SUBCELLULAR LOCALIZATION OF MULTIPLE FORMS OF STARCH PHOSPHORYLASE IN PLANT LEAVES DURING DEVELOPM ENT

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Indian J. Plant PhYSIOI., Vol. XXV, No.4. pp. 31 -323 Decem er

SUBCELLULAR LOCALIZATION OF MULTIPLE FORMS OF

STARCH PHOSPHORYLASE IN PLANT LEAVES DURING

DEVELOPM ENT

ANIL KUMAR AND G. G. SANWAL Department of Biochemistry

Lucknow University Lucknow 226007. U. P.• India (Received: August 11, 1981 ; Revised: December 16, 1981)

SUMMARY

Four multiple forms (enzymes A, B,

e

and

oj

of starch phosphorylase (Ee 2.4.1.1) in plant leaves were detected by polyacrylamide gel electrophoresis with the following electrophoretic mobilities (cm/2t h) in the respective order: 1.9, 2.2,3.2 and 4.2. In Marigold leaves, one starch phosphorylase, enzyme B, was present throughout the maturation stage and was localized in the soluble fraction. Cabbage leaves, contained four multiple forms of the enzyme (enzymes A, B, C and D) in young leaves Which decreased to one (enzyme C) in the mature leaves. !n cabbage. the enzyme detected in mature leaves (enzyme C) was in the soluble fraction whereas, in the young leaves three of the four (enzymes A, B and OJ were partially localized in the chloro­ plast and partially in the soluble fraction. Potato leaves contained three starch phosphorylases (enzymes B,

e

and 0), one (enzyme B) was partially localized in the chloroplast and partially in the soluble fraction while the other two (enzymes C and 0) were in the soluble. fraction throughout maturation. Two enzymes (enzymes B and e) were present in the brinjal leaves. one (enzyme B) was partially localized in the chloroplast and partially in the soluble fraction while the other (enzyme C) was totally in the soluble fraction. Arhar leaves contained two enzymes (enzymes C and OJ throughout maturation, one (enzyme C) was partially localized in the soluble fraction and the other (enzyme 0) was entirely in the soluble fraction. Turnip leaves had one enzyme (erzyme B) which was localized in the soluble fraction throughout maturation.

INTRODUCTION

Multiple forms of starch phosphorylase (SP) exist in plant tissues (Khanna

et al..

1971; Kumar and Sanwal. 1977; Shivaram, 1976). Chen et al. (1970)

demonstrated that in Xanthium leaves, the number of SP isoenzymes decreased from

four to one with leaf maturity. Singh and Sanwal (1975) demonstrated two and

three forms of SP in young and mature banana fruit, respectively. Earlier, we

demonstrated different forms of SP in banana leaves during development (Kumar

and Sanwal, 1977). Several workers have shown the presence of SP in chloroplasts

(Defekete, 1968; Gerbrandy and Verleur, 1971) and interpreted its involvement

In

starch biosynthesis. In this communication we report our results on the multiple

forms and subcelh,ilar localization of SP in marigold, turnip, cabbage, brinjal, arhar

and potato leaves during their development.

318

KUMAR AND SANwAL

MATERIALS AND METHODS

Leaf' tissue.

Marigold (Tagites erecta). turnip (Brassica rapa), brinjal

(Solanum melongena), arhar (Cajanus indicus), cabbage (Elephantopus scabar) and

potato (Solanum tuberosum) leaves were collected from plants grown in the depart­

mental garden and used for the assay.

Isolation of chloroplast.

Leaf tissue, 10 g was cut into small pieces and

homogenized at 0-4°C in a glass bowl of a Waring blender using 70 ml homogeni­

zing medium consisting of 0.01 M Tris-HCI buffer pH 7.5, 0.02 M 2-mercaptoetha­

nol, 0.5% (wt/vol) PVP (MW 40,000), 5 mM MgClz and 0.5 M sucrose. The

homogenate was fIltered through two layers of muslin and the volume made up to

100 ml. The filtrate was designated as enzyme homogenate. Subsequently this

homogenate was centrifuged at

1500xg

for 10 min in a refrigerated centrifuge to

sediment the chloroplasts. After decanting the supernatant the chloroplast fraction

was washed with the isolation medium and centrifuged to obtain the chloroplasts.

The two supernatants were combined. The particulate fraction was suspended in

10

011

of the isolation medium using a hand operated glass homogenizer.

