Plant Science, 55 (1988) 87-92 87 Elsevier Scientific Publishers Ireland Ltd.

EFFECT OF KINETIN (6.FURFURYLAMINOPURINE) ON CHANGES IN MEMBRANE LIPIDS IN RELATION TO GROWTH OF ISOLATED COTYI.EDONS OF VEGETABLE MARROW ( C U C U R B I T A P E P O L).

YASH PAUL and SHAVILA GUPTA

Department of Biochemistry, Punjab Agricultural University, Ludhiana-141004, (India)

(Received June 26th, 1987) (Revision received October 16th, 1987) (Accepted December 3rd, 1987)

Kinetin (6-furfurylaminopurine) treatment resulted in the enhanced formation of total polar lipids, total phospholipids and total glycolipids in the isolated cotyledons of vegetable marrow (Cucurbita pepo L.). The content of phosphatidic acid, phos- phatidyl glycerol and diphosphatidyl glycerol also rose markedly with kinetin treatment. The increase in the content of DGDG was more pronounced compared to MGDG and even more conspicuous on treatment with kinetin. There was a spectacular rise in the carotenoid and chlorophyll content. The results indicated the accelerated formation of plant cell organelles particularly photosynthetically active chloroplasts from heterotrophic leaf on treatment with kinetin.

Key words: vegetable marrow (Cucurbitapepo L.); membrane lipids; kinetin; carotenoid; chlorophyll

Introduct ion Materials and methods

Cytokinins are known to induce cell expan- sion growth in isolated cotyledons of oil rich seeds [1,2]. Such cotyledons undergo lipolysis during germination with simultaneous increase in the level of reducing sugars [2-4]. During such an expansion growth, an increase in the activities of glyoxysomal enzyme [3], Calvin cycle enzymes [5] and enzymes of sucrose metabolism [4,6] have been reported. Cytokinins have also been shown to increase membrane constituents of chloroplasts particularly carotenoids and chlorophylls [7-9]. Except for a few reports on membrane lipids in isolated cotyledons of cucumber [10] and squashmelon [2] on treatment with cytokinins, not much work has been done on this aspect. Thus the present investigation was undertaken to establish the effect of kinetin on changes in different mem- brane lipids constituents in relation to growth of isolated cotyledons of vegetable marrow (Cucurbita pepo L.) Var I.

Seeds of vegetable marrow (Cucurbita pepo L.) were procured from the Department of Veg- etable Crops, Landscaping and Floriculture, Punjab Agricultural University, Ludhiana (Punjab), India.

Preparations of cotyledons and treatment with kinetin, have been reported in our previous communication [4]. All the experiments were run under dark incubation for 5 days at 30 + l°C except for chlorophyll in which continuous white light was given.

Estimation of different polar lipids, carotenoid and chlorophyll content

Freshly drawn cotyledons were immersed for 5 min in boiling isopropanol and the total polar lipids were extracted and purified from them according to the methods ofFolch et al. [11]. The purified lipids were dried under vacuum at 40°C and redissolved in chloroform to known volume. The small aliquot was evaporated to a constant

0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

88

weight in a pre-weighed glass dish to deter- mine the amount oflipids. The solvent partition method of Nichols [12] was used for separation of polar lipids and non-polar lipids. The total phospholipids were estimated according to Ames [13] and glycolipids with the method of Roughan and Batt [14]. The phospholipids from the total polar lipids were separated by two- dimensional thin layer chromatography using solvent system, chloroform/methanol/water (65:25:4, by vol.) in the first direction followed by chloroform methanol: 7N NH 3 (65:25:4, by vol.) in the second direction [15]. Individual glycolipids were separated according to the pro- cedure ofPohl et al. [16] employing solvent sys- tem, acetone/benzene/water (91:30:8, by vol.), and individual phospholipids [13] and glycoli- pids [14] were then determined. Total caroten- oids were estimated according to Longo et al. [17] and chlorophyll content according to Fran- cis et al. [18].

Resu l t s

As reported in a previous communication [4], 25 ppm kinetin strongly stimulated the increase in size and fresh-weight and thus this concent- ration was used to study polar lipid changes, carotenoid and chlorophyll content in this com- munication. Kinetin treatment enhanced an increase in the amount of polar lipids and decreased the content of non-polar lipids (Table I). The polar lipids increased from 0.81 mg cot -I

1600

1400

"T 1200

o 1000

800

A 600 B

500

~---~ 400

,j," 300

-"v"'/1~ 200

' ' , , 100

#

/ /

600 ' , , , . , 0 1 2 3 4 5 0 1 2 3 4

D o y s .-

F i g . 1. Changes in the level of total phosphol ipids and glycolipids dur ing incubat ion in da rk (A) - o - , control; -- • -- ; k inet in for phospholipids. (B) - o - , control; -- • -- , k inet in for glycolipids.

