Planta (Berl.) 81, 16--27 (1968)

Studies on the Hormonal Relationships of Algae in Pure Culture

I I . The Effect of Potent ia l Precursors of Indole-3-Acetic Acid on the Growth of Several Freshwater Blue-Green Algae

MrazA RASHID ~IViAD and ALAN WINTER Department of Biology, University of York, York, England

Received February 16, 1968

Summary. The effect of potential precursors of Indole-3-acetic acid (IAA) on the growth (increase in dry weight) of Anacystis nidulans, Chlorogloea ]ritschii, Phormidium /oveolarum, Nostoc muscorum and ToIypothrix tenuis have been in- vestigated under as sterile conditions as possible.

Tryptamine showed a marked stimulation of growth indicating its possible conversion to IAA. Tryptophan at a hormonal concentration promoted growth in only 1 species, Chlorogloea /ritschii. Indole stimulated the growth of Chlorogloea /ritschii, Nostoc muscorum and Tolypothrix tenuis. Anthranilic acid promoted the growth of Nostoc muscorum, it failed to stimulate the growth of Chlorogloea/ritschii. Anthranilonitrile promoted the growth of Nostoc muscorum and Chlorogloea/ritschii. fl-Alanine promoted the growth of Nostoc muscorum, Tolypothrix tenuis and Chloro- gloea/ritschii at hormonal concentrations.

Introduction

I t has previously been shown tha t blue-green algae respond to Indole-3-Acetie acid (IAA) in a manner analogous to higher plants (AHMAD and W I ~ T ~ , 1968). Cultures of blue-green algae can be main- tained in axenic state so tha t any apparent response by the algae is not due to the presence of bacteria. The response of algae to exogenous t ryptophan has been investigated by DAVrDSON (1950) with Fucus sporelings in aseptic conditions. There was no evidence for its conversion to IAA, whereas DAo (1956) using Acetabularia mediterranea and HUSTED]~ (1957) using Vaucheria sessilis found a clear indication tha t t ryptophan acted in a manner compatible with its conversion to IAA. However, no sterile precautions were taken. ScgI]~WEg (1967) found tha t in green, brown and red marine algae t ryptophan was readily converted to IAA in non-sterile conditions, algae maintained in sterile conditions showed a low conversion rate.

In view of the controversy about the role of t ryptophan as a pre- cursor of IAA in higher plants (LIBB]~T et al., 1966; WrNT~R, 1966; T~rfMA~ et al., 1968) it seems important to examine the response of blue-green algae to t ryp tophan and other potential precursors of IAA.

Studies on the Hormonal l~elationships of Algae in Pure Culture 17

Materials and Methods

Organisms The following five blue-green algae maintained in a bacteria-free state were

employed in the present study. a) Anacystis nidulans. A cultm'e of this organism was kindly supplied by Prof.

FoGo, Westfield College, University of London. b) Chlorogloea/ritschii. The bacteria-free culture of this alga were also supplied

by Prof. FOGG. c) Phormidium ]oveolarum. Cambridge culture collection No. 1462/1. d) Nostoc muscorum. Cambridge culture collection No. 1453/8. e) Tolypothrix tenuis. A culture of this organism was supplied by Prof. WATn-

NAreS, Seijo University, Tokyo, Japan.

Methods Culture conditions, growth measurements, preparation and maintenance of

experimental cultures were identical with those described previously (AHMAD and WL~T~R, I968).

Chemicals Compounds tested for their ability to act as precursors of IAA were obtained

from K & K Laboratories, Inc., Plainview, New York, U.S.A. They were dissolved in pyrex-distilled warm water except anthranilic acid which was dissolved initially in i % KOH. Additions of compounds under test were m~de to the basM medium. The test medium was filter sterilized using 'Millipore filters' (Millipore Corporation, Bedford, Mass., U.S.A.) with a pore diameter of 0.45 tz in a sterile cabinet.

Expression o/Results The results are expressed in mgm. dry weight. The standard error of the mean

is shown graphically where appropriate.

