Planta

Planta (1982)156:433-440

9 Springer-Verlag 1982

Inhibition of elongation growth by two sesquiterpene lactones isolated from Helianthus annuus L. Possible molecular mechanism

Otmar Spring and Achim Hager Institut fiir Biologie I der Universit/it, Auf der Morgenstelle 1, D-7400 Tfibingen, Federal Republic of Germany

Abstract. Two sesquiterpene lactones belonging to

the germacranolides were isolated from the leaves and stems of H e l i a n t h u s a n n u u s L. Their formation in the plant is light-dependent. Both sesquiterpene lactones (SL) strongly inhibit indole-3-acetic acid (IAA)-induced elongation growth of A r e n a s a t i v a L. coleoptile segments and H e l i a n t h u s a n n u u s L . hypocotyl segments. Both SL do not, however, inhibit acid-induced growth nor growth triggered by fusicoccin at all. In the presence of dithiothreitol (DTT), the inhibitory effect of SL in the A r e n a segment-test can be completely neutralized. This can be attributed to the binding of DTT to both SL. Using thin-layer-chromatography it could be shown that the inhibitors build adducts with SHrich compounds, e.g., cysteine, glutathione, mercapto-ethanol, and DTT, whose Rf-value significantly differs from those of the primary substances. If the coleoptile segments are first treated with an inhibitor and the inhibitor is subsequently washed out, close to normal elongation growth can be induced by adding an IAA-solution. If the segments are simultaneously treated with inhibitor and IAA, no notable growth can be initiated for an extended amount of time, after the removal of both substances and the anewed addition of IAA. Fusicoccin, however, can immediately neutralize the induced growth inhibition. The same irreversible inhibition is observed when 2,4-dichlorophenoxyacetic acid (2,4-D) is used: If coleoptile segments are treated with an inhibitor plus 2,4-D or an inhibitor plus 3,5-dichlorophenoxyacetic acid (3,5-D), respectively, IAA-induced growth after removal of the substances can only be observed by Abbreviations: 2,4-D = 2,4-dichlorophenoxy-acetic acid; 3,5-D = 3,5-dichlorophenoxy-acetic acid; DTT=dithiothreitol; FC = Fusicoccin; GA 3 = gibberellic acid; IAA = indole-3-acetic acid; MES=2-(N-morpholino)-ethane sulfonic acid; SL=sesquiterpene lactone(s)

those coleoptiles which had previously been treated with the non-auxin, 3,5-D plus an inhibitor. Based on these results, a possible mechanism describing how the inhibitor functions is discussed. The binding of an auxin to an auxin receptor sets a SHgroup free (possibly due to a change in the conformation of the receptor); a site is given to which the inhibitor can bind irreversibly (via a S-bond). The IAA-receptor-inhibitor-complex is then no longer able to initiate elongation growth. If auxin is not present, no lasting bond between the inhibitor and the receptor can occur, since the essential SH-group remains masked. The inhibitor can be washed out again. Consequently, the SL's have to be able to intervene at the beginning of the IAAinduced reaction sequence, while the following steps remain uninfluenced, i.e. namely, the active excretion of protons into the cell wall compartments, which is directly induced by fusicoccin and causes elongation growth. Key words: Auxin receptor Elongation growth - Sesquiterpene lactone.

- Helianthus

Introduction

The sesquiterpene lactones are natural compounds, which are often found in the abundant species of the compositae plant family (Hegnauer 1964; Heroult 1971; Heroult and Sorm 1969). These compounds are partially characterized by their cytotoxic, antimicrobial, phytotoxic, and cytostatic effects (Spring and Hager 1982; Willuhn 1981; Powell and Smith 1980; Lee et al. 1977a, 1977b; Ogura et al. 1978; Kupchan et al. 1970). Their value for the plant still remains largely unknown (Kalsi et al. 1977, 1981; Rodriguez et al. 1976; 0032-0935/82/0156/0433/$01.60

434

O. Spring and A. Hager: Inhibition of growth by sesquiterpene lactones

H

4'

0

o

CH~

C

(fr. 6). The sesquiterpene lactones were found in fraction 5 (compound I) and fraction 6 (compound

yH3

II

CH3 ~,

c

7 l

0

] I HOJ

Fig. 1. Sesquiterpene lactones from H e l i a n t h u s

0 Ang

0

(I) ~_Niveusin C (Ohno et al. 1980; Herz and Kumar 1981; Spring et al. 1981), (II) = 15-Hydroxy-3-dehydro-desoxyfruticin (Spring et al. 1982) annuus.