(7.4%) to 1.39 mg cot -1 (21.6%) and non-polar lipids decreased from 10.14 mg cot -1 (92,2%) to 4.92 mg cot -1 (76.5%) in control. The corres- ponding values with kinetin treatment were 1.86 mg cot -1 (41.6%) for polar lipids and 2.60 mg cot -1 (58.2%) for non-polar lipids after 5 days incubation in dark.

The amount of total phospholipids and total glycolipids (Fig. 1) also increased during dark incubation but the effect was more with kinetin treatment. The value of phospholipids increas- ed from 167/~g cot -1 to 880/~g cot -1 in control and to 1224/~g cot -1 with kinetin treatment. The corresponding value for total glycolipids reached its maximum i.e. 327/~g cot -1 and 593 ~g cot -1 from 167/~g cot -1. The amount of phos- phatidic acid, phosphatidyl glycerol and diphos-

Table L Changes in the content of polar and non-polar lipids of vegetable-marrow cotyledons with kinetin in dark.

Incubation Polar lipids Non-polar lipids time (days) mg cot-1 %age total lipids mg cot- I %age totallipids

C a T a C T C T C T

0 0.815 0.81 7.4 7.4 10.14 10.14 92.2 92.2 1 0.93 0.95 9.4 10.1 8.93 8.49 90.5 90.1 2 1.02 1.16 11.3 13.7 7.96 7.32 88.6 86.4 3 1.16 1.35 14.5 23.0 6.78 4.48 85.2 76.4 4 1.24 1.62 18.3 30.1 5.39 3.66 79.6 69.3 5 1.39 1.86 21.6 41.6 4.92 2.60 76.5 58.2

~C, Control; T, Treated. bAverage of duplicate analysis.

89

340

300

260

T 220

,,4o 100

60

20,

A

I I | I I

1 2 3 4 5

B

0 1 2 3 4 S

DAYS

0 1 2 3 4 5

F i g . 2. C h a n g e s in t h e c o n t e n t of i n d i v i d u a l phospho l i p id s . (A) - • - , control ; - o - , k i n e t i n for p h o s p h a t i d i c acid. (B) - • - , control ; - o - ; k i n e t i n for p h o s p h a t i d y | glycerol. (C) - • - , control ; - o - , k i n e t i n for d i p h o s p h a t i d y l glycerol.

phatidyl glycerol (Fig. 2) increased in control and effect was much enhanced with treated cotyledons. Phosphatidic acid increased from 15 /~g cot -1 to 153 fig cot -~ in control and to 334 fig cot-1 in the t reated cotyledons. The amount of phosphatidyl glycerol rose from 2 ~tg cot -1 to 130 /~g cot -~ in control and corresponding value in t reated cotyledons (275 ~g cot -1) was much enhanced aRer 5 days of incubation in dark. There was also a spectacular rise in the content of diphosphatidyl glycerol in t reated cotyledons (167/~g cot -1) as compared to the control (69/~g cot -z) after 5 days of incubation. On the other hand, almost negligible decrease in the content of major phospholipids (PC, PE and PI) was noticed and their decrease slightly enhanced on t rea tment with kinetin (data not shown).

MGDG was the major and DGDG generally the minor glycolipid at all stages of development during incubation (Fig. 3). Both fractions increased with period of incubation and the increase was even more enhanced with kinetin treatment. MGDG increased from 149 ~tg cot -~ to 214 fig cot -1 in the control and to 296/~g cot -~ in the treated cotyledons. The rise in the level of DGDG was spectacular as compared to MGDG both in control and t reated cotyledons.

The carotenoid and chlorophyll contents were used as indicators of cytokinin action on the formation of photosynthetic apparatus. The carotenoid level (Fig. 4) increased from 0.25/~g cot -~ to 4.15/~g cot -1 in the control. The amount

of total chlorophyll increased from 2.30 mg cot- 1 to 16.78 mg cot -1 in the control and to 36.74 mg cot -1 in the treated cotyledons (Fig. 5).

D i s c u s s i o n

An enhanced formation of total polar lipids (Table I) on t rea tment with kinetin under dark incubation may be as a result of an accelerated

280 . " p /

/ . /

240 ,~- / /

200

11 // 160 ~ /

"r ,,

12C / D /

80

40

I I . I

- - Days =

F ig . 3. Changes in the level of ind iv idua l glycel ipids dur- ing incubat ion in dark. (A) - o - , control; (B) -. • -- , ] r~e t i n for MGDG; ( C ) - o - , control; (D) -- • -- , k ine t in for DGDG.

9 0

8 /e

6 / 7

¢.) /

2

1 2 3 4 5 - - Days

F i g . 4 . C h a n g e s i n c a r o t e n o i d l e v e l d u r i n g i n c u b a t i o n i n

dark. - o - , c o n t r o l ; -- • - - , k i n e t i n .