Results

1. The E//ect o/Tryptamine

The resul ts of va ry ing concent ra t ions (10-~~ -s M) of t r y p t a m i n e in steri le condi t ions on the growth of Anaeysti8 nidulans, Chlorogloea /ritschii, Phormidium /oveolarium, Nostoc muscorum and Tolypothrix tenuis compared with the g rowth of algae in the control are summar ized in Fig. 1.

a) Anaeystis nidulans. I t is ev ident f rom Fig. 1 A t h a t A. nidulans responded to exogenous t r y p t a m i n e a t a concent ra t ion of 10 -6 M, the d r y weight increased b y 31 per cent, this is s ignif icant a t the 5 per cent level of p robab i l i ty . Higher concent ra t ions of t r y p t a m i n e inh ib i ted the growth of the alga.

b) Chlorogloea /ritsehii. The m a x i m u m increase in d r y weight for this alga was ob ta ined in 10 -s M t r y p t a m i n e t r ea t ed cul tures (Fig. 1 B). This increase was a s t imula t ion of 86 per cent over the control. This is s ignif icant a t the 1 pe r cent level of p robab i l i ty . A t 10 -3 M t r y p t a m i u e

2a P l an t a (Berl.), Bd, 81

18 M. ]~, Altl~AD and A, W I s T ~ :

4.00

350

300

25O

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r', 150

|

100 �9

|

o /j jt / \oj

/~

10 q0 10-9 10- 8 10 -? 10-6 1(3 5 1E a 10 -3 Molar concentration of tryptemine

Fig. 1. Effect of different concentrations of Tryptamine on the growth of Anacystis nidulans, Chlorogloea [ritschii, Phormidium [oveolarum, Nostoc muscorum and Toly- pothrix tenuis. �9 �9 A Anacystis nidulans, �9 - - - �9 B Chlorogloea ]ritschii, , - - v C Phormidium loveolarum, o . . . . o D Nostoc muscorum, D - - [ ] E Toly~othrix

tenuis

growth was a lmost comple te ly inhibi ted . Other concent ra t ions showed no difference f rom the control.

c) P h o r m i d i u m [oveolarum. The cul tures of P . [oveolarum t r e a t ed with t r y p t a m i n e concentra t ions showed no signif icant growth p romot ion (Fig. 1 C). Here too 10 -s M t r y p t a m i n e m a r k e d l y inh ib i ted the growth of the ~lga.

Studies on the Hormonal Relationships of Algae in Pure Culture 19

d) Nostoc muscorum. The growth effect shown by t ryptamine on N. muscorum is shown in Fig. 1 D. High concentrations of t ryptamine (10 -s-~ 10 -4 M) produced an inhibition of growth. Tryptamine had a wide range of growth promotive concentrations on N. muscorum. There was an increase in dry weight in t ryptamine concentrations from 10-5--10 -1~ M. The maximum growth was obtained in l0 -7 M tryptamine. This increase is 128 per cent over the control and is significant at the 1 per cent level of probability.

e) Tolypothrix tenuis. The maximum growth of T. tenuis was ob- tained in 10 -a M tryptamine, this was 68 per cent higher than the control and is significant at the 1 per cent level of probability. Lower concen- trations of t ryptamine 10 -9- - 10 -6 M were also growth promotory, 10 -a M inhibited the growth almost completely.

2. The El/set o/Tryptophan

The effect of t ryptophan on the growth of Anacystis nidulans, Chlorogloea /ritschii, Phormidium /oveolarum, Nostoc muscorum and Tolypothrix tenuis is shown in Fig. 2.

a) Anacystis nidulans. Fig. 2A shouts the effect of t ryptophan on the growth of A. nidulans. I t will be seen that there is no promoting effect on growth. 10 -4 and 10 -s M t ryptophan produced an inhibitory effect.

b) Chlorogloea /ritschii. Tryptophan produced a highly significant increase in dry weight (142 per cent at l0 -~ M) on this alga (Fig. 2B). I t is of interest to note the narrow concentration range which is effective. Higher concentrations of t ryp tophan inhibited the growth of the alga.

c) Phormidium/oveolarum. The effect of t ryptophan on P./oveolarum is shown in Fig. 2 C. There is a significant increase (at the 5 per cent level of probability) in dry weight (30 per cent) at 10 -a M over the control. Other concentrations produced no significant effect.