Kefeli and Kadyrov 1971; Kefeli 1977). A compound was found in Helianthus annuus, which not only had a remarkable inhibitory effect on elongation growth in the sunflower itself, but also in various different plants. This compound was identified as a sesquiterpene lactone with a molecular weight of 378 (Krauss 1971). Structural determination by spectroscopic measurements (Spring et al. 1981) revealed the compound to be an 0~-methylene-7-1actone with an angelic acid side chain (I, Fig. 1), the structure of which is identical to a compound (Herz and Kumar 1981) isolated from Helianthus maximiliani and one which Ohno and Mabry (1980) isolated from Helianthus niveus (Niveusin C). Beside this compound (I), a further sesquiterpene lactone (II) was isolated and its structure analyzed (II, Fig. 1) (Spring et al. 1982). Structurally, it is very similar to desoxyfruticin (Herz and Scharma 1975) as well as to tagitinin C (Baruah et al. 1979). In this report isolation methods are described, and, furthermore, the effect of these compounds on the growth of coleoptile segments and sunflower hypocotyls, the interaction of these compounds with IAA and fusicoccin, and the effect on the ATP-dependent proton pumps of microsomal membranes isolated from maize coleoptiles is investigated. Materials and methods

Extraction and isolation. Seedlings of Helianthus annuus var. giganteus were grown in a greenhouse. Young leaves and stems of three - week-old plants were harvested, extracted in boiling ethanol, homogenized, and filtered. The filtrate was evaporated in vacuo and the residue extracted by ether. The crude extract was chromatographed by CC (Polygosil 60-4063), eluted with petrol (60-80 ~ C) 100% (fraction 1), petrol-CHC13 v:v 1:1 (fr. 2), CHC13 100% (fr. 3), CHC13-EtOH 49:1 (fr. 4), CHC13-EtOH 19:1 (fr. 5), and CHC13-EtOH 9:1

I0. Further purification was performed by thin layer chromatography (TLC) (CHzC12-Ac-EtOAc , 5:4: 1). The inhibitors were analyzed by bioassay and by coloring with Ehrlich's reagent in TLC. The germacranolide (I) could be crystallized from CHC13, giving colorless crystals (mp. 79 ~ C); (n), a light yellow oil could not be induced to crystallize. Arena straight growth test. Etiolated deleafed Arena coleptile segments 5 to 6 d old were used in the experiments. Segments of 10 mm in length (apical 2 mm sections discarded) were incubated in 5raM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.85, for 4 h at 30 ~ C. Each assay tube contained 5 ml test solution (auxin plus inhibitor in different concentrations) and 10 coleoptile segments. The average length was compared with segments from samples without inhibitor, but with or without auxin. The time course of growth inhibition was investigated with an inductive displacement transducer (Philips PR 9314-01) in the same test solutions. Assay tubes contained 25 ml 5 mM MES buffer (pH 5.85, 30 ~ C) and five coleoptile segments prepared in the same manner as for the Arena straight growth test. The preparation of vesicles from maize coleoptiles and the determination of ATP-dependent acidification within the vesicles have been described in preceding papers (Hager et al. 1980; Hager and Helmle 1981). Results

Inhibition of auxin-induced elongation growth in the Arena segment test by (I) and (II). Indole-3-acetic acid (IAA) induced elongation growth in l-cmlong coleoptile segments is already inhibited at 5 and 10 gM of (II) and (I), respectively (Fig. 2). Complete growth inhibition is achieved at concentrations of between 150 and 300 gM; substance (II) displays stronger inhibitory activity. 2,4-D (20 gM)-induced elongation growth of Arena coleoptile segments is influenced similarly. The inhibition brought about by (I) and (II) (100gM), respectively, is approximately 75%. Under the same conditions the weak elongation growth induced by gibberellic acid (20 gM) is 82.5% inhibited. IAA (10 gM)-induced growth, however, remains unaffected, if the assays with 100 gM (I) and (II), respectively, are treated with dithiothrei-

