Aquatic Botany, 13 (1982) 73--85 73 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

E N V I R O N M E N T A L A N D G R O W T H R E G U L A T O R E F F E C T S O N H E T E R O P H Y L L Y A N D G R O W T H O F P R O S E R P I N A C A I N T E R M E D I A ( H A L O R A G A C E A E )

MICHAEL E. KANE and LUKE S. ALBERT

Department of Botany, University of Rhode Island, Kingston, RI 02881 (U.S.A.)

(Accepted for publication 19 November 1981)

ABSTRACT

Kane, M.E. and Albert, L.S., 1982. Environmental and growth regulator effects on heterophylly and growth of Proserpinaca intermedia Haloragaceae). Aquat. Bot., 13: 73--85.

Effects of five growth regulators GAs, AMO-1618, ABA, IAA and kinetin on growth and leaf development of aerial forms of Proserpinaca intermedia Mack. were investigated. Plants were cultured in different photoperiods (8, 10, 12, 14 and 16 h light) and at different temperatures (15 and 27°C) for 35 days. Untreated plants maintained in photo- periods of 14 h and greater at 27°C grew erect, produced lanceolate-serrate leaves and flowered. Plants subjected to shorter photoper iods grew prostrate, developed small dis- sected leaves and remained vegetative. Leaf form and growth habit were modified by low temperature. Plants cultured at 15°C grew prostrate and developed dissected leaves regardless of photoperiod. ABA, IAA or kinetin treatments had no effect on growth or leaf form. GA 3 application to plants cultured under short days induced a growth habit and leaf form similar to that observed in plants cultured under long days. Low tempera- ture induced prostrate growth and resulting dissected leaf formation was counteracted by GA 3 application.

Plants which were made gibberellin deficient following application of AMO-1618 pro- duced dissected leaves under long days (14 h). It is concluded that the morphological changes observed in Proserpinaca are caused by changes in endogenous hormonal levels. Their action is initiated by photoper iod through phytochrome and is modulated by tem- perature.

INTRODUCTION

A q u a t i c a n g i o s p e r m s d i s p l a y s o m e o f t h e m o s t v a r i a b l e g r o w t h a n d deve l - o p m e n t a l p a t t e r n s o b s e r v e d in h i g h e r p l a n t s . W h i l e t e r r e s t r i a l p l a n t s e v o l v e d a n d a d a p t e d t o su rv ive a n d r e p r o d u c e in d r y d e s i c c a t i n g e n v i r o n m e n t s , a q u a t i c a n g i o s p e r m s , e s p e c i a l l y a m p h i b i o u s spec i e s , f r e e d f r o m th i s c o n - s t r a i n t , h a v e e v o l v e d t o d i s p l a y a c o m b i n a t i o n o f a n c e s t r a l l a n d a n d a q u a t i c c h a r a c t e r i s t i c s t h a t p r o d u c e a f l e x i b i l i t y o f a d a p t a t i o n u n i q u e in t h e p l a n t k i n g d o m . In c e r t a i n i n s t a n c e s th i s f l e x i b i l i t y o f g r o w t h has r e s u l t e d in

0304-3770/82[ 0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company

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noxious aquatic weed problems (Grace and Wetzel, 1978). Within the Water- milfoil Family, Haloragaceae, members of the amphibious genus Proserpinaca {Mermaid weed) and certain species of the genus Myriophyllum adapt to seasonal changes through the production of distinct morphological forms including distinct alterations in leaf morphology on the same plant (hetero- phylly) (Davis, 1956; Wallenstein and Albert, 1963; England and Tolbert, 1964; Schmidt and Millington, 1968).

Wallenstein (1963) has described the phenology of Proserpinaca paIustris L. in detail. This heterophyllous species, an inhabitant of marshes and ephemeral ponds, alternates between an emergent and submergent form. The erect emergent form (adult aerial phase) appears in spring and persists through early fall. Plants grow erect, produce lanceolate-serrate leaves that are helically inserted with flowers borne in the leaf axils. In the fall, P. palustris grows prostrate, and forms branches at the base of each plant. Under these conditions each shoot consists of short internodes and small dis- sected leaves that do not flower. The leaves of this form are inserted helically but show a dorsiventral symmetry (aerial juvenile phase). When the plants become submerged, their shoots become erect and their leaves are no longer appressed to the stem. Subsequently, pectinate leaves consisting of long fili- form divisions are produced (submerged juvenile phase). Proserpinaca grows submerged throughout the winter until early spring when the lanceolate- serrate leaf form (adult submerged phase) is produced below the surface prior to emergence. If aerial plants become submerged again by heavy summer rains, these shoots revert to the production of the submerged leaf form until the shoot tips become aerial again (Wallenstein, 1963).