40

32 i

i

i I

i

i I

i i

J

Days *

F i g . 5. Changes m ch lo rophy l l con ten t d m ~ , ~ i ncuba t i on in l ight . - o - , contro l ; -- • - - , k i ne t i n .

7 24

o c. 16 £

mobilisation of total lipids and triacylglycerols. The enhanced breakdown of lipids and triacylglycerols on t reatment with kinetin has been reported earlier in squashrnelon cotyle- dons [2] and vegetable marrow cotyledons [4]. The conversion of a portion of stored lipids (triacylglycerols) into membrane lipids viz. phospholipids and glycolipids may be required for the formation of membranes during germi- nation. An increase in the synthesis of polar lipids particularly phospholipids was stimu- lated with BA in cucumber cotyledons [10]. The increase in the level of total phospholipids, total glycolipids, individual phospholipids viz. phos- phatidic acid, phosphatidylglycerol and diphos- phatidylglycerol and individual glycolipids viz.

MGDG and DGDG in control and their increased formation in treated cotyledons under dark (Fig. 1 -Fig . 3), again depict the role of kinetin in the accelerated formation of mem- branes during development of plant cell organelles. An enhanced synthesis of total phos- pholipids, total glycolipids, MGDG and DGDG has been reported in isolated squashmelon cotyledons [2] on t reatment with kinetin. The accelerated development of plastids [9] in detached pumpkin cotyledons and plastids, microbodies and mitochondria [19-20] in excised watermelon cotyledons has been indi- cated on t reatment with cytokinins. The forma- tion of glyoxysomes was observed during incu- bation in excised watermelon cotyledons [21]. Phosphatidyl glycerol is the only major phos- pholipid of chloroplastic membranes [22] of higher plants, its much enhanced formation on treatment with kinetin shows the role of this phospholipid in the accelerated formation of chloroplastic membranes. The accelerated development of plastids on t reatment with cytokinin has been reported in pumpkin cotyle- dons [9]. Diphosphatidyl glycerol, being exclu- sively the major phospholipid present in inner mitochondrial membranes of higher plants [23], its increased formation in kinetin-treated cotyledons shows the role of kinetin in the accel- erated development of mitochondria. The accel- erated development of mitochondria on treat- ment with BA has been shown in watermelon cotyledons [20]. The increase in the level of phosphatidic acid, phosphatidyl glycerol and diphosphatidyl glycerol under control and their drastic increase with kinetin t reatment indi- cates its role in the continuous formation of new membrane phospholipids during growth of iso- lated cotyledons of vegetable marrow. The source for the biosynthesis of these membrane phospholipids seems to be triacylglycerols, as almost negligible decreases in the level ofphos- phatidyl choline, phosphatidylethanolamine and phosphatidyl inositol take place during incubation in dark (data not shown). Thus, it seems probable that phosphatidic acid is formed from triacylglycerols as the action of lipases, glycerol kinase and acyl transferases, which is

then converted to phosphatidylglycerol and diphosphatidylglycerol utilizing enzymes CDP- diacylglycerol synthetase and diphosphatidyl glycerol synthetase. The activation of lipases with kinetin treatment have already been reported earlier in the vegetable marrow cotyle- dons leading to enhanced lipolysis oflipids dur- ing incubation in dark [4]. Glycerol can be expected to be incorporated into phosphatidic acid, phosphatidylglycerol and diphos- phatidylglycerol after conversion to Sn-Glycerol 3-phosphate with enzyme glycerol kinase. Bar- ton and Stumpf [24] and Cheniae [25] have reported high activity of this enzyme in plant tissues synthesizing phospholipids. All other enzymes preceding the synthesis of phos- phatidylglycerol and diphosphatidylglycerol are known to be present in plant tissues syn- thesizing phospholipids [26].

The amount of MGDG and DGDG increased and an increase was even more pronounced with kinetin t reatment (Fig. 3). It is thus shown that increases in these lipids (being major com- ponents of chloroplastic membranes [27],) shows their role in the accelerated development of chloroplasts. The differentiation of prolamel- lar membranes in excised cucumber cotyledons with BA has been reported [10]. The increase in the amount of total carotenoids (Fig. 4) and total chlorophylls (Fig. 5) under light incubation on treatment with kinetin again possibly shows its role in the accelerated development of plastids leading to the formation of photosynthetically active chloroplasts from heterotrophic leaf. An increase in the amount of carotenoids has been reported earlier from our laboratory in isolated squashmelon cotyledons with kinetin treat- ment [2]. The much increased formation of chlorophyll content under light along with kine- tin probably shows that light and cytokinin interact to trigger the biosynthesis of chlorophyll through induction of various enzymes involved in the chloroplastogenesis. Dei [28-30] has reported that BA treatment accelerated the synthesis of chlorophyll in iso- lated and attached cucumber cotyledons. Lew and Tsuji [31] have suggested that BA abolishes the lag phase by triggering the stimulation of~-

91

amino levulinic acid formation and cytokinin has other stimulatory effects on chlorophyll pro- duction after longer incubations.