00

O. Spring and A. Hager : Inhibition of growth by sesquiterpene lactones I

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I

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c

1.2

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0.6 0./~ 10

20

50

100

300

/uM Inhibitor

0.2

Fig, 2. Inhibition of IAA (10 gM)-induced elongation growth of 1 cm long Arena coleoptile segments by (I) and (II) after 4 h of incubation at 30 ~ C. Elongation of 2.0 ram/segment is defined as 0% inhibition

100

MES pH 5.85

Citrat pH 3.5 I 1T (100~uM) (100,uM)

Fig. 4. Growth of 1 cm long Arena coleoptile segments in a 2-(N-morpholino)ethane sulfonic acid (MES)-buffer (4 mM, pH 5.85), b citrate buffer (4 raM, pH 3.5), c citrate buffer and (I) (100 I~M) and d citrate buffer and (II) (100 laM) after 4 h of incubation at 30 ~ C. Acid-induced growth is not influenced by (I) or (II)

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+ IAA(10,uM) Fig. 3. Inhibition of 1AA (10 pM)-induced elongation growth of 1 cm long coleoptile segments through (I) and (II) (100 pM). Neutralization of inhibition through dithiothreitol (DTT) (500 IxM), incubation for 4 h at 30 ~ C. Elongation of 2,0 ram/ segment is defined as 100% growth

tol (DTT) (500 gM). Thus, DTT can neutralize growth inhibition completely (Fig. 3).

Non-inhibition of acid-induced growth by (I) and (II). Unlike auxin-induced growth, acid-induced growth (Hager 1962; Hager et al. 1971; Cleland 1971) is not influenced by sesquiterpene lactones from Helianthus at all. Neither (I) nor (II) bring about a change in elongation growth of the segments at a concentration of 100 gM (Fig. 4).

Time-course of growth inhibition of coleoptile segments by (I). Inhibition of auxin-induced growth

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I (100 ~uM)

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Fig. 5. Time-course of elongation growth of i cm long Arena coleoptile segments after addition of IAA (10 pM) and (I) (100 gM) respectively

after addition of sesquiterpene lactones occurs after a lag phase of 15 to 20 min (Fig. 5). The slow onset of this effect is observed in Arena coleoptile segments as well as in hypocotyl segments of Helianthus annuus. Additions of (I) (100 gM) leads to a nearly complete growth standstill after approximately 120 min. Figure 6 shows the time-course of growth after the addition of (I) (I 50 gM) at different points in

436

O. Spring and A. Hager: Inhibition of growth by sesquiterpenelactones I

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Fig. 6. Time-course of elongation g r o w t h o f 1 cm long coleop-

tile segments when (I) (150 gM) is added at different points of time before and after the addition of IAA (10 gM)

_

Reversibility of the inhibitory effect. If Arena coleoptile segments are incubated in MES-buffer together with the inhibitors (100 pM) for two hours and subsequently transferred into a medium without inhibitors for 40 rain, addition of IAA induces strong growth. Thus, the inhibitor can be rinsed out. However, if the segments are incubated in a solution containing inhibitor (100 gM) and IAA, no growth is observed after transfer into a medium without inhibitors and subsequent addition of IAA. Sixty to 120 min after the addition of IAA, increased growth gradually starts again (Fig. 7). In an analogous experiment it can be shown

I

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time before and after treatment with IAA (10 gM). Addition of the inhibitors less than 30 rain before treatment with IAA leads to nearly complete suppression of auxin-induced elongation growth. A second addition of IAA or other growth hormones, e.g. 2,4-dichlorophenoxyacetic acid, (2,4-D) or gibberellic acid (GA3) can no longer stimulate growth. However, the addition of citrate buffer (4 raM; pH 3.5) leads to immediate, uninhibited acid-induced growth. These results clearly show that the membranes are not unspecifically affected by the inhibitors (I) and (II), e.g., through binding to SH-groups. The permeability of the membranes remains unchanged and the turgor pressure within the cell unimpaired, since normal acid-induced growth is observed upon a reduction of the pH-value within the cell wall compartment. The inhibitors must therefore directly intervene in the auxin-induced reaction sequence.

