Institute of Physiological Botany, University of Uppsala, Uppsala, Sweden
Morphogenetic Effects of Phenylacetic and p-OH-Phenylacetic Acid on the Green Alga Enteromorpha compressa (1.) GREV. in Axenic Culture LISBETH FRIES
and
STIG ÄBERG
With 2 figures Received 7 November 1977 . Accepted 17 March 1978
Summary In axenic culture Enteromorpha compressa forms tubular sporelings which develop into sm all bulbous thalli. Phenylacetic acid or p-hydroxyphenylacetic acid in conc. 10-7-10-5 M induces the thalli to stretch into real tubes. Kinetin, 100 ,ug/l has a similar effect which is enhanced by the combination with some of the phenylacetic acids. Phenylacetic acid was identified by GS-MS technique in wild-growing Enteromorpha as the first species among the green algae. The effects are strongly bound seasonally and are thus coupled to the activity of another unspecified growth regulator.
Key words: Green alga, Enteromorpha, p-hydroxyphenylacetic acid, auxin, kinetin.
benzoic
acid,
phenylacetic
acid,
Introduction After some time in axenic culture many members of the family Ulvaceae develop small pincushion-like stages (PROVASOLI, 1958; PROVASOLI and PINTER, 1977; FRIEs, 1971, 1973, 1975). Many experiments have been made to reestablish the natural morphology in Ulva and Enteromorpha species by addition of different wellknown growth regulators. Work has been especially intensive in Ulva (PROVASOLl et al., 1977). In the following we will describe how thalli in an early stage are influenced by some growth regulators.
Material and Methods Enteromorpha plants were collected on the Swedish west coast and pieces of thin thallus tubes were treated with antibiotics to obtain axenic plants (FRIES, 1963). The species was classified by Dr. CARL BUDING as E. compressa (L.) GREV. In bacterized stage E. compressa is very varying. It can form single tubes or thalli with Abbreviations: GC = gas-liquid chromatography; MS = mass-spectrometry; PAA phenylacetic acid; p-OHPAA = p-hydroxyphenylacetic acid; IAA = indolylacetic acid. Z. Pjlanzenphysiol. Bd. 88. S. 383-388. 197'1,
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several branches. In axenic conditions it forms sm all moss-like tufts built up by uniseriate, branched filaments (FRIES, 1975). A few tubular fronds can be produced on the moss-like plants but they soon stop growing and form protrusions on the surface. These protrusions can loosen and grow into new plants where the tubes are reduced to small morel-like formations consisting of conglomerates of sm all vesicles of a size less than 1 mm. When placed in natural daylight and 20°C swarms of spores were formed in some flasks after vernal equinox and until early June. A few of the spores developed into tubular sporelings. If left in the original thick mat of sporelings they soon lost their colour and died or were grown over by «mossplants». Young tubular sporelings transferred into flasks, one per flask, developed into small morel-like bulbous plants. Y oung sporelings such as these were used as inocula. The alga was cultivated in artificial seawater ASP6 F2 (FRIES, 1977) in 100 ml Pyrex flasks containing 25 ml medium. All additions were dissolved in water, autoclaved and added to the sterilized medium. The experiment was placed in an incubator at 17°C with light for 18 h per day from a lightbank containing 5 tub es (Philips, 2XTL29, 2XTL55, 1 XTL05) giving 15-19.5 W 1m 2 • The experimental series comprised 6 paralleIs. The experiments were run from April to June during two consecutive years. Algal material for MS-determination was collected on 7 August 1977 on the Swedish west coast from rocks exposed to the open sea and in an area with little population influence. The algae belonged to the Enteromorpha intestinalis group and therefore E. compressa was included but not examined further. The material was deepfrozen immediately and stored at -18°C for 7 weeks. Deepfrozen material of E. sp. was grinded with liquid nitrogen and extracted with twice their volume of acetone for 24 h in +4 oe. The algae were filtered and extracted a second time with 80 % acetone-water for 15 min in an ultrasonic bath (Branson Instr. Co. Stamford Conn. USA) under cooling to 30 oe. The two acetone extracts were pooled. For further extraction we followed the method described by WIGHTMAN (1977). The acidic ether fraction was minimized and an extract corresponding to 65 g alga was transferred to a microsilylation vessel, concentrated to dryness and further exposed to phosphoruspentoxide to remove every trace of water. BSA (= bis [trimethylsilyl] acetamide) 10,u1 and MeCN 20,u1 were added and the entire mixture heated at 60°C for 15 min and subjected to GC-MS (PEDERSEN 1976). The analyses of the trimethylsilyl derivatives so obtained were carried out on a LKB 9000 mass spectrometer coupled to agas chromatograph described by BERGSTRÖM (1973). 1.2 m X 4 mm i.d. silanized glass column packed with 3 % (w/w) SE 30 on 80/100 mesh acid-washed DMCS (= dimethyldichlorosilane)-treated Chromosorb W was used. The detector was heated to 240 oe. The pre-column was packed with the same material. Helium was emloyed as the carrier gas at a flow of 25 ml min- 1 •
