The Effects of 2,4-D on Lipid Mobilisation in Germinating seeds of Cucurbita pepo L.

Botany Department, The University, Bristol, BS8 1UG, U.K.

The Effects of 2,4-D on Lipid Mobilisation in Germinating Seeds of Cucurbita pepo 1. N. ]. PINFIELD and

o.

A. F. YOUSIF';·)

With 4 figures Received February 7, 1980 . Accepted March 6, 1980.

Summary During 12 days incubation in water, neutral lipid in the cotyledons of germinating marrow seeds fell markedly, while both polar lipid and soluble sugar levels in the embryonic axes increased. These changes were largely inhibited by incubation in 5 X 10-6 M 2,4-D, which in addition caused some reduction in the rate of increase in the activities of the enzymes isocitrate lyase and lipase observed in the water controls. Radioisotope experiments, however, indicated that some aspects of primary respiratory metabolism continued to occur actively in seeds incubated in 2,4-D, indicating some specificity of 2,4-D action. The changes associated with lipid mobilisation occurred late in the incubation sequence and it is suggested that the observed effects of 2,4-D on these processes are the result of more fundamental changes induced at an earlier stage in the germination sequence. It seems likely, therefore, that these effects on lipid metabolism are among a number of secondary responses to 2,4-D. Key words: 2,4-D, lipid mobilis.aion, seeds, Cucurbita.

Introduction Seeds of Cucurbita pepo are non-dormant and about 60 % of the fresh weight of the mature seeds is lipid, much of which is triglyceride (YOUSIF, 1977). The usual morphological changes associated with germination are seriously impaired by incubation of the seeds in dilute solutions of 2,4-D, resulting in abnormal cell growth and organ development (YOUSIF, 1977; YOUSIF et al., 1979). The germination of lipid-rich seeds has been shown to involve, among other processes, the consumption of lipid reserves by their conversion in the storage organs to sugars, followed by translocation of these sugars to the embryo or embryonic axis (CHING, 1972; THOMAS and AP REES, 1972). In addition, lipase (MuTo and BEEVERS, 1974; HUANG and MOREAU, 1978) and isocitrate lyase activity (HOCK and BEEVERS, 1966; CHING, 1970) have been shown to increase during germination in many species. Little has been reported on the effects of 2,4-D on lipid mobilisation, ';.) Present address: Plant Propagation Unit, Sudan Gezira Board, Barakat, Sudan.

z. Pjlanzenphysiol. Bd. 99. S. 215-224.1980.

216

N.

J.

PINFIELD and O. A. F. YOUSIF

although there are some reports of changes in oil content of the seeds of some species following 2,4-D application (RIEs, 1976). In addition, SELL et a!. (1949) reported a decrease in the rate of both sugar accumulation and lipid loss in the internodes of young kidney bean seedlings as a result of 2,4-D treatment. In studies involving 2,4-D it is necessary to distinguish carefully between the effects of lethal and sub-lethal doses on growth and development of the treated plants, a sub-lethal dose being one which modifies development without suppressing it completely (YOUSIF, 1977; YOUSIF et a!., 1979). In the present investigation the effects of such a sub-lethal dose, namely 5 X 10- 6 M 2,4-D, on some of the metabolic sequences concerned with the utilisation of lipid reserves during germination have been studied.

