p-Hydroxyphenylacetic acid and other phenolic compounds as growth stimulators of the red alga Porphyra tenera

Plant Science Letters, 6(1976) 299--307 299 © ElsevierScientific Publishing Company, Amsterdam-- Printed in The Netherlands

p-HYDROXYPHENYLACETIC ACID AND OTHER PHENOLIC COMPOUNDS AS GROWTH STIMULATORS OF THE RED ALGA P O R P H Y R A T E N E R A

LISBETH FRIES Institute of Physiological Botany, University of Uppsala, Box 540, Uppsala (Sweden) and HIDEAO IWASAKI Faculty of Fisheries, Mie University, Tsu (Japan) (Received November 10th, 1975) (Revision receivedand accepted February 9th, 1976)

SUMMARY Growth stimulations were obtained in axenic cultures of Porphyra tenera KjeUm. (Rhodophyta) in the "Conchocelis phase" by several simple phenolic compounds. Caffeic acid, syringaldehyde, 4-methyl catechol and 2,5
INTRODUCTION Many simple phenolic compounds have been found to affect the growth and morphology of some red algae in axenic culture. Growth stimulation of far more than 100% has been obtained with phenolic substances of both C6-C3 and C6-C1 types [1--3]. Many species in the Floridae contain high Abbreviation: IAA, indole-3-acetic acid.

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amounts of brominated phenolic compounds while in the Bangioideae no such brominated compounds have been detected [4]. As the strongest growth stimulation with phenols was obtained in Goniotrichum alsidii (Zanard.) Howe, a species belonging to the latter group, it seemed advisable to confirm these observations by experiments with other species lacking bromophenols. Accordingly an investigation was undertaken with Porphyra tenera Kjellm. (Bangioideae) in the "Conchocelis phase". It was started at the Institute of Algological Research, Hokkaido University, Muroran, Japan, and completed in Uppsala. M A T E R I A L AND METHODS

Porphyra tenera was brought into axenic culture in the "Conchocelis phase" by the second author [5] and has been maintained in his laboratory. The nutrient medium, ASP 12NTA previously prepared for Porphyra by Iwasaki [5] was used. Like sea water, the medium had a pH of 8.3. To make it more comparable to natural sea water, the bromide content was increased to 814 ~gatoms/l, even though Iwasaki [6] had found 60 pg-atoms/1 more favourable for the growth of Porphyra. The preliminary experiments were performed in glass tubes containing 10 ml of medium. Later, 100 ml Pyrex flasks with 25 ml medium were used. The phenols were dissolved directly in distilled water and the solutions were sterilized by filtration through a membrane filter, pore size 0.2 #m (Sartorius, GSttingen), and added separately to the autoclaved nutrient medium. To minimize the transfer of substances leaking from old plants in the inoculum flasks, only one young plant of the "Conchocelis phase" was transferred per experimental flask or tube. To obtain essentially equal growth in the experimental series the inoculation was made "across the series" and the flasks were placed randomly under light banks containing warm white tubes, Philips 33, with the addition of one incandescent bulb, and giving an irradiance of 22--25 W/m 2 . Three different combinations of daylength and temperature were used: (1) 10°C and 10 h light, which is favourable for thallus formation and growth in non-axenic cultures; (2) 15°C and 15 h light, which gives good Conchocelis growth [7] ; and (3) 20°C and 20 h light which has been found to be favourable for axenic cultivation [5]. The axenic Porphyra grows extremely slowly. The second author needed 7 months in his previous axenic experiments to obtain weighable amounts of dry matter, even when algae from all flasks in a series were weighed together. To enhance growth the daily light period was made as long as possible, 20 h, since 4 h darkness had been found long enough for the response to phenolic compounds in Goniotrichum [1]. Where possible the mean dry weight, ± standard error, of the plants from the 6 separate flasks in a series is given as the measure of growth. However, in some experiments the weights so obtained were so small that the dried

301

plants from six flasks in one series had to be weigl~d together. The radii of spore plants were also used as a growth parameter. RESULTS