d) Nostoc muscorum. There is an increase in dry weight at 10 -s M and 10-4M t ryptophan (Fig. 2D). The maximum increase at 10-4M t ryptophan was 50 per cent over the control. Lower concentrations produced no significant effect.

e) Tolypothrix tenuis. Tryptophan produced a slight increase (17 per cent) at 10 -5 M which is barely significant at the 5 per cent level of probabili ty (Fig. 2E). Other concentrations produced very little effect. I t is of interest to note tha t none of the concentrations under test were inhibitory to growth.

3. The Growth E//ect o/ Indole The results of this series of experiments are summarized in Fig. 3. a) Chlorogloea/ritschii. There is a maximum' increase in dry weight

of 148 per cent at 10 -~ M Indole (Fig. 3A). Higher concentrations of

2*

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10-10104 10-s 10-7 10-6 lo-s 10-~ 10-3 Molar concentrcltion of tryptophQn

Fig. 2. Effect of different concentrations of tryptophan on the growth of Anacysti8 nidulans, Chlorogloea ]ritschii, Phormidium ]oveolarum, Nostoc muscorum and Toly- ~otttrix tenuis. �9 �9 A Anacystis nidulans, �9 . . . . . B Chlorogloea {ritschii,

v - - v C Phormidium [oveolarum, o . . . . . D Nostoc muscorum, ~ - - D E Tolypothrix tenuis

Indole (10-% 10 -4 M) were inhibitory to growth, lower concentrations have no significant effect, a l though there is small increase in dry weight at 10 -6 and 10 -7 M.

b) Nos toc m u s c o r u m . There is a significant increase of 72 per cent in d ry weight at 10 _9 M (Fig. 3B). Concentrations of Indole of 10 -~ M and higher were inhibi tory to growth.

Studies on the Hormonal Relationships of Algae in Pure Culture 21

bOO

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0 1040 10 -9 10 -8 10 -7 10 -6 10 5 10 -4 0 .3 Molar concentration of indole

Fig. 3. Effect of different concentrations of ~ndole on the growth of Chlorogloea ]r i tschi i , Nos toc m u s c o r u m and ToIy!oo~hrix tenuis . �9 . . . . . A ChIorogloea ]r i tschi i ,

o . . . . o B Noetoc m u s c o r u m , = ~ C T o l y p o t h r i x tenui8

c) Tolypothrix tenuis. Indole showed growth promotion on T. tenuis (Fig. 3C). The maximum growth was obtained at 10 -4 M which was 45 per cent higher than the control and is significant. Other concen- trations of Indole namely 10-~--10-sM also produced a stimulatory effect on the growth of T. tenuis. Concentrations lower than 10-~ N[ produced no significant effect.

Indole had no effect on the growth of Anacystis nidulans and Phormidium /oveolarum except that at 10 -s M growth was almost completely inhibited.

2 b Planta (BerL), Bd. 81

22 M. I~. Am~AD and A. WInTeR:

4. The El/ect o/Anthranilonitri le

The response to this compound by blue-green algae is shown in Fig, 4. a) Chlorogloea/ritschii, There is a highly significant increase in dry

weight of 142 per cent over the control at 10-6M Anthranilonitrile (Fig. 4A). 10 -s M Anthranilonitrile produced an inhibiting effect on the growth of C. ]ritschii, concentrations of 10 -7 M and less have no effect.

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"~ 100

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Fig. 4. Effect of different concentrations of Anthranilonitrile on the growth of Chlorogloea /ritschii, and Nostoc muscorum. �9 . . . . . B Chlorogloea ]ritschii,

o . . . . o A 5Tostoc muscorum

b) Nostoc muscorum. 10 -s M Anthranilonitrile produced a growth inhi- biting effect on N. muscorum (Fig. 5 B). There is a 30 per cent increase in dry weight at 10 -4 M over the control, this is significant at the 5 per cent level of probability. Other concentrations produced no effect.

There was no significant effect on the growth of Phormidium ]oveolarum or TolyTothrix tennis at physiological concentrations.