Material and Methods Seeds of marrow (Cucurbita pepo L., cultivar. Medullosa) were soaked in distilled water for 4 hr and transferred to 9 cm. Petri dishes lined with a single thickness of Whatman no 1 filter paper moistened with 10 ml of either distilled water or aqueous 2,4-D solution. Unless otherwise stated,S X 10-6 M 2,4-D was used throughout these investigations. 20 seeds were placed in each dish, and dishes were incubated for up to 12 days at 20 ± 2 DC in darkness. Germination was recorded daily throughout this period with samples of 100 seeds, but for other determinations seed samples were removed at intervals during the incubation period and the seeds divided into cotyledons and embryonic axes for subsequent analysis. Soluble sugars and reducing sugars were determined by the anthrone method (YEMM and WILLIS, 1954) and the arsenomolybdate method (NELSON, 1944) respectively, and these determinations were made on 80 % ethanol extracts of the plant tissues prepared as described by STOBART and PINFIELD (1970). Total lipid was extracted by the method of DAVIES and PINFIELD (1980), and neutral and polar lipid fractions were obtained by phase separation between petroleum ether and 95 % methanol (STOBART and PINFIELD, 1970). Quantitative determinations of these fractions were made by the charring method of MARCH and WEINSTEIN (1966). Determinations of both lipase (glycerol ester hydrolase, E.C. 3.1.1.3) and isocitrate lyase (threo-D-isocitrate glyoxylate lyase, E.C. 4.1.3.1) activities were made from acetone powders prepared as described by STOBART and PINFIELD (1970). Extracts were made with 0.05 M potassium phosphate buffer pH 7.5 at 0 DC. Lipase was determined at pH 7.0 with tween 20 as substrate, by the titration method of NYMAN (1965) and isocitrate lyase at pH 6.85 as described by PINFIELD (1968). Radioisotope feeding experiments were conducted in 100 ml Erlenmeyer flasks with centre wells, as described by BRADBEER and COLMAN (1967), except that CO 2 released was absorbed in 2-phenylethylamine. Samples of 1 g cotyledons or 0.5 g embryonic axes were incubated for 6 hr at 20 ± 1 DC in 5 ml potassium phosphate buffer at pH 6.0 containing 1 {lCi [2_14C] acetate. At the end of this period the tissue was washed free from excess radioisotope, and ethanol-soluble materials and lipids were separated as described by STOBART and PINFIELD (1970). Individual constituents of the ethanol soluble fraction were separated by paper chromatography (BRADBEER and COLMAN, 1967) and radioactivity in all samples was determined as described by DAVIES and PINFIELD (1979).

Results Fig. 1 shows the effects of 5 X 10- 6 M 2,4-D on germination of marrow seeds over a 12 day incubation period. By the 10th day 80 Ofo germination was observed Z. PJlanzenphysiol. Bd. 99. S. 215-224. 1980.

Effects of 2,4-D on lipid mobilisation

217

100

90

80

70

c:

60

0

.~

c:

E

Q;

50

Cl ~

40

30

20

10

Incubation time (days)

Fig. 1: The effect of 2,4-D on germination of marrow seeds incubated at 20°C in the dark 6--6 water control 0----0 5 Y 10-6 M 2,4-D and 0---0 5 X 10-5 M 2,4-D.

in the water control, but germination was impaired in 5 X 10-6 M 2,4-D, and as early as the 2nd day percentage germination in these samples was significantly lower (P < 0.05) than that in the corresponding controls. A maximum of 58 % germination in 5 :< 10- 6 M 2,4-D was reached on the 10th day. Only 40 Ofo of the seeds sown in 5 X 10- 5 M 2,4-D germinated during the incubation period, and in most of these the radicle showed marked necrosis a few hours after its emergence and further development was suppressed. Incubation in 5 X 10- 4 M 2,4-D prevented germination entirely. Fig. 2 shows the changes in neutral lipid in cotyledons from seeds incubated in water or 2,4-D for periods up to 12 days. In the control series neutral lipid fell from an initial level of about 500 mg per 10 cotyledon pairs to less than 300 mg by the 10th day, and more rapidly during the next two days to about 120 mg. In cotyledons from 2,4-D treated seeds these decreases were not observed, and although some fluctuations occurred during the early part of the incubation period, amounts of neutral lipid present after 12 days were similar to those found at the beginning of the experiment. Z. Pjlanzenphysiol. Bd. 99. S. 215-224. 1980.

218

N.