Some of the compounds most active in Goniotrichum alsidii were chosen as representative of the phenolic substances to be tested [3]. The effects of the two cinnamic acid derivatives, caffeic acid and syringaidehyde, as well as the C4~1 compound 4-methyl catechol were thus tested in amounts of 4.10-7--4.10-s M (Table I). Growth stimulation was obtained with all these substances and confirmed the preliminary results we obtained with tube cultures at 16°C and 14 h light in the Institute of Algological Research, Muroran. Two substances, p-hydroxyphenylpyruvic acid and 2,5~lihydroxyphenyl acetic acid (homogentisic acid), which are natural products of higher plants and algae and have a central position in the biosynthesis of many phenolic derivatives [8], influence the growth and morphology in Goniotrichum [3]. When tested on Porphyra (Table II) under long or short day conditions, p-hydroxyphenylpyruvic acid gave an increase of growth up to 100% at low temperature at a concentration of 4 . 1 0 -4 M, while the effect at 20 ° was not statistically significant. Homogentisic acid was active over a wider range, and an optimal increase in growth was obtained with a concentration of 4 . 1 0 -4 under long day conditions. No thaili were formed during the experiment. Some other phenolic substances such as phenylacetic acid, p-hydroxyphenyl. acetic acid, and p-phenyloxyacetic acid, known to give auxin effects in higher plants [9,10] were also tested. The first two substances could be of special TABLE I GROWTH OF "CONCHOCELIS STAGE" OF P O R P H Y R A TENERA AS AFFECTED BY SOME SIMPLE PHENOLIC COMPOUNDS Nutrient used: ASP~2NTA with 814 ~g-atoms Br/l. 20°C, 20 h light. Incubation time: Expt. I: 80 days; Expt. H: 90 days.

Expt.

Substance added

Dry weight of algae from 6 flasks (mg) Conc. in medium of substance added

I H I II II

Caffeic acid Caffeic acid Syringaldehyde Syringaldehyde 4-Methylcatechol

0

4*10-7M

4.10-6M

4.10-5M

0.6 0.4 0.6 0.4 0.4

1.2 0.6 0.6 0.6 1.2

1.8 0.8 1.0 0.8 1.0

2.6 1.2 0.6 0.1 0.6

Analysis of variance gave F-values (regression of growth compared to concentration) significant at 0.05 for caffeic acid and syringaldehyde.

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TABLE II GROWTH STIMULATION ON "CONCHOCELIS" PHASE OF P O R P H Y R A T E N E R A BY p-HYDROXYPHENYLPYRUVIC ACID AND 2.5-DIHYDROXYPHENYL ACETIC ACID (HOMOGENTISIC ACID) UNDER D I F F E R E N T LIGHT AND TEMPERATURE CONDITIONS Incubation time 60 days Temperature and day length

Substance added

Dry weight of algae from 6 flasks in mg Concentration in medium of substance added

10°C 10 h

20°C 20 h

p-Hydroxyphenylpyruvic acid 2.5-Ditlydroxyphenylacetic acid p-Hydroxyphenylpyruvic acid 2.5-Dihydroxyphenylacetic acid

0

4.10-7M

4.10-~ M

4.10-5M

0.8

1.3

1.6

0.8

0.8

1.5

1.2

1.5

0.8

0.9

1.3

0.5

0.8

1.5

1.9

1.3

Differences among concentrations of p-hydroxyphenylpyruvic acid were significant at the 0.1 probability level and differences among concentrations of 2.5-dihydroxyphenylacetic acid approached significance of the 0.05 level as revealed b y analysis of variance and F-test.

TABLE III INFLUENCE ON THE GROWTH OF "CONCHOCELIS PHASE" OF P O R P H Y R A T E N E R A BY SOME PHENYLACETIC ACIDS COMPARED TO THAT O F I A A Light conditions 15 h. Temperature: 15°C. Incubation time 70 days. Substance added

Mean dry weight (rag) of algae from 6 flasks Conc. in medium of substance added

Phenylacetic acid Phenyloxyacetic acid p-Hydroxyphenylacetic acid Indole-3-acetic acid

0

4"10-7M

4-10-~ M

2-10-5 M

0.18+0.06 0.18+0.06 0.18±0.06 0.18±0.06

0.30±0.07 0.26±0.1 0.27.±0.06 0.20±0.05

0.32±0.1 a 0.14±0.0 0.25±0.04 0.15±0.05

0.46±0.10

S.E., 0.04; LSDo.os, 0.12; HSD0.05, 0.21. a Significantly different from control at P < 0.05.