The natural growth pattern of Proserpinaca has also been observed under experimental conditions. Davis (1956) found that heterophylly in several species of Proserpinaca is controlled by photoperiod, temperature and sub- mergence. In addition, Wallenstein (1963) and Wallenstein and Albert (1963) showed that the general morphological expression of the aerial forms is con- trolled by photoperiod and modified by temperature and the growth regulator gibberellic acid (GA3). It was observed that leaf shape and stem orientation were affected by photoperiod; long-day treatments (16 h) caused the formation of plants with lanceolate-serrate leaves and erect shoots while short-day treatments (8 h) yielded plants with small dissected leaves on prostrate stems. It was found (Davis, 1956) that temperature can modify the overall photoperiod responses of the long-day aerial form. Continuous low temperatures (7--16°C) increased the degree of leaf dissection on plants grown in long days, similar to those produced on plants grown in short days. Wallenstein (1963) reported that 1.0 gg GA3 applied to the shoot tips con- verted the prostrate short-day form into plants which grew erect and which developed a leaf orientation and form similar to the long-day form. These results were confirmed by Davis (1967).

In the present study changes in stem elongation, growth habit and leaf form of P. intermedia Mack., another heterophyllous representative of the

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genus (Fig. 1), were examined following application of growth regulators under different photoper iodic and temperature regimes. The primary objec- tive of this s tudy was to fur ther define the roles of endogenous growth regulators and environmental factors controlling growth and development in heterophyllous aquatic plants.

Ih 0.06 0.14 0.27 0.36 0.50 0.78 Fig. 1. Typical range of variation in leaf form observed in P. intermedia with Index of Heterophylly (Ih) values indicated below each leaf form. Dissected leaf form on the ex- treme left is typical of the submerged form. Lanceolate-serrate leaf form on the extreme right is typical of the long-day aerial form.

METHODS

Plant culture

Shoot cuttings (5.0 cm tips) ofP . intermedia were made from plants grow- ing in a small freshwater marsh located in the Great Swamp Management Area, West Kingston, Rhode Island. These tips were rooted in a mixture of perlite and tap water under a 16 h photoper iod provided by a mixture of 'Cool White' f luorescent tubes and incandescent bulbs at a photon flux den- sity of 20 pE m -2 s -1 (400--700 nm). Temperature was maintained at 27°C. After 12 days, when the cuttings were from 6.0 to 7.0 cm in length, they were transplanted individually into separate 5.7 cm diameter peat pots con- taining a 3:1 mixture of pot t ing soil and perlite. These peat pots were placed in plastic trays (15 per tray) into which was added 600 ml of a complete modified Shive's nutr ient solution (Albert and Wilson, 1961) supplemented with 0.6 g 1-1 'Sequestrene 330Fe ' (Geigy Chemical Co.). Nutrient solutions were changed every 5 days. Aerially grown plants were used in all experi- ments.

Growth regulator treatments and experimental conditions

The effects of natural as well as synthetic growth regulators were tested. Plants were treated singularly with aqueous preparations of the following growth regulators: gibberellic acid (GA3) (Sigma Chemical Co.); AMO-1618 (Calbiochem); indole 3-acetic acid (IAA) (Mann Research Lab.); abscisic acid (ABA) (Sigma Chemical Co.); and kinetin (Sigma Chemical Co.). Solutions

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of each growth regulator of different concentrations were prepared in de- ionized-distilled water containing 0.01% Tween-20 (v/v) by serial dilution. Growth regulators were applied to shoot tips as 10 pl drops using micro- pipets. Plants were treated at 5-day intervals for the first 20 days of each experiment. Results were expressed as the total microgram (pg) amounts of growth regulators applied during the experimental periods. Following appli- cation of the growth regulators, trays containing the treated plants and con- trols were placed in clear polyethylene enclosures constructed in three growth chambers (Sherer Gillette Models CEL 25-7 and CEL 37-14). Rela- tive humidity was maintained between 81 and 90% within these enclosures. Plants were cultured in photoperiods of 8, 10, 12, 14 or 16 h at a photon flux density of 140 pE m s s -~ (400--700 nm) provided by a mixture of 'Cool White' fluorescent tubes and incandescent bulbs. Temperatures were main- tained at 15 or 27°C. Each combination of photoperiod and temperature consti tuted a separate experiment with all combinations of photoperiod and temperature being tested. Experiments were 35 days in duration.

