaves of peas, sunflowers and broad beans were wilted for a few hours so that their fresh weight was reduced to 80-60 Ofo of that of the original and Z. Pflanzenphysiol. Bd. 81. S. 43-56. 1977.

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K. DORFFLING,

J. STREICH, W. KRUSE and BARBEL MUXFELDT

afterwards rewatered, the opening of the stomata was clearly delayed in relation to the disappearance of the water deficit (fig. 1, 2, 3). This delay may be called the «after-effect » of water stress on stomatal openin g in accordance with STALFELT (1955) and FISCHER (1970). It differs in length from species to species and is also affected by the duration of the wilting period. In broad bean leaves wilted for four to five hours it lasted three to six hours, but only two hours when the stress period lasted for one hour (fig. 3). The recovery of leaf turgor, measured as the increase in

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Fig. 1: Pisum sativum. Changes in leaf weight, ABA content and stomatal opening during and after a tWO hours wilti ng period. Mean of eight experiments. Ver tical lines: st an da rd deviation of the mean value. ABA content of controls (unw ilt ed leaves) : 42 ngl100 mg fre sh weight (FW).

Z. Pflanzenphysiol. Bd. 81. S. 43-56.1977.

ABA and the after-effect of water stress

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weight, began immediately upon transfer of the wilted leaves to water. Especially in pea, full turgidity was regained within one hour (fig. 1). However, after this period the stomata were still completely closed. This indicates that the after-effect of stress on stomatal opening is not due to the persistence of leaf water deficit. While there was no correlation between recovery of leaf turgor and stomatal opening with the three mesophytic species, a clear correlation was found to exist between stomatal behaviour and endogenous content of ABA. In all three species, the amount of ABA increased during the wilting period and even during the first period

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of the recovery. As wit h the duration of th e after-effect, the maxima l ABA level in a leaf appears to be depen dent on the duration of the stress (fig. 3 and unpublished results of J. STREI CH) . The decrease in the ABA content after transfer to water was most rapid in pea, slower in sunflower and slowest in broad bean. Throughout the duration of the experiment, the ABA level of unwi lted leaves did not change significantly. The hygrophytic species Tradescantia, Men yanthes and Men tha behaved quite diff erently (figs. 4, 5, 6, 7). In these species, a dela y in the opening reaction of the stomata was not observable, although the reduction of fresh weight was in the same magnitude as in the three species described above . Moreover, the ABA level did not change significantl y in Meny anthes, wher eas in T radescantia and in Mentha it Z. Pjl:mzenphysiol. Bd. 81. S. 43-56. 1977.

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actually decreased during the wilting and (in Tradescantia) even during the post-wilting period to less than 50 % of the original concentration. To test the possibilit y that th e water deficit obtained after a wilting per iod of only thre e hours is below the threshold level which is necessary to induce an increase in the ABA (ZABADAL, 1974; BEARDSELL and COHEN, 1975), the stress period was prolonged in T radescantia. Even after a wilting per iod of 23 h, (leaf-weight loss of nearl y 30 0/0) the leaves of Tradescantia recover ed rapidly, the stomata opened without a delay and the ABA level decreased (fig. 6). 24 h after being transferred to water the ABA level in the leaves attained the pre-wilting level. Z. Pflanzenphy siol. Bd . 81. S. 43-56. 1977.

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The effect of exogenously applied ABA on the duration of the after-effect and on the endogenous ABA level The possibility of modifiying the duration of the after-effect by the addition of exogenous ABA was investigated with Pisum (fig. 8). Leaves were wilted for two hours as described and subsequently transferred to tap water. As a consequence of water stress, the ABA content increased about threefold and the stomata opened with a delay of nearly one hour measured from time zero. ABA 10- 4 M added to the tap water at time zero for one hour maintained complete stomatal closure for at least three hours. Then the stomata began to open very slowly. 5 X 10- 5 M ABA resulted in a slow opening after two hours. With 2 X 10- 5 M ABA the stomata began to open Z. Pjlanzenphysiol. Bd. 81. S. 43-56. 1977.