5. The E]ject o 1 An$hranilic Acid on the Growth ol 1Vostoc muscorum and Chlorogloea jritschii

The results of this experiment are shown in Fig. 5. a) Chlorogloea/ritschii. The effect of Anthranilic acid on the growth

of C. ]ritschii is shown in ~ig. 5A. Higher concentrations 10-s--10 -a M of Anthranilic acid produced an inhibitory effect on the growth of

Studies on the Hormonal Relationships of Algae in Pure Culture 23

C. / r i t sch i i . There was a small but insignificant increase in dry weight of 16 per cent over the control at 10 -9 M Anthranilic acid.

b) Nostoc muscorum. Higher concentrations 10-7--10 -a M of Anthra- nilic acid produced an inhibitory effect on the growth of N . m u s c o r u m (Fig. 5B). There is a significant increase of 32 per cent over the control in dry weight in cultures treated with 10 -s M Anthranilic acid. There is no effect of concentrations lower than this on h r. muscorum.

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10- 8 10- 7 10 -5 10-5 10 -4 10-3 0 I I

o Io -'o io ~ Molar concentration of anthronilic acid

Fig. 5. Effect of different concentrations of Anthranilie acid on the growth of Chlorogloea ]ritschii and Nostoc muscorum. �9 . . . . �9 A Chlorogloea /ritschii,

o - - - o B N o 8 t o c ~ u s c o r u m

6. The E]]ect o / f l - A l a n i n e

fl-Alanine was selected in order to compare the effect of a " typ ica l" amino acid with other Indole compounds (WItTilY, 1966). The results of this series of experiments are presented in Fig. 6.

a) Chlorogloea ]ritschii. fi-Alanine produced some growth promotion at 10 -6 M (Fig. 6A). There is an increase of 25 per cent in dry weight over the control, this is barely significant at the 5 per cent level of probability. Higher concentrations 10 - z - - 10 -3 M were growth inhibitory whereas lower concentrations produced no effect.

b) Nostoc muscorum. Fig. 6 B shows the effect of fl-Alanine on the growth of N. muscorum. A significant increase of 62 per cent in dry

24 NL ~.AHNIAD and A. WINTER:

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~ 250 C31

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150

100

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0 104010 -8 10 -8 10- ? 10 -6 10 -5 t # 10 -3 Molar concentration of ~8 alanine

Fig. 6. Effect of different concentrations of fl-ilanine on the growth of Chlorogloea [ritschii, Nostoc muscorum and Tolypothrix tenuis . . . . . . �9 A Chlorogloea ]ritschii,

. . . . . o B Nostoo muscorum, = ~ C Tolyloothrix ten'~is

weight was obtained at 10- 6 M. 10 -8 M fl-Alanine inhibited the growth whereas lower concentrations produced no significant increase in dry weight.

c) T o l y p o t h r i x t enu i s . There is an increase in dry weight of 38 per cent over the control at 10-SM fl-Alanine (Fig. 6C). 10-7---10 -5 M fl-Alanine produced significant increases in dry weight. No inhibition was observed at any concentration.

Studies on the Hormonal I~elationships of Algae in Pure Culture 25

A~acystis nidulans showed no response to added fl-Alanine. Phor- midium/oveolarum responded only at 10 -a M where there was a small but significant increase in growth.

Discussion

In the present study all the blue-green algae responded to tryptamine added to basal medium except Phormidium ]oveolarum which did not respond at all. The present results of the effect of tryptamine on Ana- cystis nidulans, Chlorogloea ]ritschii, Phormidium ]oveolarum, Nostoc muscorum and Tolypothrix tenuis can be compared with the results of IAA on these algae (AItMAD and WI~T~g, 1968). The optimum growth promoting concentration of IAA and tryptamine is the same (10-~M) for Anacystis nidulans. Chlorogloea [ritschii responded well to added tI.yptamine but the optimum growth promoting concentration is very much lower (10 -s M) than for IAA (10 -5 M). Phormidium ]oveolarum did not respond to added tryptamine, although it responded to IAA (AItMAD and WI~TEI~, 1968). In Nostoc muscorum the optimum concentration is l0 -7 M for both tryptamine and IAA, however the tryptamine treatment showed a much higher increase in dry weight than did the IAA treatment. Tolypothrix tenuis responded to added tryptamine. Here the optimum growth promoting concentration was higher (10-4M) than for IAA (10 -~ M). It is interesting to note that only heterocystous-nitrogen fixing forms (Chlorogloea /ritschii, Nostoc muscorum and Tolypothrix tenuis) of blue-green algae responded well to added tryptamine in the basal medium.