J. PINFIELD and

O. A. F.

YOUSIF

500

c:

o

-g

>.

o

~

"-

200

0>

E

u

~ ~

'ijj

100

Z

oL-----~----_r----~----_,------r_--~

a

4

6

8

10

12

Incubation time (days)

Fig. 2: Changes in neutral lipid of cotyledons taken from marrow seeds incubated in water (6) or 5 X 10-6 M 2,4-D (0) for various periods. The result of parallel determinations of polar lipid in the embryonic axes are shown in Fig. 3. Marked differences became apparent by the 8th day, at which stage the amounts of polar lipid from axes of water-incubated seeds was substantially greater than those extracted from the corresponding 2,4-D-treated material. These differences were accentuated in longer treatments, and after 12 days polar lipid levels in the embryonic axes of water control samples were 7 times greater than those for the corresponding 2,4-D treatments. Since the increases in polar lipid in embryonic axes occurred during the later stages of incubation presumably represent increased synthesis of membrane material during renewed growth of the axis, it seems clear that 2,4-D seriously impairs some parts of the metabolic sequences which bring about these increases. Polar lipid in the cotyledons and neutral lipid in the embryonic axes were also determined, but the amounts present were small, and although subject to fluctuation, showed no consistent changes or differences between water and 2,4-D treatments during the incubation period. Z. Pjlanzenphysiol. Bd. 99. S. 215-224. 1980.

Effects of 2,4-D on lipid mobilisation

219

25

20

oL-----r-----r-----r-----r-----r---~ o 2 8 10 12 8

Incubation time (days)

Fig. 3: Changes in polar lipid of embryonic axes taken from marrow seeds incubated water (6) or 5 )! 10- 6 M 2,4-D (0) for various periods.

III

Fig. 4 shows the changes in soluble sugars in cotyledons and embryonic axes from seeds incubated in water or 2,4-D for various periods. In the cotyledons of the water controls there was a rapid fall in soluble sugars between the 2nd and 4th days, resulting in a halving of the amount present from an initial value of 40 mg per 10 cotyledon pairs. Subsequently levels remained unchanged for the remainder of the incubation period. Soluble sugars in the cotyledons of 2,4-D-treated seeds also showed a decline between the 2nd and 4th days, but the decline continued subsequently and by the 12th day soluble sugar in the cotyledons of 2,4-D-treated seeds was only one third that in the water control. In the embryonic axes of water-treated seeds changes in soluble sugars were characterised by a large increase between the 8th and 10th days of incubation. An increase was also observed at this time in the 2,4-D-treated material, but this was much smaller and by the 12th day the amounts present were only about one third of those in the corresponding water controls. Determinations of reducing sugars showed that amounts of these components never exceeded 2 mg/IO cotyledon pairs or 5 mg/20 embryonic axes, and although changes were observed during incubation, it is clear that these quantities are wholly inadequate to explain the large changes in soluble sugars which occurred during this period. Z. Pflanzenphysiol. Ed. 99. S. 215-224. 1980.

220

N.

J. PINFIELD and

O. A. F.

YOUSIF

50 120

I>.

100

A

40

(/)

(/)

Q)

c:

x

'" .11

0

"0 Q)

>. (5

"~

c:

~ I>.\/~I>.

'iii

c.

0

S-

.s

0>

b--\

20

a;

0>

::>

(/)

Q)

:0 ::> "0

10

(/)

0

0

80

0

30

~ E Q)

0

~O>

60

.§.

a;

0>

::>

40

(/)

Q)

:c::>

.--0

"0 (/)

20

0 0

2

4

6

8

10

12

0

4

2

8

6

10

12

Incubation time (days)

Incubation time (days)

Fig. 4: Changes in soluble sugars in cotyledons (A) and embryonic axes (B) taken from marrow seeds incubated in water (,6) or 5 X 10-6 M 2,4-D (0) for various periods.