Results and Discussion (1969) found a strong stimulation on Enteromorpha sp. still in the tubular stage by a few ml of the medium where bacterized or axenic thalli of E. linza had grown. We tried the effect of the culture medium where a secondarily contaminated E. compressa had grown and developed thalli. One ml from the contaminated medium added to a flask with a sporeling initiated normal thallus formation (Fig. 1). If the contaminated medium was autoclaved the sporlings grew out into a bulbous stage similar to the control but bigger and many new plants were formed from protrusions on these fronds. There were thus strong indications that E. compressa had lost its ability to produce a growth regulator after a certain period in bacteria-free culture. CHANDRAMOHAN (1971) showed that bacteria living on the surface of E. intestinalis could synthesize BERGLUND
z.
Pjlanzenphysiol. Bd. 88. S. 383-388. 1978.
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IAA from tryptophan and SCHIEWER (1967) had found previously that only traces of added tryptophan were transferred into IAA in macroalgae, when their bacterial flora was suppressed by antibiotics. This growth regulator could thus be suspected to
a Fig. 1: E. compressa growing in ASP6 F2. a. No addition: Bulbous thalli. b. Nutrient medium from a secondarily contaminated E. compressa culture added, 1 ml per flask: Tubular, branched thalli. Incubation time 48 days. All algae from the six parallels in one series were transferred for photographing to a Pe tri dish with a diameter of 7 cm.
represent a mInimum factor in axenic Enteromorpha cultures. IAA was given to young sporelings in the same concentrations that PROVASOLI (1958) has found to influence growth of Vlva, i. e. 10-100 pg .1- 1 • It was also combined with kinetin, 100 pg .1- 1 • When 4.10- 7 M IAA was given alone it induced normal thallus growth in some experiments, but the results were very erratic. PAA and p-OHPAA have long been known (HANSEN, 1954) to give auxin effects in higher plants, but it is onlY in recent years they have been identified as naturally occurring substances in these plants (OKAMOTO et al., 1967; WIGHTMAN and RAUTHAN, 1974). About the same time they were identified in the brown alga Vndaria pinnatifida (ABE et al., 1974). The effects of these phenylacetic acids were tested in concentrations of 4· 10- 7_4 . 10- 5 M either alone or in combination with kinetin 100 pg' 1- 1 (Fig. 2). The experiment was run du ring May. In the control flasks the sporelings developed into small bulbous thalli. When P AA or p-OHP AA were given separately apparent effects could be noted in all concentrations used. Kinetin enhanced the effects further. In all se ries there were sporelings which grew out into the bulbous type but were bigger than the control plants. In certain flasks the plants stretched to comparatively coarse tubes. The phenylacetic acids thus acted as growth regulators. In one flask with p-OHP AA in combination with kinetin a thallus of 12 cm was produced, but the wall-cells were so loosely connected that the thallus fell apart when transferred from the flask to the Petri dish for photogaphing. This might indicate how these phenylacetic acids are acting. When the experiments were repeated later in summer only bigger bulbous thalli developed into the P AA series, but the spring results were confirmed during the following spring. Z. Pflanzenphysiol. Bd. 88. S. 383-388. 1978.
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LISBETH FRIES
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5TIG ÄBERG
0
KInetin
~.
100~g/l
4'10- 7
. CV
----..'
r
f. No kinetin
.
."
~
4.10- 6
4'10- S M
P-OHPAA
..(
PAA
• ~
P-OHPAA
PAA
Fig. 2: Morphogenetic effects of PAA and p-OHPAA in conc. 4· 10-7 M-4' 10-5 M added separately or in combination with 100 ,ug kinetin per litre. Incubation time 30 days. For photographing, see Fig. 1.