Electrophoresis.

Polyacrylamide gel electrophoresis was carried out

(Weber and Osborn, 1969) but without SDS employing 6% gel. Tris-HCI (0.05 M,

pH 7.5) was used as the electrode as well as gel buffer. Electrophoresis was done

in the cold and 8 mA current per tube for 2!- h. After electrophoresis, gels were

removed and incubated overnight in a mixture consisting of 0.02 mM NaF. 0.02

M Tris-maleate buffer pH 6.0. 0.3% starch (freshly prepared) and 5 mM glucose-1­

P04. Gels were stained with 12 reagent (Krisman. 1962). Place (s) with SP activity

became dark on staining.

Enzyme assay. SP was assayed in the direction of polysaccharide synthesis

as described earlier (Kumar and Sanwal. (1981).

Protein determination.

Proteins were determined according to Lowry

et al. (1951) and modified by Khanna et al. (1969) using bovine serum albumin as a

standard.

RESULTS AND DISCUSSION

As determined by polyacrylamide gel electrophoresis. four multiple forms of

SP were observed during maturation of plant leaves. The electrophoretic mobility of

these multiple forms is shown in Table II. The multiple forms have been numbered

according to their increasing electrophoretic mobility.

PLANT STARCH PHOSPHORYLASB

319

TABLE I. Suggested forms of SP

Multiple forms of the enzyme Electrophoretic mobility (cm/2t h)

---~---A 1.9

B 2.2

-

C 3.2

\

o

4.2

- - -

---­

Electrophoresis was carried out for 2! h at constant current using 8 mA current per tube. The length of the gel was

J

0 em.

TABLE

II.

Distribution of multiple forms of SP in leaves of differing maturity

Plant Multiple form

Supernatant (1500xg)

Tagites erecta

Young 6

Mature B

B rassica rapa

Young B

Mature B

Solanum melongena

Young B

B,C

Mature B

B,C

Caianus indicus

Young C

C,O

Mature

C

C,O

Elephantopus scabar

Young

A. B,O

A, B, C, 0

Mature C

Solanum tub~rosum

Young B . S, C, 0

Mature B B, C, 0

potato. In contrast, in cabbage, the number of multiple forms changes from four

to one during maturity similar to Xanthium leaves (Chen et aI., 1970).

There is no specific rule for the change in the multiple forms of SP during

development of plant leaves. PerhapsSP might be involved either in starch biosyn­

thesis or degradation. Some workers.CGerbrandy et al., 1975) have observed a

. change in isozymic pattern due to proteolysis. We believe that changes in the

pattern of SP during development may

be

dependent on the physiological state

of the leaves.

I

.~

320

KUMAR AND SANWAL

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0'"

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c' c...

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d'

d"

e

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eO<

,

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f"

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PLANT STARCH PHOSPHORYLASE

₃₂₁

TABLE III. Distribution of starch phosphorylase in isolated chloroplast fractions

. _ - - - - _ .

Tissue Young leaf Mature leaf

Enzyme Protein SP act Enzyme Protein SP act activity (mgfg (nkatslmg activity (mgfg (nkats! (nkatsf tissue) protein) (nkatsl tissue) mg pro­

-

1

gtissue) g tissue) tein)

.--~---...

Marigold

Enzyme homogenate 2.53 10.00 0.25 2.53 5.63 0.45

..

•

1500"g particulate fraction 0 1.00 0 0 0.63 0

1500xg supernatant 2.22 8.75 0.25 2.53 5.00 0.51 Turnip leaves

Enzyme homogenate 3.03 15.63 019 4.55 21.25 0.21

1500"g particulate fraction 0 1.75 0 0 2.50 0 1500xg supernatant 2.63 13.03 0.20 4.24 17.50 0.24 Brinjalleaves

Enzyme homogenate 2.02 7.50 0.27 2.53 12.50 0.20 1500xg particulate fraction 0.35 1.00 0.35 0.40 1.63 0.25 1500xg supernata nt 1.62 6.25 0.26 2.02 10.00 0.20 Arhar leaves

Enzyme homogenate 3.03 11.88 0.26 3.54 21.25 0.17 1500xg particulate fraction 0.40 1.38 0.29 0.40 2.50 0.16 1500xg supernatant 2.53 10.63 0.24 3.03 18.75 0.16 Cabbage leaves