This indicates that the large changes in amounts of sugar present must be caused by fluctuations in nonreducing components, and chromatographic separations demonstrated that the major one was sucrose. Table 1 shows the changes in lipase and isocitrate lyase activity which occurred in the cotyledons of seeds incubated for various periods in water or 2,4-D. Isocitrate Table 1: Changes in the actIVity of lipase and isocitrate lyase in cotyledons of marrow seeds incubated for various periods in water or 2,4-D. Isocitrate lyase activity is given in .umoles substrate converted mg protein- l min-I, and lipase as acid equivalents produced mg protein-l 30 min-I. Soluble protein varied from 40 to 45 mg. g-l acetone powder. Values represent the means of 3 separate determinations for isocitrate lyase and four for lipase. Incubation time (days)

2 4 6 8 10

Isocitro.te lyase activity

Lipase activity

2,4-D

H 2O

2,4-D

HP

2.0 2.4 3.1 2.6 8.4

2.0 2.2 3.3 9.0 23.0

2.9 3.4 3.3 3.3 3.7

2.7 3.8 3.9 4.7 5.5

Z. PJlanzenphysiol. Bd. 99. S. 215-224. 1980.

221

Effects of 2,4-D on lipid mobilisation

lyase activity in the cotyledons of water incubated seeds increased markedly in the later stages of the incubation period. These increases became apparent by the 8th day and by the 10th day a 10-fold increase in activity over that present on the second day was observed. In the 2,4-D-treated material this increase in activity was largely suppressed, although after 10 days incubation a slight increase was observed, resulting in about one third the enzyme activity observed in the corresponding water control. Changes in lipase activity in cotyledons during a 10 day incubation of seeds in water were very small, although by the 10th day the activity was approximately twice that present at day 2. Incubation of seeds in 2,4-D totally inhibited this small increase. Incorporation of HC into ether-soluble and ethanol-soluble fractions and also into released CO~ of cotyledons from water or 2,4-D treated seeds following a 6 hr feeding with [2_14C] acetate is shown in Table 2. The consistently high figures for incorporation into ethanol-soluble constituents even following 2,4-D treatment suggest that many intermediary metabolic sequences were unimpaired by 2,4-D. Chromatographic separation of the ethanol-soluble components has shown that the bulk of HC incorporated into this fraction appeared either in the TCA cycle acids malate and citrate, with smaller amounts in fumarate and succinate, or in the amino acids glutamate and aspartate which arise direct from the TCA cycle by transamination. No consistent differences in labelling patterns of these constituents between the water and 2,4-D-treated material were observed. Incorporation of HC into ether-soluble components was also appreciable, indicating considerable synthesis of lipids in the cotyledons. After 7 or 10 days incubation in 2,4-D, however, this incorporation was greatly reduced when compared with the corresponding controls. This indicates that not only lipid consumption but also lipid synthesis in the cotyledons is markedly affected by 2,4-D treatment. Table 3 shows a further separation of the ether-soluble fraction from the cotyledons into polar and neutral lipids. Although, as would be expected, much of the 14C was incorporated into polar lipids, a considerable amount was also detected in the neutral lipid fraction. With the cotyledons of seeds incubated in water the

Table 2: The uptake of [2_14C] acetate and distribution of radioactivity in cotyledon slices taken from marrow seeds incubated for various periods in either water or 5 X 10-6 M 2,4-D. Slices were given a 6 hr feeding period at 22 DC and the figures are c.p.m. X 10-3 . Incubation period (days)

2,4-D

H 2O Fraction CO 2 80 Ofo ethanol-soluble materials Ether soluble materials Total upt~ke

1 18 322 45 385

4 13 307 21 341

7 11 249 42 302

10 19 330 88 437

19 286 46 351

4 11

319 27 357

7 8 283 9 300

10 10 363 12 385

Z. Pflanzenphysiol. Ed. 99. S. 215-224. 1980.

222

N.