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Fig. 7. Time-courseof elongation growth of i cm long coleoptile segments.Pre-incubationin (I) (100 gM) with and without IAA (10 gM). Transfer into pure MES-buffer(5 mM, pH 5.8) after t= 140 rain. Addition of IAA (10 gM) after t= 180 rain. On the top : growth. On the bottom: growth rate (SEM-calculation based on n = 9 independentexperiments) that after pre-incubation in a solution containing inhibitor (100 I-tM) and 3,5-dichlorophenoxyacetic acid (3,5-D) (50 ~tM), IAA-induced growth is stimulated, whereas pre-incubation in a solution containing inhibitor (100 gM) and the auxin 2,4-D (50 gM) does not initiate an immediate growth reaction after the addition of IAA (Fig. 8). The reversibility of the inhibitory effect in the Arena segment test after pre-incubation of the segments in solutions containing inhibitor (100 pM) and a) 3,5-D (50 gM), b) 2,4-D (50 gM) or c) IAA (10 gM) is summarized in Table 1.

Fusicoccin-induced growth is not retarded by the inhibitors (I) and (II). Fusicoccin, a substance isolated from fungi, induces elongation growth to quite the same extent as auxin (Marr6 1977, 1979). One supposes that this substance is responsible for the direct activation of a proton pump on the plasma membrane, leading to an increased H +-efflux into the cell wall compartment (Lado et al. 1973). In contrast to IAA-induced elongation growth, fusicoccin-induced growth is not inhibited by the

O. Spring and A. Hager : Inhibition of growth by sesquiterpene lactones

Reaction of the sesquiterpene lactones (I) and (II) with compounds possessing SH-groups. The inhibi-

1.0 ,,."

fi

tor (I) and (II) bind with free SH-groups (a kind of Michael-addition reaction), whereby they lose their potential to further react with bionucleophiles possessing sulfhydriles. An adduct formation of (I) and (II) with DTT as well as cysteine (cys), glutathione, and mercaptoethanol can be proven by thinlayer chromatography (Fig. 11). Compared to the free-substance, the adducts distinguish themselves through a markedly changed Rr-value. Mixture of inhibitor with cystin (cys cys), which possesses no free SH-group, do not reveal new bands after TLC. Likewise, mixtures of IAA and inhibitor also do not yield changed Rf-values.

IAA .." (IOjuM) .," .:" oo o 0.5 E G;

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Fig. 8. Time-course of elongation growth of 1 cm long Arena coteoptile segments. Pre-incubation in (I) (100 MM) with 3,5-D (50 gM) and 2,4-D (50 #M) respectively. Transfer into pure MES-buffer after t=120min. Addition of IAA after t = 180 rain

sesquiterpene lactones (I) and (II). The strong effect on growth, which is triggered by 2 gM fusicoccin, can be observed after a lag phase of 2 to 4 rain. (I) and (II) (100 gM each) remain ineffective (Fig. 9). Also of interest is the fact that growth retardation induced by an inhibitor can be reversed by fusicoccin, but not by IAA (Fig. 10). The inhibitors do not seem to inhibit H+-excretion into the cell wall compartment driven by an activated proton pump (Hager et al. 1971); however, they do seem to inhibit certain auxin-dependent steps leading to the activation of this pump.

Influence of (I) and (II) on the A TP-fueled, C1 dependent acidification of microsomal vesicles from maize coleoptiles. Microsomal vesicles which possibly already exist in the cytoplasm as derivatives from the ER or Golgi-apparatus, and which can fuse with the vacuole (or the plasmalemma) can be isolated from maize coleoptiles (Hager and Helmle 1981; Hager et al. 1980). They possess an ATP-fueled, C1- dependent proton pump, which seems to play an important role in growth (Hager and Helmle 1981). It has been investigated as to whether the ATP-dependent intravesicular acidification of these vesicles is influenced by (I) and (II). Only a minute unspecific decrease in H +-accumulation could be observed similar to that caused by other lipophilic substances (ca. 15-20% with 100 ~tM I or II).

