Further Investigations on the Occurrence and the Effects of Abscisic Acid in Algae

Biochem. Physiol. Pflanzen 184, 259- 266 (1989) VEB Gustav Fischer Verlag Jena

Further Investigations on the Occurrence and the Effects of Abscisic Acid in Algae A. TIETZ, U. RUTTKOWSKI, R. KÖHLER and W. KASPRIK Institut für Allgemeine Botanik der Universität Hamburg, Hamburg, F.R.G. Key Term Index: abscisic acid; stress, green algae

Summary After abscisic acid (ABA) had been detected in the green alga Stigeocloflium a much wider occurrence of the hormone in other green algae e.g. DUflaliella parva, Chlamydomoflas reiflhardii, Ulva lactuca, Draparnaldia mutabilis and Charafoetida could be demonstrated by combined GC-MS. The effect of exogenously applied ABA in the concentration range of 10- 7 -10- 5 Mon batch cultures of Stigeocloflium cf. teflue was rather smalI. 3 weeks after application, a slight reduction of growth was caused by ABA, and in some cases, senescence was promoted. In order to investigate the effects of salt stress on endogenous ABA levels batch cultures of DUflalielia parva (halotolerant ecotype) and Draparnaldia mutabilis (halophobe ecotype) were treated with various NaCl-concentrations. When 85.5 mM NaCl were added to the culture medium the ABA concentration of Draparnaldia cultures increased to the tenfold within a week. On the contrary, in DUllaliella the ABA level is lowest at 1.5 M NaCI. This salt concentration is regarded optimal for e.g. photosynthesis of DUflaliella.

Introduction Recently, the plant hormone abscisic acid (ABA) was detected by means of combined GC-

MS analysis in the green alga Stigeocloflium cf. tenue (TIETZ and KASPRIK 1986). Earlier efforts trying to demonstrate the unequivocal occurrence of ABA in algae failed (PRYCE 1972; NIEMANN and DÖRFFLING 1980), though "ABA-like activity" was found in members of the green and the brown algae (JENNINGS 1969; HUSSAIN and BONEY 1973; NIEMANN and DÖRFFLING 1980). In some reports the occurrence oflunularic acid in algae instead of ABA was discussed (PRYCE 1972; GORHAM 1977). There are only a few data available on the influence of exogenous ABA applications on the development of algae. TILLBERG (1970) observed certain effects of ABA on ATP-levels in Scenedesmus. Then, the uptake ofnitrate in Chlorella was found to increase markedly after ABA treatment (ULLRICH and KUNZ 1984), and growth of Ecklonia gametophytes was shown to be inhibited by the hormone (JENNINGS 1969).

In higher plants changes in endogenous ABA concentrations play an important role in stress physiology (TIETZ and TIETZ 1982). If ABA synthesized by algae is not merely a product of secondary metabolism but is somehow involved in regulation, the amount of endogenous ABA should be correlated with other parameters. Abbreviatiolls: ABA, abscisic acid; GC-MS, combined gas chromatography-mass spectrometry; HPLC, high performance liquid chromatography; SIM, selected ion monitoring; TLC, thin layer chromatography 17*

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In this paper we report the part of a screening programme to detect ABA in different algal species, and studies of effects of exogenous ABA on algal batch cultures . Moreover, the influence of stress factors such as increasing salt concentrations on the endogenous ABA level of two different algal ecotypes is investigated.

Materials and Methods Plant material Draparnaldia mutabilis (Roth) Cedergren (strain B 111.80) was obtained from the culture collection SAG, Göttingen . Dunaliella parva Lerche was provided by Prof. GIMMLER, Würzburg. Stigeoclonium cf. tenue KÜTZ. was taken from the culture collection of algae in Hamburg . Axenic cultures of Chlamydomonas reinhardii DANGEARD (strain 8381) were provided by Dr. YOJGT, Hamburg. Charafoetida BRAUN was harvested from a pond in the Hamburg Botanical Garden, and material of Vlva lactuca L., Ceramium rubrum (HUDS.) AGARDH, and Lamillaria saccharina (L.) LAM. was collected from the North Sea coast ne ar Sylt and Helgoland. Batch cultures Cultures were grown in glass vessels, illuminated with fluorescent tubes for 14 or 16 h per day with about 200 f.tmol photons m -2 S -I. The flasks were aerated with sterile air, and all cultures were maintained at temperatures between 21 and 25 oe. Draparnaldia and Stigeoclonium were grown in Bold's Basal Medium , BBM (NICHOLS and BOLD 1965) . DUllaliella was cultivated in a modified BBM, containing additional 87.66 g NaCI , 10 g MgS04 . 7H 20, 1.5 g KCI, and 2 g CaS04 . 2H 2 0 per litre. Chlamydomonas was cultivated under mixotrophic conditions according to YOIGT and MÜNZNER (1987).