0.28±0.07 0.30±0.09 0.20±0.0

a

303 interest as they had been isolated from the brown alga Undaria pinnatifida by Abe et al. [11,12]. As the results presented in Table III show the growthpromoting ability of phenylacetic acid on Porphyra, measured by dry weight, is evident. Iwasaki [5] found strong growth-promoting effects by IAA at a concentration of 20--50/~g/l (10 -7 M). 100 ~zg/l did n o t increase growth while 200 #g/l acted inhibitory. Lower concentrations were n o t investigated. In this experiment IAA was given at the same concentrations as the phenolic compounds for comparison. Even the lowest concentration, 4 . 1 0 -~ M (70/~g/l), was obviously too high for any growth-stimulating effect. However, the algae in the series treated with phenylacetic acid and p-hydroxyphenylacetic acid differed completely in colour and general appearance from those in the control series. The phenylacetic acid-treated plants were much redder but growth varied considerably in each series. In some flasks big red tufts grew out while in others the algal plants stayed in a small compact form. The effects were rather similar to those caused by caffeic acid in Goniotrichum [ 1]. In the spore plants phenylacetic acid caused longer threads, but they often grew curled and attached to the bott o m of the flasks. For further studies of the influence of p-hydroxyphenylacetic and caffeic acid on the morphology of the young spore plants some cultures of Conchocelis were observed for 6 weeks. Pieces of old algal material were inoculated in three series of flasks with these substances at the concentration of 2-10 -5 M in long day conditions (15°C, 15 h light). Both phenolic acids rapidly gave rise to plants much darker than those of the control series and they also caused earlier spore formation. After 6 weeks spore plants had appeared in all series and they were carefully observed and measured (Table IV). p-Hydroxyphenylacetic acid caused a strong elongation of the tips of the algal threads and comparatively short side branches (Fig. 1). In the small new spore plants the normal brownish-red colour changed to rose-pink. This change was partly due to all the less coloured new branches spreading out from the spore plants. As seen from Table IV the radius of a p-hydroxyphenylacetic acid colony is about 50% longer than that of an untreated colony. Caffeic acid gave no T A B L E IV

INFLUENCE ON CELL ELONGATIONBY P-HYDROXYPHENYLACETICACID AND CAFFEIC ACID MEASURED AS THE RADIUS OF YOUNG CONCHOCELIS-PLANTS Incubation conditions: 15°C, 15 h light for 45 days. Substance added ( 2 . 1 0 -s M)

Mean of radii from 25 alga-plants (nun)

Control p-Hydroxyphenylacetic acid Caffeic acid

0.98 + 0.05 1.57 ¢ 0.06 0.95 ¢ 0.04

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2

Fig. 1. Effect ofp-hydroxyphenylacetic acid on the morphology of monospore plants from

Porphyra tenera. (1) Control × 75. (2) An addition of 2.10-s M p-hydroxyphenylacetic acid × 75. Crosses indicate the centre of the spore plant. Incubation conditions: 15°C, 15 h light, 45 days. The photos were taken through the bottom of the experimental flasks. Mean dry weight 1.0 'mg O~ 0£ 0.4 0.; 0

•/J10I "e

I

I

I

I

10 "z

10"6

10"s

"tO"4

i

10 "3 M

p-Hydroxyphe ny tacatic acid

Fig. 2. Growth of "Conchocelis phase" of Porphyra tenera influenced by p-hydroxyphenylacetic acid in the concentration range 10-s--10 -3 M. Mean dry weight of 5 duplicates. Vertical bars: ± standard error. Incubation conditions: 20°C, 20 h light for 60 days.

measurable elongation of cellsin the concentration used but an increased formation of monospores was observed. T o elucidate the concentration range over which p-hydroxyphenylacetic acid affected Porphyra the alga was cultivated at concentrations of 10 -s to 10 -3 M of this substance. As w e can see from the curve on Fig. 2 concentrations from 10 -7 to 3.3.10 -s M increased growth. In the control series no