Data recorded

At 5-day intervals, measurements of net stem elongation (from an India ink mark made 1.0 cm below the shoot apex), growth habit and number of flowers were recorded. On the last day of each experiment a leaf 1.0 cm down from the shoot tip of each plant was removed, pressed, mounted and photographed. Leaf form (degree of dissection) was numerically evaluated by calculation of an Index of Heterophylly (Ih)- This was determined by cal- culation of the ratio of the midrib width to the width of the total blade at its widest point

I h = midrib width/total blade width (1)

RESULTS

The effects of photoperiod and temperature on stem elongation are shown in Fig. 2. Maximum stem elongation occurred in a 16-h photoperiod at 27°C. An abrupt decline in stem elongation was observed in plants cultured in less than 14 h light. In photoperiods of 12 h or less there was little difference in stem elongation between plants maintained at 15°C and those at 27°C tem- perature regimes. At 27°C the photoperiodic threshold for the production of lanceolate-serrate leaves (Fig. 3), flowering and erect growth habit was ob- served to be between 12 and 14 h light. Plants maintained at 15°C grew prostrate, remained vegetative and produced dissected leaves regardless of the photoperiod in which they were grown. Slight increases in overall leaf size were observed in plants maintained in longer photoperiods at 15°C (Fig. 3).

Treatments with ABA, IAA and kinetin had no effect on flowering, leaf

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form or stem elongation as tested (up to 50 t~g applied). However, dramatic responses were observed in GA3-treated plants. GA3 treatment greatly stimulated stem elongation of plants maintained at both 27 and 15°C (Figs. 4 and 5). Plants cultured at 27°C were more responsive to applied GA3 with respect to stem elongation when subjected to longer photoperiods. Treat- ment with 5.0 × 10 -3 pg GA3 stimulated stem elongation to a greater extent than controls, however this was only apparent in photoperiods with less than 14 h light. Treatment with 0.5 pg GA3 in 16 h light at 15 and 27°C appeared to have been optimal as plants did not grow significantly greater than those plants treated with 5.0 X 10 -1 t~g GA3. Figure 6 shows the effects of GA3 on final leaf form of plants grown in different photoperiodic regimes at 27°C. GA3 treatments partially replaced the photoper iod requirement for the pro- duction of the long day expanded leaf form in photoperiods less than 14 h. Increasing amounts of GA3 stimulated mesophyll development. The effec- tiveness by which a given amount of GA3 stimulated mesophyll expansion declined when plants were cultured in shorter photoperiods. Under a 10-h photoper iod at least 5.0 × 10 -2 pg GA3 was required to stimulate mesophyll

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80

expansion while 5 X 101 pg was required to exceed the threshold for meso- phyll expansion under an 8-h photoperiod. Different GA a threshold levels for other various growth and developmental responses to applied GAa were also noted. Under an 8-h photoperiod at 27°C, plants treated with 5.0 × 10 -3 or 5 × 10 -2 pg GA3 remained prostrate but appression of leaves to the stems was relieved. Thus, the order of response elicited by increasing amounts of applied GA3 under short days was observed to be: (1) loss of leaf appression to the stem, (2) stimulation of stem elongation coupled with (3) erect growth habit and (4) stimulation of mesophyll development. The ab- solute amounts of GA3 required for expression of each of these responses increased in plants maintained under progressively shorter photoperiods. Figure 7 depicts the effects of GA3 treatment on the final leaf form of plants maintained at 15°C. In photoperiods greater than 10 h GA3 treatments re- sulted in the development of leaves with greater mesophyll development as noted by increased I h values. The effectiveness by which a given amount of GA3 stimulated mesophyll development decreased in shorter photoperiods.

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81

Under a 10-h photoperiod the morphological response to applied GA3 was distinctly altered such that treatments only stimulated vein elongation with- out further increases in mesophyll development, resulting in the production of large dissected leaves composed of long filiform divisions. These leaves were very similar to those observed on submerged shoots (Fig. 1). The possi- bility that this altered GA3 response could be due to alterations in levels of an endogenous cell division factor was examined by simultaneous application of GA3 and kinetin in 36 combinations applied to plants cultured in a 10-h photoperiod at 15°C. None of these combinations stimulated mesophyll development {unpublished data).