Hormone extraction and purijicatioll Extraction of algal material, sampled by filtration or centrifugation, was performed with 96 % ethanol and 80 % methanol, respectively. Culture medium was adjusted to pH 3 and partitioned 3 times against ethyl ether. Further purification procedures inc1uding thin layer chromatography (TLC) on precoated silicagel plates F-254 (Merck) were done according to TIETZ and TIETZ (1985). For the last steps redistilled solvents were used in order to minimize contamination. For derivatization, sampies were transferred to 1.5 ml Reacti-vials (Pierce), taken to dryness under reduced pressure . They were then methylated either with diazomethane or with Methyl-8 (Pieree) as described in a previous paper (TIETZ and TIETZ 1985). In some cases, further purifieation of the sampie was necessary. This was performed using TLC plates developed with the solvent system ethylacetate: hexane (1: 1 v/v). Subsequently, preparative high performance liquid chromatography (HPLC) was used, especially when further analysis was by mass spectrometry. For this, a Waters HPLC model was equipped with a 300 mm LiChrosorb RP-18 (7 f.tm) column, and sampIes were eluted with a linear gradient of 30-80 % methanol in 0 .2 % acetic acid. The fractions corresponding to authentic methylated ABA were collected . Gas chromatography - mass spectrometry Quantitative determination of ABA was carried out using gas chromatography either with packed gl ass columns (OY -17) or fused silica capillary columns (BP-5, CP-Sil 5) and employing electron capture detection. SampIes were dissolved in isooetane , and aliquots of 1 ftl were injeeted . Conditions with packed columns: Injector temp . : 250°C, oven temp .: 225°C, detector temp.: 250°C, carrier gas nitrogen at 35 ml min- I . Conditions with capillary columns: On column injector tempo at the beginning 60°C, detector tempo 300 °C, oven temperature programming: till 180°C with 30°C min- I , then 1 °C min - I, carrier gas nitrogen at abou t 1 ml min - I . Peaks of ABA were identified in comparison with the retention time of authentie material (Serva, Heidelberg). For combined gas chromatography - mass spectrometry a Finnigan MAT model 8222 was

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connected to a Varian 3700 gas chromatograph, splitless injection was used. The carrier gas was helium. Electron impact ionization (70 eV) and selected ion monitoring (SIM) were employed to identify the peaks.

Results and Discussion

Occurrence oi ABA in Different Algal Species In addition to various species of the Chlorophyta, the red alga Ceramium rubrum and the brown alga Laminaria saccharina were extracted for comparison. Gas chromatograms, obtained from extracts of about 250 g fresh material, showed but small and untypical peaks corresponding to retention times comparable to those of synthetic ABA and 2-trans ABA (figures not presented). Therefore mass spectrometry could not be perforrned. This leads to the assumption that these genera possibly do not synthetize ABA or that the content of the extracts was below detection limits, at least during the season of sampling (May). On the other hand, occurrence of ABA could easily be demonstrated in species of green algae (Table 1). As an example forthe GC-MS results, in Fig. 1 selected ion monitoring (SIM) Table 1. ARA contents of algal extracts. ng ABA g-I fr. wt. cells