J. PINFIELD and o. A.

F. YOUSIF

Table 3: The distribution of radioactivity incorporated into ether-soluble material extracted from cotyledons of marrow seeds incubated for various periods in either water or 5 X 10- 6 M 2,4-D. Figures are c.p.m. X 10-3 • Incubation period (days)

H 2O Fraction Neutral lipid Polar lipid

18 26

2,4-D

7

10

19 22

37 50

19 26

7

10

3 5

4 8

HC incorporation into both neutral and polar lipids increased considerably over a 10 day period, but 2,4-D caused a marked reduction in 14C incorporation into both fractions during this time. Discussion

The conversion of storage lipid to soluble carbohydrate, which occurred in marrow seeds during germination was seriously disrupted by 2,4-D treatment. Thus, the processes resulting in losses of neutral lipid from the cotyledons and increases in polar lipid and soluble sugar in the embryonic axes, which were clearly functional in the water controls were impaired in the presence of 2,4-D. The results of HC feeding experiments, however, indicated that in the cotyledons, 2,4-D at the concentrations used had little effect on many aspects of respiratory metabolism. It appears, therefore that at appropriate concentrations 2,4-D has only localised effects on respiratory metabolism, and only a partial blockage of these sequences results from its application. Similar effects of 2,4-D on lipid mobilisation and sugar accumulation in seedling internodes have been shown by SELL et al. (1949). Although the breakdown of neutral lipid is an expected phenomenon in such a metabolic system, the incorporation of 14C into neutral lipids in the cotyledons indicates a simultaneous synthesis of these components in cotyledonary tissues, an observation also made by THOMAS and AP REES (1972). It is clear, however, that the substantial fall in neutral lipid occurring during the later stages in the water controls is the result of accelerated hydrolysis rather than decreased synthesis, since the rate of HC incorporation into these components increased considerably during the later stages of incubation. Nevertheless, 2,4-D appeared to have a marked effect on both degradative and biosynthetic aspects of neutral lipid metabolism in the cotyledons. The storage tissues of many seeds synthesise substantial amounts of membrane lipid during germination (HARDMAN and CROMBIE, 1958; DONALDSON, 1976) and it seems likely that the neutral lipid synthesis described in the present report represents the production of fatty acids, mono- and diglycerides, which are used in the formation of these polar lipids (NISHIMURA and BEEVERS, 1979).

z. Pjlanzenphysiol. Bd. 99. s. 215-224.

1980.

Effects of 2,4-D on lipid mobilisation

223

The changes in soluble sugars in the cotyledons of water incubated seeds are not wholly consistent with the findings of others, since sugar accumulation during the early stages of germination has been demonstrated in storage organs of seeds of a number of species prior to transport to the embryonic axis (CHING, 1972; AP REES 1974). It is not clear why such an accumulation was not observed in the present investigation, although one explanation would be that the translocation rate for sugars produced is very rapid in marrow cotyledons. Since the sugar content of cotyledons of seeds incubated in 2,4-D was lower than in the water controls, it could be argued that this indicates the acceleration of translocation by 2,4-D. This seems unlikely, however, in view of the reduced sugar accumulation in the embryonic axes and the reduced consumption of cotyledonary lipid in 2,4-D-treated samples. Increases in isocitrate lyase activity occurring during germination in the cotyledons of water control samples were also much reduced by 2,4-D treatment, indicating an effect on enzymic processes associated with lipid mobilisation. The effects on lipase activity assayed at pH 7 were less clearly defined, although there were indications that the small increases observed after longer incubations in water were inhibited by 2,4-D. These changes in enzyme activities, however, occurred late in the incubation period, and it seems likely that earlier events in the germination process may be affected. It it possible that one such event may be mRNA synthesis (RIEs, 1976), although in the present case such suggestions are largely speculative since it is not yet clear whether the increased enzyme activity results from activation or de-novo synthesis. Furthermore, metabolic sequences other than those concerned with storage lipid utilisation are also affected, indicating a broader spectrum of response to 2,4-D. The extent to which these metabolic changes can be linked to abnormal development patterns in germinating marrow seedlings (YOUSIF, 1977; YOUSIF et a!., 1979) is not clear. Although changes in membrane biosynthesis and function in the embryonic axes may well produce such effects directly, it is more difficult to see how reduced storage lipid mobilisation can make a direct contribution to the initiation of such abnormal growth. It is possible that the morphological effects may result, not from such metabolic events, but from a simultaneous production of ethylene in the tissues. Indeed, the metabolic changes may themselves be a product of ethylene activity (HOLM and ABELES, 1967). STEVENS et al. (1962) have, however, demonstrated that similar effects on metabolic processes could be induced by a range of 2,4-D analogues, even though these compounds failed to produce any of the morphological abnormalities characteristic of 2,4-D treatment. The results of the present report indicate that at the concentration employed, 2,4-D, although not a universal inhibitor of metabolic activity, does appear to operate at more than one site. It seems likely also that the changes in lipid metabolism following 2,4-D treatment are secondary effects resulting from more fundamental changes induced by 2,4-D at an earlier stage in the germination sequence. Z. P/lanzenphysiol. Ed. 99. S. 215-224. 1980.