These results indicated that application of GA3 to short-day or low tem- perature grown plants partially mitigated the requirement for specific environmental conditions for growth and development. The possibility arose that these environmental conditions of temperature and photoperiod were in turn acting by altering the endogenous hormone balance such that short days or low temperatures resulted in the morphological forms of Proserpinaca ob- served due to decreased endogenous gibberellin levels. This appeared plausible

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as plants presumably made gibberellin deficient when treated with an in- hibitor of gibberellin synthesis, AMO-1618 (Cathey, 1964; Douglas and Paleg, 1974), produced dissected leaves when maintained in a noninductive 14-h photoper iod at 27°C (Fig. 8). Plants treated with AMO-1618 in amounts from 5.0 × 10 -2 to 5.0 pg at 15°C (Fig. 9) produced leaves decreased in overall size when compared to controls. Trea tment with more than 5.0 pg AMO-1618 proved to be toxic, especially to plants maintained under longer photoperiods.

DISCUSSION

The data presented in this paper indicate that photoper iod, temperature and gibbereUic acid have related effects on leaf shape and on stem and leaf orientation of the amphibious angiosperm P. in termedia. Gibberellic acid p romoted vegetative growth associated with long days while plants were cultured under short days. Low temperature induced prostrate growth habit

83

and formation of dissected leaves under long days, an effect opposite to that obtained with long days at 27°C or short days plus gibberellic acid. Similar tempera- ture effects on leaf morphogenesis have been demonstrated in both sub- merged and terrestrial forms of the amphibious buttercup, Ranunculus flabellaris Raf. (Bostrack and Millington, 1962; Johnson, 1967). However, this is the first report in which it has been demonstrated that exogenous gibberellin application can mitigate increases in leaf dissection and prostrate growth habit induced by low temperature in Proserpinaca.

The observation that the responses could be photoinduced by the ap- propriate day length followed by response expression under the opposite day length led Wallenstein and Albert (1963) to believe the phytochrome system, through photoperiod, interacted with temperature and exogenously applied gibberellic acid to control leaf shape, stem orientation and the geotropic response of the shoot. In this regard similar effects on the photoreversible control of heterophylly in Hippuris vulgaris L. have been observed following the variation of red/far red light ratios (Bodkin et al., 1980).

It might be assumed that the changes observed in growth and development in Proserpinaca reflect changes in endogenous hormone levels and their effects which are initiated through phytochrome via photoperiod and modulated by temperature. Seasonal changes in hormone levels have been observed in other aquatic plants including: CeratophyUurn demersum L. (Best and Soekarjo, 1976; Best, 1979), Utricularia vulgaris L. (Winston and Gorham, 1979) and Myriophyllum verticillatum L. (Weber and Nooden, 1976). However, correlation of changes in endogenous levels of the different hormones and their combined action on growth and developmental patterns in these plants is not completely understood.

The interpretation that alterations in gibberellin biosynthesis and/or metabolism play a role in the dramatic alterations in the leaf form in Pro- serpinaca is further supported by the observation that plants, presumably made gibberellin deficient following application of an inhibitor of gibberellin synthesis, AMO-1618, produced dissected leaves while maintained under the noninductive conditions of long days and high temperature (Fig. 8). The interaction of natural growth inhibitors such as abscisic acid cannot be ruled out. Anderson (1978) reported that abscisic acid treatment resulted in the production of the floating leaf form in a heterophyllous species of Pota- mogeton while submerged. In the data reported here, abscisic acid applica- tions (up to 50 pg) had no effect on leaf form in Proserpinaca intermedia. This difference may be due to the facts that aerially grown plants were utilized in this study and that differences in control mechanisms may exist between monocotyledonous and dicotyledonous aquatic species.

The gibberellin effect on leaf form at 15°C (Fig. 7) was striking, especially under a 10-h photoperiod. In photoperiods of 14 h and greater at 15°C, gibberellin treatments stimulated mesophyll development primarily by cell division. In shorter photoperiods this GA3 response was altered such that leaf development occurred by vein elongation without further mesophyll develop-

84

men t . Such e longa t ion o f vein t issue is usual ly a t t r i b u t e d to the ac t ion o f endogenous aux in (Went, 1951) or e thy lene ( C o o k s o n and Osborne , 1978) . I t is in teres t ing to n o t e t h a t this resul t ing leaf f o r m was very similar t o t h a t p r o d u c e d on s u b m e r g e d shoo t s (see Fig. 1).