Draparnaldia 60d

4.75

Dunaliella 60d, 1.5M NaCl

40.0

Chlamydomonas 5d

not tested

ng ABA 1-1 g-I fr. wt. culture medium

15.0 130 95.7

Ulva

0.44

not tested

Chara

4.1

not testet

and mass spectrum of ABA from Dunaliella medium extract and Chara cell extract are shown. Fragments mJz 278 (M+), 190 (base peak), 162, 134, and 125 correspond to those of authentie methylated ABA and 2-trans ABA (TIETZ et al. 1979; TIETZ and KASPARIK 1986). The results obtained from the other green algae were quite similiar and are therefore not described in detail. The contents of ABA and 2-trans ABA did not differ considerably. This might be due to isomerization of ABA by irradiation with light during cultivation and subsequent purification of the extracts (PLANCHER 1979), although care had been taken to avoid bright light during extraction and purification. Contradictory to the above assumption is the high 2-trans ABA concentration in extracts from Chara thalli, forrnerly growing in a rather dark pond of the botanical garden. As far as the biosynthetic pathways of ABA are under discussion (CREELMAN and ZEEV AART 1984) it might be true that algae also produce endogenous 2-trans ABA maybe via carotenoids and 2-trans xanthoxin (NONHEBEL and MILBORROW 1987). With the exception of Chlamydomonas algal material was not axenic, but judged from microseopie observation there were only very few bacteria present. Since ABA could be BPP 184 (1989) 3/4

261

CHROMA10GRAM MAP ABA CHARA

~lC.

-

-- --

-

---

1-H ,<

-

-

~----

-

MASS 27B - :---- -- - 260

:A --

222

---1

JllI

---1

190 162 134 125

-~ 900

850

134 125

sm

950

190

100.0 HASS SPECIRUH CHARA SCAN 888-879

RIC.CHROMA1OGRAM MAP DUNAllEllA ABA

I-ABA

-

i

,

I-ABA

- --~--

MASS 278

--~fi!

i

900

950

SCAN

190

125 100.0

162

,ll~ T :I :r li 5

140

-

i

850

134 125

50.0

120

--

-~ ~-~~--

100.0 100.0 HASS SPEClRUM OUNAllEllA SCAH 8BO - 875

50D

M/Z 100

-

2

T T

H' 2t6 2~0 271l

160 180 200 220 240

260 280

1.1~ 300

MIZ 100

120 140

50.0

162

160

175

2j5

180 200

~F

246 260

~8

Z20 240 260 280 300

Fig. 1. Reconstructed ion current (RIC) , selected ion monitoring of major fragment ions, and mass spectra of Chara and Dunaliella extracts, showing the presence of ABA.

detected in non-axenic as weil as axenic algal batch cultures, and as different genera of macro algae from the same sampling place may or may not contain ABA, contamination of extracts by ABA of bacterial or experimental origin is considered most unlikely. At the beginning of the research programme, it was not possible to detect ABA in cultures of Fritschiella tuberosa and Draparnaldia plumosa (TIETZ and KASPRIK 1986). However, preliminary results of recent experiments indicate that , under different culture conditions, ABA mayaIso be produced by these two genera. Thus abscisic acid seems to occur more often in green algae than had been assumed be fore . Perhaps most if not all green algae are capable of synthesizing ABA. Effects 0/ Exogenous ABA Applications Batch cultures of Stigeoclonium cf. tenue were grown in ABA concentrations from 10- 7 to 10- 5 M for 3 weeks. Growth ofthe alga was measured as fresh and dry weight accumulation. Probable changes in cell size were analysed by microscopic measurements. Growth of Stigeoclonium was only slightly affected by the hormone. In some experiments dry weight accumulation was decreased to a minor extent especially with 10- 5 M ABA. Lower ABA concentrations led to a small increase in growth, but on the whole, there were no significant differences (Fig. 2). Microscopic measurements of cell length and cell breadth revealed small differences between ABA - treated cultures and the controls (Fig. 3). In many cases the cell width (as in senescent cultures) was somewhat increased by ABA as weH as the number of dead cells. Up to now, there is no explanation for the weak effect of exogenously applied ABA on 262

BPP 184 (1989) 3/4

• Control o 10- 7 ABA

600

..,

.&:

··Gi

=-

A

400

D

10- 6 ABA 10- 5 ARA

"0

er>

E

200

O-;-==------.--------.--------r'

o

7

doys

14

21

Fig. 2. Riomass production in Stigeoclonium batch cultures after application of 10- 7 to 10- 5 M ARA, measured as dry weight accumulation.