]:IUSTEDE (1957) showed that formation of zoospores in Vaucheria sessilis was stimulated by tryptamine as well as IAA, this indicates a possible conversion of tryptamine to IAA. It is well known that trypt- amine can be converted to IAA in higher plants. Wr~'rv.xr (1966) clearly demonstrated that tryptamine possesses a growth activity comparable with IAA in apical Avena coleoptile sections.

Earlier studies on higher plants suggested several ways for the con- version of tryptamine to IAA. K ~ T E ~ and MAN~ (1952) suggested the presence of an enzyme capable of converting tryptamine to IAA in pea- seedlings, the possibility of such an enzyme in blue-green algae is not known. Another suggestion is the activity of tryptophan decarboxylase (STOWS, 1959) but the evidence for the presence of this enzyme indicates that it is not at all widely distributed in higher plants nor in micro- organisms (P~LEu and Swow]~, 1966a, b).

The effect of tryptophan is interesting in view of the fact that out of 5 blue-green algae tested only one responded to added tryptophan at a hormonal concentration, namely Chlorogloea ]ritschii. The growth of Chlorogloea /ritschii was greatly promoted by the presence of l0 -5 M

26 M. 1~. A~MAT) and A. WII~Tm~:

t ryptophan in the basal medium. The optimum response to IAA was also at the same concentration (AxMAD and WI~TE~, 1968). This result may be regarded as indicating a conversion of t ryptophan to IAA by the alga. The stimulation of growth of Phormidium foveolarum at 10 -~ M tryptophan is purely a nutritional rather than a hormonal effect.

This alga seems to be incapable of converting t ryptophan to IAA but it does respond to added IAA (AI~AD and WINTmL 1968). The growth of Nos~oc muscorum was also stimulated by added tryptophan but this effect too could be accounted for as purely a nutritional effect. There was no significant increase in dry weight in ~olypothrix lenuis as a result of t ryptophan addition. Thus these results indicate that Ana- cystis nidulans, Phormidium ]oveolarum, 1Vestee muscorum and Toly- pothrix tenuis are incapable of converting t ryptophan to IAA whereas Chtorogloea/ritschii may be capable of doing so.

Our observations on Anacystis nidulans, Phormidium /oveolarum, Nostoc muscorum and Tolypothrix tenuis are in agreement with the observations of DAVIDSO~ (1950) on Fucus sporelings and similar studies on higher plants that t ryptophan is not a precursor of IAA (LI~BERT et al., 1966; WI~TEI~, 1966 and T ~ A ~ et al., 1968).

The activity of Indole on the growth of Chlorogloea ]ritschii, Nostoc muscorum and Tolypothrix tenuis point to a possible involvement in the biogenesis of IAA, although its presence has not been reported from algae. I t has been reported from higher plants (STOWE, 1959) and was suggested as a potential precursor of IAA in higher plants by WI~Tm~ (1966). I t greatly increased the growth of Chlorogloea ]ritschii, Nostoe muscorum and Tolypothrix tenuis and seems to possess an IAA like activity on these algae.

Anthranilic acid promoted the growth of Nostoc muscorum where as it failed to stimulate the growth of Chlorogloea ]ritschii. Anthranilonitrile accelerated the growth of Nostoc muscorum and Chlorogloea ]ritschii. Its growth promoting activity is already known on higher plants (WII~Tm~., 1966).

In our experiments with fi-Alanine, the growth of Nostoc museorum, Tolylpothrix tenuis and Chlorogloea/ritschii was promoted at a hormonal concentration. In other algae fl-Alanine acts at a nutrient concentration level. Long ago BOYSEI~-J~NsE~ (1931, 1932) reported experiments that indicated that certain amino acids act as precursors of IAA in Asper. gillus niger cultures. The evidence for the conversion was based on activity in the Arena curvature test. This work has never been followed up with a biochemical investigation. Alanine, however, was not one of the amino acids that appeared to act as a IAA precursor.