The s t r iking d e v e l o p m e n t a l p las t ic i ty shown b y P. intermedia and P. palustris, and by o the r aqua t ic angiosperms , can be e n c o m p a s s e d by the con- cep t o f ' phase change ' (Brink, 1962) w h e r e b y : " D u r i n g d e v e l o p m e n t perpe- tual ly e m b r y o n i c apical mer i s t ems in m a n y higher p lants unde rgo a m o r e or less a b r u p t swi tch in po t en t i a l f r o m a juveni le to an adul t t y p e o f g r o w t h " . By focus ing on the mechan i sm(s ) involved in con t ro l l ing phase change a ful ler insight in to d e v e l o p m e n t t h a t m a y be appl icable to p lants in general can be gained. H e t e r o p h y l l o u s aquat ic or a m p h i b i o u s ang iospe rms are ideal p lan ts for such s tudies since the d e v e l o p m e n t a l r e sponse o f h e t e r o p h y l l y is clearly expressed and can be quant i f ied .

ACKNOWLEDGEMENTS

The au thor s wish to t h a n k Glen T h u r s b y w h o assisted in the p r e p a r a t i o n o f the plates .

REFERENCES

Albert, L.S. and Wilson, C., 1961. Effect of boron on elongation of tomato root tips. Plant Physiol., 36 : 244--251.

Anderson, L., 1978. Abscisic acid induces formation of floating leaves in the hetero- phyllous aquatic angiosperm Potamogeton nodosus. Science, 201: 1135--1138.

Best, E., 1979. Growth substances and dormancy in Ceratophyllum demersum. Physiol. Plant., 45: 399--406.

Best, E. and Soekarjo, R., 1976. Seasonal effects in the hormonal control of growth in the submerged aquatic macrophyte Ceratophyllum demersum. Physiol. Plant., 38: 249--256.

Bodkin, P.C., Spence, D. and Weeks, D., 1980. Photoreversible control of heterophylly in Hippuris vulgaris L. New Phytol., 84: 533--542.

Bostrack, J.M. and Millington, W.F., 1962. On the determination of leaf form in an aquatic heterophyllous species of Ranunculus. Bull. Torrey Bot. Club, 89: 1--20.

Brink, A., 1962. Phase change in higher plants and somatic cell heredity. Q. Rev. Biol., 37 : 1--22.

Cathey, H.M., 1964. Physiology of growth retarding chemicals. Annu. Rev. Plant Physiol., 15: 271--303.

Cookson, C. and Osborne, D., 1978. The stimulation of cell extension by ethylene and auxin in aquatic plants. Planta, 144: 39--47.

Davis, G.J., 1956. The effects of certain environmental and chemical factors on hetero- phylly in aquatic angiosperms. Ph.D. Thesis, University of North Carolina, Chapel Hill, NC, 59 pp.

Davis, G.J., 1967. Proserpinaca: photoperiodic and chemical differentiation of leaf devel- opment and flowering. Plant Physiol., 42: 667--668.

Douglas, T. and Paleg, L.G., 1974. Plant growth retardants as inhibitors of sterol biosyn- thesis in tobacco seedlings. Plant Physiol., 54: 238--245.

85

England, W. and Tolbert , R.J., 1964. A seasonal s tudy of the vegetative shoot apex of Myriophyllum heterophyllum. Am. J. Bot., 51: 349--353.

Grace, J. and Wetzel, R., 1978. The product ion biology of Eurasian Watermilfoil (Myrio- phyllum spicatum L.): A review. J. Aquat. Plant Manage., 16: 1--11.

Johnson, M., 1967. Temperature dependent leaf morphogenesis in Ranunculus flabellaris. Nature, 214: 1354--1355.

Schmidt, B.L. and Millington, W.F., 1968. Regulation of leaf shape in Proserpinaca palustris. Bull. Torrey Bot. Club, 95: 264--286.

Wallenstein, A., 1963. Modification of morphology, anatomy, and physiology of Proser- pinaca palustris L., an aquatic angiosperm, by photoper iod and other environmental factors. Ph. D. Thesis, University of Rhode Island, Kingston, RI, 181 pp.

Wallenstein, A. and Albert, L.S., 1963. Plant morphology: its control in Proserpinaca by photoperiod, temperature, and gibberellic acid. Science, 140: 998--1000.

Weber, J. and Nooden, L., 1976. Environmental and hormonal control of turion germina- t ion in Myriophyllum verticillatum. Am. J. Bot., 63: 936--944.

Went, F.W., 1951. The development of stems and leaves. In: F. Skoog (Editor), Plant Growth Substances. University of Wisconsin Press, Madison, WI, pp. 237--298.

Winston, R. and Gorham, P., 1979. Roles of endogenous and exogenous growth regulators in dormancy of Utricularia vulgaris. Can. J. Bot., 57: 2750--2759.