During cell expansion growth, continuous supply of reducing sugars are needed to prom- ote accelerated development of plant cell organelles on t reatment with kinetin. The enhanced formation of reducing sugars from storage lipids has been reported in the same seed material earlier through accelerated increase in the activities of enzymes involved in the conversion of storage lipids to carbohyd- rates [4]. Thus, it is evident from this study and the earlier one [4] that cell expansion growth is as a result of accelerated breakdown of lipids and their conversion to sugars leading to the accelerated formation of various plant cell organelles, particularly photosynthetically functional chloroplasts from heterotrophic leaf.

Acknowledgements

The authors are thankful to Dr. Sudarshan Singh, Senior Biochernist-cum-Head, Depart- ment of Biochemistry for providing facilities during the course of this study.

References

1 D.S. Letham. Plant Physiol., 25 (1971) 391-396. 2 Y. Paul, S. Kaur and B.N. Sharma, Plant Sci., 41

(1985) 193-195. 3 O. Servettaz, F. Castesi and C.P. Longo, Plant

Physiol., 70 (1982) 1634-1636. 4 Y. Paul and S. Gupta, Plant Sci., 53 (1987) 29-34. 5 B.M.R. Harvey, B.C. Lui and R.A. Fletcher, Can. J.

Bot., 52 (1974) 2581-2586. 6 G.L. Salerno, Physiol. Plant., 64 (1985) 259-264. 7 J. Guern and C. Leonod (Eds.), Metabolism and

molecular activities of cytokinins, Springer-Verlag, Heidelberg, New York, 1981, p. 287.

8 K. Haru, and S. Hiroshi, Physiol. Plant., 55 (1982) 247-254.

9 C. Le Pabic, N. Farineau and J~ Roussax. Z. Pflanzen- Physiol., 111 (1983) 261-272.

10 H.V. Davies and J.M. Chapman, Ann. Bot., 53 (1964) 65-72.

11 J. Folch, M. Lees and G.H. Sloane-Stanley, J. Biol. Chem., 226 (1957) 497-509.

12 B.W. Nichols, Lab. Pract., 13 (1964) 299-305. 13 B.N. Ames, Methods Enzymol., 8 (1966) 115-118.

92

14 P.G. Roughan and R.D. Batt, Anal. Biochem., 22 (1968) 74-88.

15 Y. Paul, B.N. Sharma and I.S. Bhatia, Indian J. Agric. Sci., 42 (1972) 435-436.

16 P. Pold, H. G l u e and H. Wagner, J. Chromatogr., 49 (1970) 488--493.

17 G.P. Longo, C.P. Longo, G. Rossi; A vitale and M. Pid- retti, Plant Sci. Lett., 12 (1978) 199-207.

18 H.W. Francis, F.B. David and M.D. Roberts, Experi- ments in Plant Physiology, Van Nostrand Reinhold Co., New York, 1985, p. 55.

19 G.P. Longo, M. Pedretti, G. Rossi and C.P. Longe, Planta, 145 (1979) 209-217.

20 G.P. Longo, M. Pedretti, G. Rossi and C.P. Longo, Plant Sci. Lett., 14 (1979) 213-223.

21 T. Kagawa, D.I. McGregor and H. Beevers, Plant Physiol., 51 (1973) 66-71.

22 J.L. Harwood, and J.L. Russel, Lipids in plants and microbes, London, George Allen and Unwin, Boston and Sydney, 1984. p. 42.

23 J.L. Harwood and J.L. Russel, Lipids in plants and microbes, London, George Allen and Unwin, Boston and Sydney, 1984, p. 44.

24 E.J. Barron, and P.IC Stumpf, Biochim. Biophys. Acta., 60 (1962) 329-337.

25 G.M. Cheniae, Plant Physiol., 40 (1965) 235-243. 26 T.S. Moore, Jr., Annu. Rev. Plant Physiol., 33 (1982)

235-259. 27 R. Douce (Ed.), Plant Organelles, E. Reid, E. Horwood.

Ltd., New York, 1977, p. 47. 28 M. Dei, Plant Sci. Lett., 30 (1983) 251-258. 29 M. Dei, Physiol. Plant., 62 (1984) 521-526. 30 M. Dei, Physiol. Plant., 64 (1985) 153-160. 31 R. Lew and H. Tsuji, Plant Physiol., 69 (1982) 663-867.