The results of this study and the previous one in this series (AHMAI) and ~VI~Tm~, 1968) demonstrate tha t blue-green algae like higher plants

Studies on the Hormonal Relationships of Algae in Pure Culture 27

reac t in a comparab le manne r to I A A and to po ten t i a l I A A precursors , thei r g rowth is inh ib i t ed b y re l a t ive ly high and p r o m o t e d b y re l a t ive ly low concent ra t ion of the compounds . I t is also clear f rom the presen t s tudies t h a t t r y p t o p h a n is no t un iversa l ly a precursor of I A A bu t the resul ts f rom Chlorogloea/ritschii indica te t h a t i t m a y act as a precursor in cer ta in species. The resul ts are compat ib le wi th t r y p t a m i n e p lay ing a m a j o r role in the biogenesis of I A A .

De ta i l ed biochemical inves t iga t ions of the responses of blue-green algae to I A A and to precursors of I A A are now underway .

This work was supported by a grant from the Agricultural Research Council of Great Britain.

L i t e r a t u r e

AHMAD, M. 1%., and A. WII~TER: Studies on the hormonal relationships of algae in pure culture. 1. The effect of Indo]c-3-Acetic acid on the growth of bhie-green and green algae. Planta (Berl.) 18, 277 (1968).

BOYSEI~-J:ENSEI% P.: lJber Bildung eines Wachstumsregulators durch Aspergillus niger. Biochcm. Z. 289, 243 (1931).

- - Uber die Bildung and biologische Bedeutung des Wachstumsregulators bei Aspe~yillus niger. Biochem. Z. 250, 270 (1932).

DAO, S. : Apropos de Faction du tryptophane sur l' Acetabularia mediterranea. C.R. Acad. Sci. (Paris) 243, 1552 (1956).

DAu F. :F. : The effects of auxin on the growth of marine algae. Amer. J. Bet. 37, 501 (1950).

HUSTEDE, H. : Untersuchungen fiber die stoffliche Beeinfhissung der Entwickhing Yon StigeocIonium [alklandicum and Faucheria sessitis dutch Tryptophan- abk6mmIinge. Biol. Zbl. 76, 555 (1957).

KENT~N, R. H., and P. J. G. MANN: The oxidation of amines by pea seedlings. Biochem. J. 50, 360 (1952).

LIBt~ElaT, E., S. WICtt]Sl~, U. SCmEWER, H. Rlse]~, and W. KAISER: The influence of epiphytic bacteria on auxin metabolism. Planta (Berl.) 68, 327 (1966).

PEnLEY, J. E., and B. B. STOWE : On the ability of Taphrina de]ormans to produce indoleacctic acid from tryptophan by way of tryptamine. Plant Physiol. 41, 234 (1966a).

- - - - The production of tryptamine from tryptophan by Bacillus cereus (KVT). Biochem. J. 100, 169 (1966b).

SOmEW~R, U. : Auxinvorkommen und Auxinstoffwechsel bei mchrzelligen Ostsee- algen. II. Zur Entstehung yon Indol-3-Essigs~inre aus Tryptophan, unter Berfick- sichtigung des Einfhisses tier marinen Bakterienflora. Planta (Bcrl.) 75, 152 (1967).

STow~, B. B.: Occurrence and metabolism of simple indoles in plants. Fortschr. Chem. org. Naturstoffe 17, 248 (1959).

TmMANN, K, V., M. GROe~OWSJ<A, and P. N. AVAD~ANI: The role of tryptophan as an IAA precursor. In: Biochemistry and Physiology of Plant Growth Sub- stances. Ottawa, Canada: The 1%unge Press (in press).

WIN~Eg, A. : A hypothetical route for the biogenesis of IAA. Planta (Berl.) 71, 229 (1966).

Dr. A. WINTER Dep~rtmcnt of Biology, University of York Heslington, York, England