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after about one hour, but more slowly than in the leaves not tr eat ed with ABA; they did not open as widely as in th e controls. 10- 5 M ABA had a weaker effect than 2 X 10- 5 M ABA and 10- 6 M ABA was ineffective . The treatment of the stressed leaves with ABA did not affect the upt ake of wa ter (results not shown) . Application of ABA not only delayed stomat al opening, but also increased the ABA content of the leaves. Th e increase and subsequent decrease were in direct correlation to sto matal behaviour. Discussion A dela y in the opening reaction of the stomata after water stress is observable in the leaves of those species (pea, sunflower and bro ad bean), in which the level of Z. Pflanzenphysiol. Ed. 81. S. 43-56. 1977.

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J. STREICH , W. KR USE an d B ARBEL MUXFELDT MENTHA AQUATICA

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Fig. 7: Ment ha aquatica . Change s in leaf weight , ABA content and stomatal opening during and after a wilting period of 30 min utes. T yp ical expe riment s presented. ABA conte nt of control s (unwi lted leaves) : 300 ng/lOO mg FW.

ABA incre ases du ring the wilting period. Th e after-effect of stress on stomatal op ening lasts on e to five hours. This dela y is similar to th e time delay STALFELT (1955) found in experiments with Rumex acetosa and BEARDSELL and COHEN (1975 ) found with corn, but it is shorte r than in the experiments report ed upon by FISCHER et al. (1970) and by ALLAWAY an d MANSFIELD (1970 ). Because turgor recove ry, mor e quickly, the persisten ce of leaf water deficit cannot be responsible for thi s phenomenon. The dur at ion of th e after-effect is directl y corre lated to the time cour se of the ABA increase and decrease. BEARDSELL and COHEN (1975), work ing wit h corn and Sorghum, recentl y obtai ned results simila r to those described here. Sho rt peri ods of stress ha d no effect on th e ABA level, and no delay in the open ing reaction was observab le. Ex tending th e stress peri od up to th ree hours resulte d in a st rong inc rease of th e ABA level which continued even af ter rewatering an d restor ation of tu rgor. Sto mata l opening was delaye d until the ABA level had decreased to a lower level (their fig. 7). Even so, Z. Pjlanzenphysiol. Bd. 81. S. 43-5 6. 1977.

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Fig. 8: Pisum sativu m. Eff ect of ABA applied to wilted leaves at time zero for 1 hour on the open ing of the stomata and on the level of ABA in th e leaves. Control: wilt ed leaves treate d with wa ter at t ime zero. Abov e: mean of th ree experiments. Below: mean of 7-13 experiments.

th ey concluded that «the after-effect of stress cannot be attributed directly to high residual levels of ABA ». Th e view that the ABA level is responsible for the stomatal delay is further support ed by the experiments with exogenously applied ABA. Th e dela y in stomatal opening can be extended by exogenous application of ABA, which is tak en up by the leaves and increases the endogenous ABA level. The dur ation of this «induced» after-effect depend s On th e concent ration of the ABA applied. Even with T radescant ia, in which the stomata begin to open immediately after wat er str ess, a delay in sto mata l opening can be induced by exogenous ABA. The fact th at stomat a of M eny anthes, Tradescant ia and of M entha also close after short stress, although the ABA level does not increase, clearly indicates that an Z. Pjlanzenphy siol. Bd. 81. S. 43-5 6. 1977.