Table 1. Arena-segment test. Elongation growth in % after 4 h (t 180-420) of incubation in indole-3-acetic acid (IAA) (10 gM) at 309 C. Elongation of 2.0 mm/segmeut is defined as 100%. Segments were pre-incubated in (I) (100 gM) and a) 3,5-dichlorophenoxyacetic acid (3,5-D) (50 ~tM), b) 2,4-dichloropheuoxyacetic acid (2,4-D) (50 MM) or c) IAA (10 gM). The nearly complete inhibition of IAA-induced growth (t 180420 min) only takes place in those segments, which were pre-incubated in a solution containing inhibitor and auxin Sample

t (rain) 0-60

Control

60-120

120-180

Buffer only

Growth

(%) 180-420 Buffer + IAA

100 • 14 48 • 13

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Buffer + I + 3.5-D

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Buffer + IAA

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Buffer + IAA

5•

c

Buffer + I

Buffer + I + IAA

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1• 6

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438

O. Spring and A. Hager: Inhibition of growth by sesquiterpene lactones I

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Fig. 9. Time-course of elongation growth of 1 cm long Arena coleoptile segments after addition of fusicoccin (FC) (2 gM) and (I) (100 gM)

I

+Ior[

Fig. 11. Thin layer chromatography of (I) and (II) with different sulfhydrilic substances as well as cystin and IAA (after preceding incubation in phosphate buffer, pH 7.5, for 30-60 min). Solvent : CH2C12-Ac-EtOAc, 5 : 4:1 (V: V : V)

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320 400 t (min)

Fig. 10. Time-course of elongation growth of 1 cm long Arena coleoptile segments. 1) Control without inhibitor, IAA (10 pM) after t = 100 min. 2) Inhibition of IAA-induced growth through (I) (100 pM) and reversal of inhibition through fusicoccin (1 IaM)

Discussion Both elongation growth inhibitors isolated from leaves and stems of Helianthus annuus belong to the sesquiterpene lactones. Related compounds have also been found in other composites (Ohno and Mabry 1980; Herz and Kumar 1981; Baruah et al. 1979). The compounds (I) and (II) display a strong inhibitory effect on auxin-induced elongation growth in A r e n a coleoptiles (Fig. 1) as well as Helianthus hypocotyl segments, whereby compound (II) distinctly inhibits more than (I) and other sesquiterpene lactones, e.g., heliangine (Shibaoka 1961) or vernolepin (Sequeira et al. 1968). Unlike auxin-induced growth, acid-induced growth is not influenced. Acid-induced growth which can also take place under anaerobic conditions (Hager 1962; Hager et al. 1971) is caused by an increase in the plasticity of the cell wall; turgor then leads to cell elongation. Since acid growth cannot be inhibited by either (I) or (II), it is certain that these inhibitors do not inhibit auxin-induced growth by changing the permeability of the membrane or decreasing the turgor. A further important result regarding the operating mechanism of the inhibitors is revealed through the experiments with fusicoccin. This substance isolated from fungi induces an equally strong elongation growth as IAA, however, in contrast to IAA growth is independent from protein synthesis (Bates and Cleland 1979). One assumes that fusicoccin (FC) directly effects certain proton pumps of the membrane (Marr~ 1977). These H +-pumps should bring about a pH-de-

