Control of Amylase Synthesis in Cotyledons of Germinating Peas: Examination of the Possibility of Osmotic Regulation

Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan

Control of Amylase Synthesis in Cotyledons of Germinating Peas: Examination of the Possibility of Osmotic Regulation Y. MOROHASHI and K. VENO With 4 figures Received May 8, 1979 . Accepted September 5, 1979

Summary Amylase synthesis was severely retarded in detached cotyledons of germinating peas

(Pisum sativum L., cv. Alaska 7), while it was markedly promoted in attached ones. The

concentration of solutes was higher in the former cotyledons than in the latter ones. Even though the solute concentration of attached cotyledons was elevated by addition of external maltose to the level of that of detached ones, amylase synthesis was not inhibited. The enzyme synthesis in attached cotyledons was curtailed only by maltose concentrations that must be regarded as unphysiological. These findings are discussed in relation to osmotic regulation of amylase synthesis in pea cotyledons. Key words: amylase synthesis, osmotic regulation, seed germination, Pisum sativum.

Introduction VARNER and colleagues (YOUNG et aI., 1960 ; VARNER et aI., 1963) observed that many hydrolytic enzymes cannot develop in excised pea cotyledons during germination and suggested that there is an important interaction between the embryonic axis and the cotyledon. A number of investigators have attempted to elucidate the mechanism for the interaction from the viewpoint that the development of enzymes may be dependent on some embryonic-axis factor(s), which is supposed to be phytohormone(s). However, available information on hormonal control of development of amylase activity in pea cotyledons is rather confusing. VARNER et ai. (1963) reported that gibberellin could replace the axis. In contrast, SPRENT (1968) could not demonstrate any promotive effect of gibberellin on amylase formation. It was also reported that zeatin or zeatin riboside plus gibberellin could bring about a full replacement of the action of the axis (LOCKER and ILAN, 1975). On the other hand, it was reported that water stress inhibits enzyme synthesis in germinating seeds (JONES, 1969; JONES and ARMSTRONG, 1971) and that hydrolysis products, which accumulate in the endosperm of germinating barley seeds, actually

z. P/lanzenphysiol. Bd. 96. S. 303-310. 1980.

304

Y. MOROHASH[ and K.

VENO

function to regulate osmotically the production of hydrolytic enzymes by aleurone layers (ARMSTRONG and JONES, 1973). A similar mechanism is possible for the effects of removal of the axis on the development of amylase in pea cotyledons: that is, hydrolytic products would accumulate in excised cotyledons, resulting in water stress and then inhibition of amylase synthesis. However, Ithere is no information on the possibility of osmotic regulation of enzyme synthesis in pea seeds. The present study was carried out to examine such a possibility.

Material and Methods Seeds of peas (Pisum sativum L., cv. Alaska 7) were obtained from Asgrow International Corp., Kalamazoo, Michigan. Husked seeds were sterilized for 10 min in 0.1 % solution of sodium hypochlorite, washed in water and then imbibed in water at 28° for 4 h. After imbibition, the two cotyledons of a seed were separated from each other. The embryonic axis remained on one of the cotyledons. Samples of 25 cotyledons (either with [attached] or without [detached] the embryonic axis) were put in 12 em Petri dishes containing 20 ml of wat,er or 5 X 10-6 M cycloheximide (CH) solution. Incubation was in darkness at 28°. The above-mentioned procedures were all carried out under aseptic conditions. After incubation, the cotyledons were homogenized with a mortar and pestle in 0.05 M Tris-maleate buffer (pH 7.2) (1 ml buffer for each cotyledon). The homogenate was centrifuged at 10,000 g for 10 min and the supernatant was used as the enzyme preparation. The activity of amylase was measured according to the method of BERNFELD (1955). The osmolarity of hydrolysis products in the cotyledons was determined by freezing point depression. Thirty cotyledons were homogenized in a mortar and pestle with sand and 10 ml water. The homogenate was squeezed through four layers of gauze and centrifuged at 25,000 g for 10 min. The supernatant was used for the measurement. Evaporation and enzymatic hydrolysis were minimized during preparation of the solution by performing all operations at low temperatures (2-4°).