~mr-------~====~------~ 25

20 15

~i~\ i

10

O~~p · ·~··~~~··~··~-~ · ·~·~-~~

10-7 10-6 ABA concentroti on (M 1

10- 5

Fig. 3. Microscopic measurements of cell breadth and cell length in 3 weeks old Stigeoclonium cultures, growing in 10- 7 to 10- 5 M ARA. Bars represent standard deviation of the mean value.

Stigeoclonium cultures. One reason might be that the metabolism of this alga, which produces a considerable amount of ABA and may even trans form it as rapidly, renders the organism rather insensitive to exogenous applications of the hormone. In this context it would be interesting to know, if metabolie degradation of ABA in algae follows the same pathways as in higher plants, wh ich is via phaseic acid and dihydrophaseic acid (TIETZ et al. 1979). Recently a different metabolie pathway was observed in the fungus Ceratocystis (KETTNER and DÖRFFLING 1987). Salt Stress and Endogenous ABA Levels Assuming that ABA plays a physiologie al role in algae, at least in part comparable to that in higher plants, stress should influence ABA metabolism . Therefore two different ecotypes of algae were exposed to osmotical stress by treating them with various concentrations of NaCI in the medium. Draparnaldia mutabilis, wh ich is not salt tolerant, was already markedly inhibited in growth by salt concentrations between 51.3 and 85.5 mM NaCl. In addition, bleaching of chlorophyll was observed. During aperiod of 8 days after NaCI-application, a BPP 184 (1989) 3/4

263

ABA ng

1000

o

culture medium (ng/l) • cells (ng/g Ir. wt.) o

800

600

400

200

I

• 0.0513

0.0171

0.0855

M KaCI

Fig. 4. ABA content in cells and culture medium of Draparnaldia mutabilis (halophobe ecotype) after application of NaCI.

ABA ng

200 160 120 80 o culture medium (ng/l) • cells (ng/g Ir. wt.)

40

0.5

1.0

1.5 M KaCI

2.0

2.5

3.0

Fig. 5. ABA content in cells and culture medium of Dunaliella parva (halotolerant ecotype) after application of NaCI.

marked increase of ABA levels in the medium and in the cells was detected (Fig. 4). The high levels in the medium compared to those in the cells might indicate that osmotic stress in this species perphaps induces leaching of ABA by affecting membrane properties. The stress periods could not be prolonged successfully, because the algal cells were injured seriously. On the contrary, in cultures of Dunaliella parva. a halotolerant alga which occurs in extremely saline habitats, lowest ABA contents in cell as weIl as in the medium were found at about 1.5 M NaCI (Fig. 5). At this NaCI concentration Dunaliella shows optimal CO r 264

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assimilation under experimental conditions (GIMMLER et al. 1981). Lower as weH as higher amounts of salt lead to an increase ofthe ABA contents. But although the concentrations of salt were much higher compared to the Draparnafdia cultures, changes of ABA levels in the culture medium of Dunaliella are rather smaH. This may indicate that Dunalielfa is weH adapted to extreme saline environments. In contrast with the report of GIMMLER et al. (1981), in our experiments biomass production in Dunaliella cultures decreased with increasing salt concentration. These contradictory results may depend on different culture conditions, e.g . aeration. GIMMLER et al. (1981) employed 1.5 % CO 2 whereas in our experiments air was used. In higher plants, beside ABA concentration, so me other parameters are affected by stress, for example proline levels . This amino acid, which obviously protects proteins from dehydration, also accumulates in green algae under osmotic stress conditions (BORNMANN 1987). This coincidence indicates that there are certain similarities in stress metabolism between lower and high er plants and that ABA in algae mayaiso be somehow involved in regulatory processes of stress . Of course, algae lack stomata and thus a conspicuous target of ABA cannot be observed, but, e.g. at the level of transport across membranes one should expect metabolic pathways, wh ich could be influenced by ABA in the context of algal stress resistance.