305 spore plants developed while in the other series a varying number of flasks contained such plants. This erratic development of spore plants explains the high variations in the dry weights of algae obtained from the different flasks in a series. The spore plants had the same appearance as in the previous experiment even though it was run at 20°C and 20 h light. The influence of p-hydroxyphenylacetic acid was thus evident in an increase in dry weight, in elongation of the algal threads, and in richer spore formation. A study on the influence of phenylacetic acid on the dry weight was also performed and statistically sure increases were noted at concentration of 10-7--10 -5 M but also at 10 -s M. The effects seem to be influenced by the irradiance value and the quality of light. Further investigation on the effect of this substance are under progress in our laboratory. DISCUSSION Some influence on growth has been obtained in Porphyra by all the phenolic compounds tested under different laboratory conditions and temperature schedules. Cinnamic acid derivatives as well as p-hydroxypyruvic acid and p-hydroxyphenylacetic acid were active at some concentration within the range of 10-7--3.3 •10 -s M and sometimes over an even wider range. These active substances all have one feature in common, a hydroxy group in the paraposition to the side chain. This type of phenol is known as cofactor of peroxidases and has especially been studied in connection with IAA oxidase. As the existence of IAA in marine multicellular algae has been called in question by Buggeln and Craigie [15] an interpretation of the growthstimulating effects as indirectly an IAA effect must be postponed until this problem of the IAA presence is solved definitely. However, both 2,5-dihydroxy. phenylacetic acid and phenylacetic acid lack the parahydroxy group, and despite that increase growth of Porphyra. This has also been observed previously in Goniotrichum alsidii [ 3 ] . Some recent investigations provide a more varied interpretation of the effects with phenolic compounds on living plants. In the plant cell many enzyme systems, well separated, are working simultaneously, whereas the metabolism of similar phenolics can follow different pathways. By small changes in pH the same enzyme system can give quite different final products [14,16]. In higher plants p-hydroxyphenylpyruvic acid was found to be transformed into homogentisic acid. In higher plants as well as in the green alga, Chlorella pyrenoidosa, and the blue-green alga, Anacystis nidulans, homogentisic acid was shown to be a precursor of plastoquinones or tocopherols [8]. There is thus a possibility that the favourable effects on the growth of Porphyra by these two phenolic acids depend upon increased formation of some of these substances. Labelled phenylacetic acid and p-hydroxyphenylacetic acid on the other hand were not detected as being incorporated into the molecules of plastoquinones or tocopherols [8].

306 Phenylacetic acid was known to act as a h o r m o n e in auxin tests long before it was isolated as a natural substance from Phaseolus seedlings by Okamoto et al. [17] and considered as a new plant auxin by Wightman [18]. In addition to phenylacetic acid, Abe et al. [11,12] isolated p-hydroxyphenylacetic acid, from Undaria, a substance which had previously been shown to exhibit h o r m o n e activity in different biotests [10,19]. Our experiments indicate that p-hydroxyphenylacetic acid as well as phenylacetic acid have auxin-like effects on red algae. The elongation of the apical part o f the threads on addition of p-hydroxyphenylacetic acid was especially striking, though this acid also brought about a richer monospore formation. Both acids are active over a much broader concentration range in Porphyra than is IAA. This is especially obvious with p-hydroxyphenylacetic acid with an optimal effect at 10 -7 M but still without any toxic effect at a concentration as high as 10 -3 M. Generally the phenolic acids are toxic to unicellular algae at concentrations > 10 -4 M [20,21] and this holds also true for red algae [3]. The effects of these phenolic acids are not limited to Porphyra; other macroalgae in axenic culture are affected, too. Goniotrichum alsidii and Nemalion rnultifidum are stimulated by additions in the same concentration range as for Porphyra though not so strongly. Growth of young plants from the brown alga, Ectocarpus fasciculatus, is increased by up to 40% during the first stage of development (Fries, unpublished results). The question arises: are these phenolic compounds also produced by the algae? The Undaria material used for analyses by Abe et al. [11] was not free from bacteria. In some green algae, bacteria seem to determine the morphology of the algae [22,23]. Some bacteria are able to transform caffeic acid into p-hydroxyphenylacetic acid [24] and fungi are able to break down phenylalanine to phenylacetic acid [25]. Under natural conditions the investigated substances ought to be available to the algae by activity of marine bacteria or fungi. The isolation o f 3,5-dibrominated derivatives o f p - h y d r o x y p h e n y l a c e t i c acid and p-hydroxyphenylpyruvic acid from the red alga Halopytis incurens [26] might speak in favour of an algal production hypothesis. This problem as well as the possibility o f interactions between these phenolic compounds and other growth stimulators are now under investigation in the first author's laboratory. ACKNOWLEDGEMENTS

This investigation was supported by grants from the Japanese Society for Promotion of Science and from the Swedish Naturv~ Science Research Council. For advices about statistical problems and careful L-evision of the English text we are indebted to professor Lorentz Pearson, Rexburgh, U.S.A.

307

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