Results and Discussion Amylase activity in attached cotyledons of peas increased rapidly after the 2nd day, while the activity in detached ones increased only slightly (Fig. 1). The development of amylase activity in attached cotyledons was severely inhibited by CH-treatment (Fig. 2). Therefore, the increase in the enzyme activity seems to be dependent on de novo synthesis of the enzyme. These results suggest that the presence of the embryonic axis is necessary for the synthesis of amylase in germinating pea cotyledons. Similar results were obtained with pea cotyledons incubated in Petri dishes (VARNER et aI., 1963; LOCKER and hAN, 1975). In contrast, YOMO and VARNER (1973) observed with pea cotyledons incubated in Erlenmeyer flasks that amylase activity was higher in excised cotyledons than in normal ones. BRYANT and HACZYCKI (1976) attributed the conflicting results to the conditions under which cotyledons were incubated. According to them, incubation in Petri dishes has inhibitory effects on seedling growth and enzyme development due to a smaller internal air space, as compared with incubation in Erlenmeyer flasks with a large Z. Pjlanzenphysiol. Bd. 96. S. 303-310. 1980.

Control of amylase synthesis

305

7.5 0:::

E ~ c: 0

~

... ..!!

>.

0

u

....... CD

III

0

5.0

"0 E III

..!! 0

E 2-

>-

I-

>

2.5

IU



...J

>-

::E
0

4

3

2 GERMINATION TIME

(days)

Fig. 1: Changes in amylase activity in attached and detached cotyledons of peas during germination. Each point is the mean value of three determinations.

internal space. However, under the conditions of the present experiments (Petri-dish culture), seeds germinated well and seedlings grew normally. Further investigations are necessary for the elucidation of this point. One of the possible explanations for active synthesis of enzymes in attached cotyledons is that some factor(s) (presumably phytohormone[s]) transported from the axis into the cotyledon is responsible for induction of the enzyme synthesis (VARNER et aI., 1963). However, no convincing result has been obtained concerning the effects of growth substances on amylase development in pea ootyledons (see Introduction). We treated detached pea cotyledons with indoleacetic acid, gibberellic acid, kinetin, benzyladenine (5 X 10-8 and 5 X 10-5 M) or zea:tin (10-· M); however, these showed little effeots on amylase development in the cotyledons (data not shown).

z. PJlanzenphysiol. Bd. 96. S. 303-310. 1980.

306

Y.

MOROHASHI

and K.

VENO

c

E 10

7.5

c

0

~

CD

....0>. u

""CD fII

....0 c

5.0

E fII

CD

0

E

3

>-

I-

> 2.5 t=

CH

0

<.(

W

0---

CJ)

<.(

-- -- --

... .cr

,-,' ..... .....

",IJ

....J

>-

::E

«

0

2

3

GERMINATION TIME (days) Fig. 2: Effects of cycloheximide on development of amylase in attached cotyledons. CH: cycloheximide treated, water: water control. Each point is the mean value of two determinations.

On the other hand, with regard to mechanisms of inhibition of amylase synthesis in detached cotyledons, JONES' (1969) and ARMSTRONG and JONES' (1973) observations are very suggestive. They observed that osmotic stress inhibits gibberellin-induced synthesis of amylase in aleurone layers of germinating barley seeds. It was also demonstrated (JONES and ARMSTRONG, 1971) that osmotic regulation of enzyme production actually occurs in germinating barley seeds. In detached pea cotyledons, hydrolysis products of reserve materials would accumulate more than in attached ones. Then, greater osmotic stress will be imposed on cells of detached cotyledons. From the analogy with results on experiments with barley seeds, it is expected that the stress might inhibit the synthesis of amylase in detached cotyledons also. It is

z. PJlanzenphysiol. Ed. 96. S. 303-310. 1980.