Acknowledgements We wish to thank INGE ERXLEBEN, University of Bremen, for preparing the mass spectra and Prof. H. GIMMLER, University of Würzburg, for providing cultures of Dllnaliella. Material of Chlamydomollas was kindly provided by Dr. J. VOIGT from our institute.

References BORN MANN , E.-J . : Osmoregulation bei halotoleranten Bakterien, Pilzen und Algen. Biol. Rundschau 25 , 27-34 (1987). CREELMAN, R. A. , and ZEEV AART , J. A. D . :Incorporation of oxygen into abscisic acid and phaseic acid from molecular oxygen. Plant Physiology 75. 166-169 (1984). GIMMLER, H . , WIEDEMANN, C ., and MÖLLER, E.-M. : The metabolie response ofthe halotolerant green alga Dllllaliella parva to hypertonie shocks . Berichte der Deutschen Botanischen Gesellschaft 94,

613-634 (1981). GORHAM, J.: Lunularie acid and related compounds in liverworts, algae and Hydrangea. Phytochemis-

try 16,249-253 (1977). HUSSAlN, A., and BONEY, A. D.,: Hydrophilie growth inhibitors from Laminaria andAscophyllllm. The New Phytologist 72, 403-410 (1973). JENNINGS, R . C.: Gibberellin antagonism by material from a brown alga. The New Phytologist 68,

683-688 (1969) . KETTNER , J., and DÖRFFLING, K. : Abscisic acid metabolism in Ceratocystis coerulescens. Physiol. Plant. 69.278-282 (1987) . NICHOLS, H. W., and BOLD, H. C .: Trichosarcilla polymorpha gen. et sp. nov . Journal ofPhycology 1,

34-38 (1965). NIEMANN , D. I., and DÖRFFLING, K.: Growth-inhibitors and growth-promoters in Enteromorpha compressa (Chlorophyta). Journal of Phycology 16, 383-389 (1980). NONHEBEL, H. M., and MilboITow, B. V .: Contrasting incorporation of 2H from 2H 2ü into ABA , xanthoxin and carotenoids in tomato shoots. J. Exp. Bot. 38.980-991 (1987). BPP 184 (1989) 3/4

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PLANCHER, B.: Anmerkungen zur UV -Isomerisation der Abscisinsäure. Gartenbauwissenschaft 44, 184-191 (1979). PRYCE, R. J.: The occurrence oflunularic and abscisic acids in plants. Phytochemistry 11, 1759-1761 (1972). TIETZ, A., and KASPRIK, W.: Identification of abscisic acid in a green alga. Biochem. Physiol. Pflanzen 181, 269-274 (1986). TIETZ, D., and TIETZ, A.: Less hazardous derivatization procedure for gas chromatography of plant hormone abscisic acid. Journal ofChromatography 325, 425-429 (1985). TIETZ, D., and TIETZ, A.: Streß im Pflanzenreich. Biologie in unserer Zeit 12 (4), 113-119 (1982). TIETZ, D., DÖRFFLING, K., WÖHRLE, D., ERXLEBEN, I., and LIEMANN, F.: Identification by combined gas chromatography - mass spectrometry of phaseic acid and dihydrophaseic acid and characterization of further abscisic acid metabolites in pea seedlings. Planta 147, 168-173 (1979). TILLBERG, J. E.: Effects of abscisic acid, salicyclic acid and transcinnamic acid on phosphate uptake, ATP-Ievel and oxygen evolution in Scenedesmus. Physiol. Plant. 23,647-653 (1970). ULLRICH, W. R., and KUNz, G.: Effect of abscisic acid on nitrate uptake, respiration and photosynthesis in green algae. PI. Sei. Lett. 37,9-14 (1984). VOIGT, J., and MÜNZNER, P.: The Chlamydomonas cell cycle is regulated by a lightldark-responsive cell-cycle switch. Planta 172, 463-472 (1987).

Received February 16, 1988; revisedform accepted May 27, 1988 Author's address: Dr. A. TIETZ, Institut für Allgemeine Botanik der Universität, Ohnhorststraße 18, D - 2000 Hamburg 52.

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