Dehydrogenases and Isocitrate Lyase Activity During Pollen Germination in Calotropis Procera

Pro¢. Indian Acad. Sci., Vol. 86 B, No. 6, December 1977, pp. 371-374, ~) Printed in India.

Dehydrogenases and isocitrate lyase activity during pollen germination in Calotropis procera

C P MALIK and M B SINGH Department of Botany, Punjab Agricultural University, Ludhiana 141 004

MS received 11 April 1977; revised 24 October 1977

Abstract. lsocitrate lyase (ICL) activity is described in the quiescent and germinating pollen of Calotropis procera. The behaviour of the enzyme depended upon the presence or absence of exogenous substrate. The activity of this enzyme in relation to some dehydrogenases(MDH; ICDH; G-6-PDH) is compared. Effectsof actinomycin-D and cycloheximideon the activity of different enzymes is described. The increase in the activity of ICL and G-6-PDH, during the activation phase, is attributed to their de novo synthesis. The possibility of sucrose in causing repressing effects on ICL synthesis and/or activation is brought out.

Keywords. Dehydrogenases; isocitrate lyase; Calotropis procera; pollen and pollen tubes.

1. Introduction

Mature pollen grains of Calotropis procera (Ait). R. Br (Asclepiadaceae) are aggre- gated to form poUinia and have abundant quantities of storage lipids and free amino acids but no starch (Malik and Mehan 1974). Isolated pollinia, when incubated in 0-3 M sucrose solution as basal medium germinate and pollen tubes emerge out of germpore after about 30 minutes. Malik and Singh (1976) recognised three phases of pollen tube development e.g. activation, initiation and exponential growth, while ungerminated pollen, with feeble metabolic patterns are referred to the quiescent phase. In the activation phase, the metabolic patterns promoting growth appear and this obviously involves initial changes in the catalytic complex(s). The continued growth of pollen tube depended upon three crucial factors: (i) availability of suitable substrates, (ii) biogenesis of wall polysaccharides and (iii) protein bio- synthesis. Pollen grains in this species also germinate in distilled water but the germination percentage and pollen tube elongation is much less as compared to that in sucrose medium. Presumably, germinating pollen grains utilize storage lipids by their conversion to sugars via the glyoxalate cycle. It appeared to be of consider- able interest to investigate the activity profile of isocitrate lyase (ICL), a key enzyme of glyoxalate cycle.

2. Materials and methods

Pollinia were isolated and incubated either in distilled water or 0.3 M sucrose solution at 28:~2°C. This particular concentration of sucrose was found to be optimal for Calotropis pollen germination. In each culture, 8 mg of pollinia were incubated in

371 372 C P Malik and M B Singh

6 ml of basal medium or basal medium supplemented with 5-100/zg/ml of actinomycin-D or cycloheximide. Sixty/~g/ml of Act. D or 30/~g/ml of CH used individually produced maximal inhibition of the tube growth. Consequently, these concentrations were used in subsequent experiments. Pollen germination percentage and pollen tube length wire determined. Germinating pollen were removed at desired intervals, homogenized in 0.4 ml 0.1 M phosphate buffer (pH 7.5) and kept at 0°C. After centrifugation at 5000 g for 10 rain at 4°C, the supernatent was directly used as a source of ' enzyme'. Protein was determined by the method of Lowry et al (1951). Isocitrate lyase activity was measured by recording the production of glyoxalate from threo-Ds-isocitrate using the method of Lui and Rods (1970). Glucose-6-phosphate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase (NADP dependent) activities were measured colorimetrically (Dejong et al. 1967).

3. Observations and discussion

Actinomycin-D reduced pollen germination but did not affect early growth of the pollen tube whereas cycloheximide reduced both pollen germination and pollen tube elongation.

E17 ,eL '°' ISO DH (c) E s I e ~ls E o E

O ~D ~k 1 I I

23 (b] (d} G- 6- PDH MDH

~C~H ~E:k ],7 t i I I I I i i J --'~" I I O 30 60 90 120 150180 0 30 60 90 120 150180 Stage of germination (in rain)

Figure 1. Effectsof actinomycinoD (60 ag/ml) and cyctoheximide (30/~g/ml) on the development of enzymatic activity of Calotropis procera pollen, a. Isocl~trate lyase, b. glucose-6-phosphate dehydrogenase; e. isocitrate dehydrogenase; d. malate dehydrogenase a. The activity of isocitrate-lyase in the germinating pollen treated with CH, Act-D and those incubated in sucrose or water. Note decrease in the enzyme activity following incubation in antimetabolites or sucrose after 30 rain or CH caused maximal decline. b. Isocitrate dchydrogenase activity between 30-180 rain in sucrose (control) and declines with antimetabolites e. Glucose-6-p-dehydrogenasepeak activity in control at 30 min and declines in the antimetabolites at 180 min. d. Reduced activity of malate dehydrogcnase with antimetabolites compared to the sucrose control. Enzymes in germinating C. pracera 373