Control of amylase synthesis

307

worthwhile to examine whether such mechanism actually functions in inhibiting the enzyme synthesis in detached pea cotyledons. As shown in Fig. 3, solute concentrations in detached cotyledons increased during the incubation, while those in attached ones remained at a low level. When embryonic axes were excised from attached cotyledons on day 2 or 3, development of amylase activity in excised cotyledons during the following day was severely inhibited (Fig. 4). Corresponding to this, concentrations of solutes in excised cotyledons increased markedly (Fig. 3). From these results alone, it seems that water stress resulting from accumulation of solutes in detached or excised cotyledons inhibits the enzyme synthesis in pea cotyledons, as in barley seeds. In order to examine whether or not this inference is valid, further experiments were carried out. If the inference is correct, it is expected that amylase synthesis is inhibited in attached cotyledons whose solute content is increased to a level similar no that of detached or excised cotyledons. For the purpose of increasing solute concentrations in cotyledons, attached cotyledons were transferred from water to 0.25 M maltose solution on day 2. The solute concentration and the enzyme activity in transferred

-... 0 0

E

en

120

0

E

z

0

.... « a=

I-

z

100

U

Z 0

U

.'

I

UJ

....

~

..J

, I

UJ

I

I

I

I

I

I

I

I

I

I

I

I

I

I

9 I

,

I

, I

I

I

I

I

I

I

I

I

I

I

,I

I

I

,o

.....+'

...

3

4

I

ATTACHED

0

(/)

,.,.. 2

GERMINATION TIME

(days)

Fig. 3: Changes in solute concentrations in attached, detached and excised cotyledons. Arrows indicate when excision was done and broken lines show the changes in solute concentrations in excised cotyledons. Each point is the mean value of three determinations. Z. Pjlanzenphysiol. Bd. 96. S. 303-310. 1980.

308

Y.

MOROHASHI

and K.

VENO

~

c

E

7.5

10

C

0 "'a

..... CD

>.

0

u

CD

en 0

c E

5.0

en CD 0

E .3

>-

I-

> 2.5

I()

<{

W

(f)

<{

...J

>-

::E

<{

0

2 GERMINATION TIME

4

3 (days)

Fig. 4: Effects of removal of the embryonic axis on the development of amylase. Arrows indicate when excision was done and broken lines show the changes in amylase activity in excised cotyledons. Each point is the mean value of three determinations.

cotyledons were measured on day 3. By this treatment, the solute concentration of maltose-treated cotyledons was increased to almost the same level as that in excised cotyledons (Table 1). However, amylase activity of the former cotyledons was still higher than that of the latter ones (Table 1). Furthermore, although the solute content in maltose-treated attached cotyledons was higher than that in water-treated attached ones, the enzyme activity was almost the same between the two (Table 1). Thus, even though the solute concentration in attached cotyledons was elevated to a level similar to that in excised ootyledons, development of amylase activity was not inhibited. When attached cotyledons were treated with 0.6 M maltose, the solute concentration in the cotyledons was much higher than that in excised cotyledons (Table 2). Although the synthesis of amylase was inhibited in some degree in these

z. Pjlanzenphysiol. Rd. 96. S. 303-310. 1980.

Control of amylase synthesis

309

Table 1: Solute concentration and amylase activIty in excised cotyledons and in attached ones treated with and without maltose. Attached cotyledons were incubated for the first 2 days in water and then divided into three groups. One was incubated further in water. The second one was transferred to 0.25 M maltose solution. As for the last one, the embryonic axis was removed and the excised cotyledons were incubated in water. Assays were done on day 3. The results represent the average of at least two independent experiments. Solute concentration (milliosmolar)

Attached cotyledons in water Attached cotyledons in maltose Excised cotyledons in water

Amylase activity (,umoles maltose! 15 min cotyledon) 3.09 3.03

87

112 113

1.58

Table 2: Solute concentration and amylase activIty in excised cotyledons and in attached ones treated with and without maltose. Attached cotyledons were incubated for the first 3 days in water and then treated in the same way as mentioned in Table 1. In this case, concentration of maltose was 0.6 M. Measurements were done on day 4. The results represent the average of at least two independent experiments. Solute concentration (milliosmolar)

Attached cotyledons in water Attached cotyledons in maltose Excised cotyledons in water