224

N.

J. PINFIELD

and O. A. F. YOUSIF

It is not yet clear, however, what constitutes the primary point of 2,4-D action marrow seed germination.

In

Acknowledgement We are grateful to the Sudanese Government for a grant to O.A.F. YOUSIF during the period that this research was conducted.

References BRADBEER,]. W. and B. COLMAN: New Phytol. 66, 5 (1967). CHING, T. M.: Plant Physiol. 46, 475 (1970). - In: KOZLOWSKI, T. T. (Ed.): Seed Biology vol. II, 103-218. Academic Press, London, 1972. DAVIES, H. V. and N. ]. PINFIELD: Z. Pflanzenphysiol. 92, 85 (1979). - - Z. Pflanzenphysiol. In the press (1980). DONALDSON, R.: Plant Physiol. 57, 515 (1976). HARDMAN, E. E. and W. M. CROMBIE: J. Exp. Bot. 9, 239 (1958). HOCK, B. and H. BEEVERS: Z. Pflanzenphysiol. 55,405 (1966). HOLM, R. E. and F. B. ABELES: Plant Physiol. 42,1094 (1967). HUANG, A. H. C. and R. A. MOREAU: Plant a, 141, 111 (1978). MARCH, J. B. and D. B. WEINSTEIN: J. Lipid Res., 7, 574 (1966). MUTO, S. and H. BEEVERS: Plant Physiol. 54, 23 (1974). NELSON, N.: J. BioI. Chern. 153, 375 (1944). NISHIMURA, M. and H. BEEVERS: Plant Physiol. 64, 31 (1979). NYMAN, B.: Physiol. Plant. 18, 1085 (1965). PINFIELD, N. J.: Plant a 82, 337 (1968). REES, T. AP, S. M. THOMAS, W. A. FULLER, and B. CHAPMAN: In: PRIDHAM, ]. B. (Ed.): Plant Carbohydrate Biochemistry, 27-46. Academic Press, London, 1974. RIEs, S. K.: In AUDUS, L.]. (Ed.): Herbicides, 313-344. Academic Press, London, 1976. SELL, H. M., R. W. LUECKE, B. M. TAYLOR, and C. L. HAMNER: Plant Physiol. 24, 295 (1949), STEVENS, V. L.,]. S. BUTTS, and S. C. FANG: Plant Physiol. 37, 215 (1962). STOBART, A. K. and N. J. PINFIELD: New Phytol. 69, 939 (1970). THOMAS, S. M. and T. AP REES: Phytochem. 11,2177 (1972). YEMM, E. W. and A. J. WILLIS: Biochem. ]. 57, 508 (1954). YOUSIF, O. A. F.: 2,4-dichlorophenoxyacetic acid effects on germination and seedling growth in Cucurbita. M. Sc. thesis, University of Bristol, 1977. YOUSIF, O. A. F., N. A. FIELDER, and N. J. PINFIELD: New Phytol. 82, 37 (1979). N.]. PINFIELD, Botany Department, The University, Bristol, BS8 lUG, U.K.

Z. Pflanzenphysiol. Bd. 99. S.

215~224.

1980.