O. Spring and A. Hager: Inhibition of growth by sesquiterpene lactones

crease in the cell wall, leading to an increase in plasticity and elongation growth (Hager et al. 1971; Rayle and Cleland 1972, 1977; Cleland and Rayle 1978; Hager et al. 1980, Hager 1980; Hager and Helmle 1981 ; Hager and Hermsdorf 1981). Since fusicoccin-induced growth is not inhibited by either (I) or (II), direct influence of the sesquiterpene lactones on the H +-pumps is unlikely. The fact that the ATP-fueled, Cl--dependent proton pumps, which are located on native microsomal vesicles of maize coleoptiles and which seem to play an important role in auxin-induced elongation growth (Hager et al. 1980; Hager and Hermsdorf 1981 ; Hager and Helmle 1981) are only insignificantly and unspecifically inhibited by sesquiterpene lactones (SL) (I) and (II) also supports this proposition. Therefore, it can only be assumed that (I) and (II) intervene in the auxin-induced reaction sequence before the actual activation of the H § ATPase takes place. In this connection it is important that the inhibitory effect fails nearly completely, if the SL's are removed before auxin is added. This characteristic has also been proven to hold true for vernolepine (Sequeira et al. 1968). If, however, auxin and inhibitor are added simultaneously, the inhibitory effects is not reversible even after the inhibitors have been rinsed out; in other words, unlike FC or acidic buffers, repeated addition of auxin can not subsequently initiate elongation growth for a longer amount of time. The specifity of auxin is also indicated by the fact that typical IAA-growth can be brought about in coleoptile segments incubated in a solution containing inhibitor and 3,5-D after these substances have been rinsed out, not, however, if the segments were pre-incubated in a solution containing the auxin 2,4-D and inhibitor (Fig. 7, Table 1). These results favor the proposition that the inhibitory effect of SL's must occur at a very early step of the auxin-induced reaction sequence. Such a step is the formation of an auxin-receptor-complex (Hertel 1979; Dohrmann et al. 1978; Ray 1977; Rubery 1981 ; Venis 1977). It is possible that this complex functions as the initiator of inhibition; in which case it has to be assumed that after binding of the auxin at the receptor a change in the conformation of the protein takes place and a SH-group is possibly set free, both rendering the binding of the inhibitor possible. The auxin receptor-inhibitor-complex should not be able to incite elongation growth; nor should the inhibitor be seperable from the complex. Such irreversible binding of the SL's could be explained by S-binding,

439

for it has been shown in this paper that the inhibitory effect in the Avena-biotest can be neutralized by adding DTT. Further, it has been shown that between the inhibitors (I) and (II) and amino acids possessing a free SH-group adducts are formed which can be detected by TLC (Fig. 10). This reaction can be explained by the presence of an exocyclic methylene group on the lactone ring of the molecule. It can especially react in a nucleophile methylene addition (Michael addition) with an SH-group of the amino acid, cysteine, and influence the effect of enzymes possessing an SHgroup (Kupchan et al. 1970; Lee et al. 1977a, b). This effect can be further supported by corresponding reactive side groups or other unsaturated centers of the molecules (see Lee 1977a). The complete structure of the molecule consequently influences where, on the protein and enzyme or on which subunit, a reaction with SH-groups can take place. In this respect a certain specific effect of these sesquiterpene lactones is quite possible. The importance of the SL's for the plant can be that they counteract the effect of growth-promoting substances under certain circumstances, thus causing stocky growth of the plant. In this respect, it is very remarkable that the formation of these inhibitors only takes place in the light (Hager, Fiesel and Spring, unpublished). References Baruah, N.C., Sharma, R.P., Madhusudanam, K.P., Thyagarajan, G., Herz, W., Murari, R. (1979) Sesquiterpene lactones of Tithonia diversifolia. Stereochemistry of tagitinins and related compounds. J. Org. Chem. 44, 1831-1835 Bates, G.W., Cleland, R.E. (1979) Protein synthesis and auxininduced growth: inhibitor studies. Planta 145, 43%442 Cleland, R.E. (1971) Cell wall extension. Annu. Rev. Plant Physiol. 22, 197-222 Cleland, R., Rayle, D. (1978) Auxin, H§ and cell elongation. Bot. Mag., Special issue 1, 125-139 Dohrmann, U., Hertel, R., Kowalik, H. (1978) Properties of auxin binding in different subcellular fractions from maize coleoptiles. Planta 140, 9%106 Hager, A. (1962) Untersuchungen fiber einen dnrch H§ induzierbaren Zellstreckungsmechanismus. Habil. Schrift, Naturwiss. Fakult/it, Universit/it Mfinchen Hager, A. (1980) Avena coleoptile segments: hyperetongation growth after anaerobic treatment. Z. Naturforsch. 35e, 794-804 Hager, A., Frenzel, R., Laible, D. (1980) ATP-dependent proton transport into vesicles of microsomal membranes of Zea mays coleoptiles. Z. Naturforsch. 35e, 783 793 Hager, A., Helmle, M. (1981) Properties of an ATP-fueled CI-dependent proton pump localized in membranes of microsomal vesicles from maize coleoptiles. Z. Naturforsch. 36e, 997 1008 Hager, A., Hermsdorf, P. (1981) H+/Ca 2+ antiporter in membranes of microsomal vesicles from maize coleoptiles, a s e c -

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O. Spring and A. Hager: Inhibition of growth by sesquiterpene lactones

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