In experiments performed hitherto no real additive effect of IAA and PAA or p-OHPAA has been obtained in E. compressa. This fact might be explained by the suggestion of MU.BORROW et al. (1975) that in higher plants IAA and PAA affect different sites although in some material they give similar auxin effects. The quest ion then arises whether these phenylacetic acids are naturally occurring growth regulators in Enteromorpha. As we could not produce sufficient material of axenic E. compressa in thallus stage for an analysis we had to use naturally growing algae. All marine algae have more or less bacteria as epiphytes but we collected our material from a habitat as clean as possible and tried to avoid secondary contammatlons. The GC-MS technique revealed two phenolic compounds with only one TMSi group, one at 143 °C and the other at 148 oe. By comparing with authentie sampies the former was identified on MS as benzoie acid and the latter as PAA. No p-OHP AA could be detected. That is the first time that P AA has been identified in a green alga. The role of bacteria in the P AA production has not been investigated further . We do not know whether PAA is a result of the activity of the algal cells or is produced Z. Pjlanzenphysiol. Bd. 88. S. 383-388. 1978.
Morphogenetic effects of phenylacetic acids on Enteromorpha
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by the bacteria which might also have induced production in the alga. However, when growing in their natural habitat Enteromorpha has P AA available, which most probably acts as a natural auxin. Additions of P AA influenced only tubular plants and even in combination with kinetin it did not initiate the development from moss stage into tubular stage. Apparently P AA is only one of several growth regulators necessary for normal growth in an axenic alga. Another growth regulator or regulators must be formed during the constellation of light and day length after vernal equinox. Such as substance or substances can apparently be formed by epiphytic bacteria at any time of the year, as seen from Fig. 1. From epiphytic bacteria on the green alga Monostroma sp. a substance has in fact been iso la ted - but not yet identified which can restore the tubular stage in this al ga (PROVASOLI et al., 1977). Auxin-like effects of PAA and p-OHPAA have previously been found in axenically cultivated multicellular species of red algae (FRIES and IWASAKI, 1976) and of brown algae (FRIES, 1977) and now we can also add a green species. The identification of phenylacetic acid in Rhodomela larix (KATSUI et al., 1967) and of 3,5-dibromo, p-hydroxyphenylacetic acid from Halopytis in cu rens (CHANTRAINE et al., 1973) as well as the metabolism of tyrosine to p-OHP AA in cellfree preparation of Odonthalia flocosa (MANLEY, 1977) speaks in favour of a production of the two phenylacetic acids in red algae. In the brown al ga Undaria pinnatifida (ABE et al., 1974) both acids have been indicated and later P AA in Sargassum muticum (GORHAM, 1977). The identification of P AA in a green alga supports the conclusion that at least P AA is a natural auxin generally occurring in marine algae. We thank Dr. M. PEDERSEN for valuable advice on mass spectrometry and Mrs. GUN RÖNNQUIST for valuable technical assistance. This investigation was supported by a grant from the Swedish Natural Science Research Council.
References ABE, H., M. UCHlYAMA, and R. SATO: Isolation of phenylacetic acid and its p-hydroxy derivative from Undaria pinnatijida. Agr. Biol. Chem. 38, 897-898 (1974). BERGLUND, H.: Stimulation of growth of two marine green algae by organic substances excreted by Enteromorpha linza in unialgal and axenic cultures. Physiol. Plant. 22, 1069-1073 (1969). BERGSTRÖM, G.: Studies on natural odoriferous compounds. VI. Use of a pre-column tube for the quantative isolation of natural, volatile compounds for gas chromatography/mass spectrometry. Chem. Scr. 4,135-138 (1973). BONNEAU, E. R.: Polymorphie behavior of Ulva lactuca (Chlorophyta) in axenic culture. I. occurrence of Enteromorpha-like plants in haploid clones. J. Phycol. 13, 133-140 (1977). CHANDRAMOHAN, D.: Indole acetic acid synthesis in sea. Proc. Indian Acad. Sc. Sero B. 73, 105-109 (1971). CHANTRAINE, J. M., G. COMBAUT et J. TESTE: Phenols bromes d'une algue rouge, Halopytis incurvis: acides carboxyliques. Phytochemistry 12,1793-1796 (1973).
z. Pjlanzenphysiol. Bd. 88. S. 383-388. 1978.