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B . D O RFFLIN G,

J. STREICH , W. KR USE and B ARBEL M UXFELDT

incr ease of the ABA level is not a necessary p rerequisite for th e induction of sto mat al closure. However, it can be conluded both from the natural and the induced after-effect that th e increase in the endogenous ABA level and the time cour se of its decrease are responsible for th e occur rence and dur ation of the after-effect of wilt ing on stom atal op ening. If th e time course of th e ABA decrease directl y regulates the duration of the af ter-effe ct, it should be exp ected th at th e sto mata do not begin to op en befor e the ABA cont ent has decreased below a cert ain th reshold level. The pr esent results show th at th e sto ma ta begin to op en immediately or soon after th at point in time when th e maximal ABA level is reached. Th erefore, th e th reshold level may be just below the maximal ABA con cent ration. Thi s would mean that a threefold concent rat ion of ABA in peas and sunflowers may be sufficient to maintain sto mat al closure. This conclu sion is in acco rdance with the findi ngs of KRIEDEM ANN et al. (1972) and LOVEYS and KRIED EMANN (1973) . Th e results obtai ned with bro ad beans, ho wever, canno t be explained in th is way . Stom ata of leaves which were wi lted for onl y one hour op ened at an ABA level of about 200 0/0, whereas tho se from leaves wilted for four to five hours open ed alrea dy at 500-600 % (fig. 3). Two oth er explanat ions are possible: a) the sensitivi ty of th e guar d cells to ABA is redu ced by prolonged stress or enhanc ed ABA level, consequentl y rendering the threshold level relative to concentration and duration. Evidence for chan ges in stomatal sensitivity to ABA in response to eith er pretreatment with ABA or moisture stress has also been pr esented by KRIEDEMANN et al. (1972) and more recently by RASCHKE (1975). b) reduction of the level of ABA at its active site in the gua rd cells may be acco mplished by plant processes ot her th an degrad at ion. Removal into sto rage sites where it cann ot act on th e stomata may be also invol ved (CUMMINS, 1972 ; R ASCHKE, 1975. The decrease in the ABA curve may not onl y indicate th e onset of degrad ation of th e hormone, but also th e beginning of th e proc ess of compa rt ment aliza tion, since it is possible tha t degrad ati on occurs in special compa rt ments wit hin the cells or th e leaf tissue. Com partmentalization may also exp lain the observat ion th at th e absolute amount s of ABA in unstressed leaves (given in the legends of the figures ) sometimes diff er greatly wi thin one species (in respon se to differences in culture cond itions) without having an effect on sto matal pore size. Leaves of pea seedlings, for exa mple, cultiva ted in sand, contain about 11 ng ABA /100 mg fresh weight , but thos e grown in wa ter culture ha ve about 42 ng/IOO mg. Stom at al por e size, howev er, does not differ significant ly. Th e decrea se in th e level of ABA after wa ter str ess is most certainly du e to meta bolism, not to for mation of th e glucoside. In previous exper iment s (D ORFFLING ec al., 1974) w ith th e same variet y of pea, a ra pid metabolism of ABA - 2- t4 C, which had been app lied to wi lted shoots, was observed, and tw o ext ractabl e meta bolites - probabl y dihyd roph aseic acid and phaseic acid - could be det ected. The activity of these metabolites with regard to stomatal move ments is not known . In Z. Pflanzenphysiol. Ed. 81. S. 43- 56. 1977.