The changes in the activity of isocitrate lyase during pollen germination and pollen tube elongation on sucrose (0.3 M) and distilled water are illustrated in figure la. Up to 90 rain of incubation, the activity was comparable in both the conditions. Beyond this period, pollens incubated in water showed a rapid rise in the enzyme activity, while those incubated in sucrose showed a gradual decrease. The fluctuations in activity of isocitrate lyase during different phases of pollen tube elongation were compared with some of the dehydrogenases e.g. MDH, G-6-PDH ISDH (figure lb, c and d.) During the activation phase, G-6-PDH and ICL had high activity levels. On the other hand, MDH and ISDH had comparatively lower levels of activity. In sucrose solution during the initiation phase (i.e. after 90 rain) ICL and G-6-PDH activities decreased while those of MDH and ISDH increased. Thus, ICL and G-6-PDH registered rapid increase only during the activation phase only, while MDH and ISDH consistently increased beyond 30 rain. With the addition of Act.-D (figure la), there was inhibition in the rise of isocitrate lyase and G-6-PDH. Presence or absence of sucrose in the growth medium affected the level of activity of ICL. Figure la shows inhibition of ICL activity beyond 90 rain. The inhibition was comparable with that caused by aetinomycin-D. The effect of sucrose became prominent after 30 min. The lag observed for the effect of sucrose to be evident possibly depended on the time required for the accumu- lation of sucrose or some of its derivatives in the pollen. In Calotropis activities of several enzymes markedly increase after suspension of pollen in growth medium, pollen tube emergence and its subsequent growth. For some enzymes e.g. hydrolases on inhibition of water and prior to pollen tube initiation, an increase in the activity is detected. Such an increase was associated with onset of catabolism of pollen stored substances. In addition, the activity of dehydrogenases also continued to increase for several hours (Malik and Singh 1976). Malik (1974) demonstrated a steady depletion of lipids with increased periods of incubation in Calotropis pollen. The activity profile of ICL was consistent with such a depletion. Glyoxalate cycle may play a vital role during pollen tube growth. During the quiescent phase of pollen, ICL feeble activity was associated with lack of tube initiation. However, ICL was activated following pollen germination. Such a physiological behaviour correlated with the functional attributes, since the pollens germinate only after its deposition on stigma. During the activation phase there was rapid increase in pentose phosphate shunt as suggested by enhanced G-6-P dehydrogenase activity. Such a parallelism implied not only formation of pentoses initially but also their subsequent utilization. Presence of sucrose in the incubation medium, inhibited the increase of ICL beyond 30 rain. This pointed towards the repressing effect of sucrose on ICL. It is interesting to note that beyond 90 rain the repression was nearly complete. Inci- dentally, ICL development was almost completely inhibited by actinomycin-D. This suggested that beyond 30 rain or so, ICL development depended on de novo synthesis. Sugars have been shown to repress ICL synthesis. Haigh and Beevers (1964) and Lado et al (1968) suggest that active effector causing repression of ICL, belonged to the class of phosphorylated sugars rather than other intermediates of glycolysis. 374 C P Malik and M B Stngh

References

Dejong D W, Janson E F and Alson A C 1967 Oxidoreductive and hydrolytic enzyme patterns in plant suspension culture cells. Local and time relationship; ExptL Cell Res. 47 139-156 Haigh W G and Beevers H 1964 The occurrence and assay of isocitrate lyase in algae; Araeh. Biochem. Biophys. 107 147-151. Lado P, Schwendimann M and Matte E 1968 Repression of isocitrate lyase synthesis in seeds germinated in the presence of glucose; Biochem. Biophys. Acta 157 140-148 Lowry D H, Roseborough N J, Farr A L and Randall L J 1951 Protein measurement with the folin phenol reagent; J. Biol. Chemistry 193 265-275 Lui M S T and Roels O A 1970 An improved method for determining glyoxalic acid; Anal. Bio. chem. 38 202-209 Malik C P 1974 Enzymes, in pollen development and pollen tube growth; First Indian Palynological Congress, Chandigarh, Abst Malik C P and Mehan M 1974 Pollen chemistry, enzymes and tube growth in Calotropis proeera 43rd Indian Soc. Biol. Chemists, Abst. Malik C P and Singh M B 1976 Fluctuations in dehydrogenases Activities during the development of pollen tube of Calotropisprocera; Biochem. Phys. Pflanzen 169 583-588