88

Amylase activitys (.umoles maltose! 15 min cotyledon) 7.00 5.06 3.76

186 114

Table 3: Amylase activIty in excised cotyledons and in attached ones treated with and without mannitol. Attached cotyledons were incubated for the first 3 days in water and then divided in four groups. One was incubated further in water. The second and the third ones were transferred to 0.25 M and 0.6 M mannitol solutions, respectively. As for the last one, the embryonic axis was removed and the excised cotyledons were incubated in water. Assays were done on day 4. The results represent the average of two independent experiments. Amylase activity (,umoles maltose! 15 min cotyledon) Attached cotyledons in water Attached cotyledons in 0.25 M mannitol Attached cotyledons in 0.6 M mannitol Excised cotyledons in water

6.85 6.64 5.67

3.37

z. Pjlanzenphysiol. Bd. 96. S. 303-310. 1980.

310

Y. MOROHASHI and K. UENO

maltose-treated cotyledons (Table 2), the inhibition degree was not so great as that observed in excised cotyledons. Effect of mannitol on amylase synthesis in attached cotyledons (Table 3) was qualitatively similar to those of maltose (Tables 1 and 2); little inhibition with 0.25 M maltose and mannitol, and slight one with 0.6 M maltose and mannitol. It is, therefore, likely that the inhibition of amylase synthesis by 0.6 M maltose (Table 2) is an osmotic phenomenon rather than inhibition of the end-product type. Thus, although it is possible that osmotic stress actually inhibits the synthesis of the enzyme in pea cotyledons as in barley seeds, the inhibition was brought about only under conditions of unphysiologic ally great osmotic stress. From these results, it is concluded that the failure of amylase to develop in detached cotyledons is not due to inhibition of the enzyme synthesis by osmotic stress resulting from an accumulation of hydrolytic products. It seems that in pea cotyledons amylase synthesis is controlled by a mechanism different from that reported with barley seeds (JONES and ARMSTRONG, 1971). Acknowledgement The authors wish to express their gratitude to Prof. M. NAKAJIMA, Tokyo University of Agriculture and Technology, for his kindness in carrying out experiments.

References ARMSTRONG, J. E. and R. 1. JONES: Osmotic regulation of a-amylase synthesis and polyribosome formation in aleurone cells of barley. J. Cell BioI. 59, 444-455 (1973). BERNFELD, P.: Amylases. In: S. P. COLOWICK and N. O. KAPLAN (Eds.): Methods in Enzymol. 1,149-150. Academic Press. New York, 1955. BRYANT, J. A. and S. J. HACZYCKI: Studies on the interactions between the embryonic axis and the cotyledons during germination in Pisum sativum L. New Phytol. 77, 757-760 (1976). JONES, R. L.: Inhibition of gibberellic acid-induced a-amylase formation by poylethylene glycol and mannitol. Plant Physiol. 44, 101-104 (1969). JONES, R. L. and J. E. ARMSTRONG: Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds. Plant Physiol. 48, 137-142 (1971). LOCKER, A. and I. ILAN: On the nature of the hormonal regulation of amylase activity in cotyledons of germinating peas. Plant Cell Physiol. 16,449-454 (1975). SPRENT, J. I.: The inability of gibberellic acid to stimulate amylase activity in pea cotyledons. Planta 82, 299-301 (1968). VARNER, J. E., L. V. BALCE, and R. C. HUANG: Senescence of cotyledons of germinating peas. Influence of axis tissue. Plant Physiol. 38, 89-92 (1963). YOMO, H. and J. E. VARNER: Control of the formation of amylases and proteases in the cotyledons of germinating peas. Plant Physiol. 51, 708-713 (1973). YOUNG, J. L., R. C. HUANG, S. VANECKO, J. D. MARKS, and J. E. VARNER: Conditions affecting enzyme synthesis in cotyledons of germinating seeds. Plant Physiol. 35, 288-292 (1960). Y. MOROHASHI, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan.

z. P/lanzenphysiol. Ed. 96. S. 303-310. 1980.