ABA and the after-effect of water stress

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several bioassays it has been demonstrated that phaseic acid is of low activity (LOVEYS and KRIEDEMANN, 1974). STA.LFELT (1955) has pointed out that the after-effect of water stress on stomata may be a «safety mechanism» against drought, enabling plants to recover their turgidity rapidly after a period of water stress by means of a delayed stomatal opening reaction, so that stomatal transpiration is reduced. This view is supported by the results presented. Moreover, the results provide evidence that this mechanism may be typical for rnesophytic plants such as pea, sunflower and broad bean, which grow naturally under conditions in which dryness sometimes occurs, as it does not appear to exist in hygrophytes such as Tradescantia and Menyanthes, which are adapted to humid conditions. More evidence, of course, is necessary to confirm this hypothesis; especially pertinent would be the investigation of a wide range of ecologically different plants and a variation of the stress conditions. It is in keeping with this hypothesis that rice plants (HIRON and WRIGHT, 1973) and submerged parts of the water plants Callitriche and Ceratophyllum (MILBORROW and ROBINSON, 1973) also show no or only a slight increase in their ABA level in response to water stress. The results obtained provide evidence that in mesophytic plants such as broad bean the increase of the ABA level is induced at much smaller leaf water deficit than in hygrophytes such as Tradescantia. To confirm this hypothesis, however, it is necessary to measure water deficit precisely in terms of water potential. This was not possible in the present work. References ALLAWAY, W. G., T. A. MANSFIELD: Experiments and observations on the after-effect of wilting on stomata of Rumex sanguineus. Canad.]. Bot. 48, 513-521 (1969). BEARDSELL, M. F., and D. COHEN: Relationship between leaf water status, abscisic acid levels, and stomatal resistance in maize and Sorghum. Plant Physiol. 56, 207-212 (1975). CUMMINS, W. R.: The metabolism of abscisic acid in relation to its reversible action on stomata in leaves of Hordeum vulgare L. Planta (Berl.) 114, 159-167 (1973). DORFFLING, K., B. SONKA, and D. TIETZ: Variation and metabolism of abscisic acid in pea seedlings during and after water stress. Planta (Berl.) 121,57-66 (1974). FISCHER, R. A., TH. HSIAO, and R. M. HAGAN: After-effect of water stress on stomatal opening potential. I. Techniques and magnitudes.]. expo Bot. 21, 371-385 (1970). FISCHER, R. A: After-effect of water stress an stomatal opening potential. II. Possible causes.]. expo Bot. 21, 386-404 (1970). HIRON, R. W. D., and S. T. C. WRIGHT: The role of endogenous abscisic acid in the response of plants to stress. ]. expo Bot. 24, 769-781 (1973). HSIAO, TH. c.: Plant responses to water stress. Ann. Rev. Plant Physiol. 24, 519-570 (1973). KRIEDEMANN, P. E., B. R. LOVEYS, G. L. FULLER, and A. C. LEOPOLD: Abscisic acid and stomatal regulation. Plant Physiol. 49, 842-847 (1972). LENTON, ]. R., V. M. PERRY, and P. F. SAUNDERS: The identification and quantitative analysis of abscisic acid in plant extracts by gas-liquid chromatography. Planra (Berl.) 96, 271-280 (1971). LOVEYS, B. R., and P. E. KRIEDEMANN: Rapid changes in abscisic acid-like inhibitors following alterations in vine leaf water potential. Physiol. Plant. 28, 476-479 (1973).

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K. DORFFLING,

J. STREICH, W. KRUSE and BARBEL MUXFELDT

Internal control of stomatal physiology and photosynthesis. 1. Stomatal regulation and associated chan ges in endo genous levels of abscisic and phaseic acids. Austr. J. Plant Ph ysiol. 1,407-415 (1974). MILBORROW, B. V., and D . R. ROBINSON : Factors affec ting the biosynthesis of abscisic acid. J. expo Bot. 24, 537-548 (1973). RASCHKE, K.: Stoma tal act ion, Ann . Rev. Plant Ph ysio!. 26, 309-3 40 (1975). - Simu lta neous requir ement of carbon dioxid e and abscisic acid for stomata l closing in Xanthiu m struma rium L. Pl anta (Berl.) 125,243-259 (1975). STALFELT, M. G.: The stomata as a hydr ophotic regulator of the wa ter deficit of th e pl ant. Ph ysio!. Pl ant. 8, 572- 593 (1955). WRIGHT, S. T. c.: Physiological and biochemica l responses to wilting and oth er stress conditio ns. In : Crop processes in cont rolled envir onments. Appl . Bio!. Ser. 2, p. 349-361 ; REES. A. R., COCKSHULL, K. E., H ARD, D. W., H URD, R. G. eds. London-New York: Acad. Press 1972. ZABADAL, T. J.: A water potential threshold for th e increase of abscisic acid in leaves. Pl ant Physio!. 53, 125-127 (1974).

Prof. Dr. K. DORFFLING, In stitut fiir Allgemeine Botan ik, Universitat Hambu rg, Post fach 30 27 22, D-2 000 H amburg 36, Federa l Republi c of Germany.

Z. Pjl anzenphy siol. Ed . 81. S. 43-56. 1977.