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FRIES, L.: On the cultivation of red algae. Physio!. Plant 16, 695-708 (1963). - Behovet av organiska substanser bland havets litorala al ger. Svensk naturvetenskap 1971. Ed. B. A/zelius. Swedish National Research Counci!. In Swedish with figure texts and summary in English (1971). - Requirements for organic substances in seaweeds. Bot. Mar. 16, 19-31 (1973). - Some observations on the morphology of Enteromorpha linza (L.) AG. and Enteromorpha compressa (L.) GREV. in axenic culture. Bot. Mar. 18, 251-253 (1975). - Growth regulating effects of phenylacetic acid and p-hydroxy phenylacetic acid in Fucus spiralis L. (Phaeophyceae, Fucales) in axenic culture. Phycologia 16, 451--456 (1977). FRIES, L. and H. IWASAKI: p-Hydroxyphenylacetic acid and other phenolic compounds as growth stimulators of the red alga Porphyra tenera. Plant. Sc. Lettr. 6, 299-307 (1976). GORHAM, J: Plant growth regulators in Sargassum muticum. J Phyco!. 13. Supp!. Abstr. IX. Int. Seaweed Symp. Santa Barbara 1977, 25 (1977). HANSEN, B.: A physiologie al classification of shoot auxins and root auxins. Bot. Not. 230-268 (1954). KATSUI, N., Y. SUZUKU, S. KrTAMuRA, and T. IRIE: 5,6-Dibromoprotocatechualdehyde and 2,3-dibromo-4,5-dihydroxybenzyl methyl ether. New dibromophenols from Rhodomela larix. Tetrahedron 23, 1185-1188 (1967). MANLEY, S. L.: Metabolism of tyrosine by cellfree fractions of the red alga Odonthalia /loccosa: A proposed biosynthetic pathway for brominated phenols. J. Phyco!. 13. Supp!. Abstr. IX. Int. Seaweed Symp. Santa Barbara 1977, 42 (1977). MILBORROW, B. V., J G. PURSE, and F. WIGHTMAN: On the auxin activity of phenylacetic acid. Ann. Bot. 39, 1143-1146 (1975). OKAMOTO, T., Y. ISOGAI, and T. KOIZUMI: Studies on plant growth regulators. Isolation of indole-3-acetic acid, phenylacetic acid and several plant growth inhibitors from etiolated seedlings of Phaseolus. Chem. Pharm. Bull. 15, 159-163 (1967). PEDERSEN, M.: A brominating and hydroxylating peroxidase from the red al ga Cystoclonium purpureum. Physio!. Plant. 37, 6-11 (1976). PROVASOLI, L.: Effects of planthormones on Ulva. Bio!. Bull. Mar. Bio!. Lab. 114, 375-384 (1958). PROVASOLI, L. and I. J PINTNER: Bacteria-induced polymorphism in an axenic laboratory strain of Ulva. Proc. VIII Int. Seaweed Symp. Bangor 1974. In print (1977). PROVASOLI, L., I. J. PINTNER, and S. SAMPATHKUMAR: Morphogenetic substances for Monostroma oxyspermum from marine bacteria. J Phyco!. 13. Supp!. Abstr. IX, Seaweed Symp. Santa Barbara 1977 (1977). SCHIEWER, U.: Auxinvorkommen und Auxinstoffwechsel bei mehrzelligen Ostseealgen. 11. Zur Entstehung von Indol-3-essigsäure aus Tryptophan, unter Berücksichtigung des Einflusses der marinen Bakterienflora. Planta (Berlin) 75, 152-160 (1967). WIGHTMAN, F.: Gaschromatographie identification and quantative estimation of natural auxins in developing plant organs. Plant growth regulation. Proc. 9th Int. Conf. Plant growth Substances, Lausanne 1976. P. E. PILET (Ed.). New York 1977. ' WIGHTMAN, F. and B. S. RAuTHAN: Evidence for the biosynthesis and natural occurrence of the auxin, phenylacetic acid, in shoots of higher plants. Plant growth substances 1973. Tokyo: Mirokawa Pub!. Co. ine., pp. 15-27 (1974). Dr. L. FRIES, Institute of Physiologie al Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden.
Z. Pjlanzenphysiol. Bd. 88. S. 383-388. 1978.