Enzyme homogenate 2.00 10.00 0.20 3.54 17.50 0.20 1500xg particulate fr!lction 0.45 1.75 0.26 0 2.63 0 1500xg supernatant 1.52 7.50 0.20 3.23 14.38 0.22 Potato leaves

Enzyme homogenate 4.04 17.50 0.23 5.05 15.63 0.32 1500xg particulate fraction 0.'l1 3.38 0.27 0.70 3.13 0.22 1500xg supernatant 3.03 13.75 0.22 3.54 11.88 0.30 10% homogenate was prepared using 0.01 M Tris-HCI buffer pH 7.5 containing 0.02 M 2-mercaptoethanol, 0.05% (w/v) PVP and 5mM MgCh.

Fig. 1. Multiple forms of starch phosphorylase in developing leaves. A. young marigold leaves; (a) homogenate (a') 1500 xg particulate fraction (a") 1500 xg supernatant; B, mature marigold leaves (b) homogenate (b') 1500 xg particulate fraction (b") 1500 xg superna­ tant; C. young turnip leaves (c) homogenate (c') 1500 xg particulate fraction (c") 1500 xg supernatant; D. mature turnip leaves (d) homogenate (d') 1500 xg particulate fraction (d") 1500 xg supernatant; E. young brinjal leaves (e) homogenate (e') 1500 xg particulate fraction (e") 1500 xg supernatant; F. mature brinjal leaves (1) homogenate (f') 1500 xg

particulate fraction (r') 1500 xg supernatant; G. young arhar leaves (g) homogenate (g') 1500 xg particulate fraction (g") 1500 xg supernatant; H. mature arhar leaves (h) homo­ genate (h') 1500 xg particulate fraction (h") 1500 xg supernatant; I. young cabbage leaves

322

KUMAR AND SANwAL

The enzyme activity in the case of marigold was same in both young and

mature leaves but protein concentration was less in mature leaves as compared to

that in the young leaves, so the specific activity increased in mature leaves. In

turnip leaves, the enzyme activity as well as sp act of SP was

~reater

in mature

leaves compared to 'young leaves. In brinjal leaves, the enzyme activity was greater

in mature leaves but sp act was less as compared to that in the young leaves due

to high protein concentration in mature brinjal leaves.

In arhar leaves, the enzyme

activity was greater but sp act was less in mature leaves as compared to that in the

young leaves. The enzyme activity was greater in mature cabbage leaves than in

young leaves but sp act did not change during maturation.

In potato, like turnip

leaves, the enzyme activity and sp act were greater in the mature leaves than in the

young reaves.

Clearly, there is no correlation between starch biosynthesis and distribution

of SP in chloroplasts. Presence of SP in chloroplasts may

be

due to physical adsor­

ption (ike the attachment of glycogen phosphorylase on glycogen particles (Fischer

et al.,

1971). On the basis of partial localization of SP in chloroplasts Defekete

(1968) and Peavey

et al.

(1977) have hinted at the role of SP in starch biosynthesis.

ACKNOWLEDGEMENTS

We thank the University Grants Commission, !\lew Delhi for financial assis­

tance. The authors wish to thank Professor Joe H. Cherry, Purdue University,

U. S.

A. for suggestions and going through the manuscript.

REFERENCES

Chen, S. L., Towill. L. R. and Lowenberg. J. R. (1970). Isozyme patterns in developing Xanthium leaves.

Physiol. Plantarum. 23 : 434-43.

Defekete. M. A. R. (1968). Die Rolle der phosphorylase in stoff wechsel der starke in den plastiden.

Planta. 79: 208-21.

Fischer. E. H., Helmeyer, L. M. G. Jr. and Has~hke, R. H. (1971). Phosphorylase and control of glycogen degradation. CUff. Top. Cell Reg., 4: 211-51.

Gerbrandy. Sj. J. and Verleur, J. D. (1971). Phosphorylase isoenzymes: Localization and occurrence in different plant organs in relation to starch metabolism. Phytochemistry. 10: 261-66.

Gerbiandy. Sj: J .. Shanker, V., Shivaram, K. N. and Stegemann, H. (1975). Conversion of potato phospho­ rylase isoenzymes. Phytochemistry, 14: 2331-33.

Khanna, S. K., Mattoo. R. L., Viswanathan, P. N., Tewari, C. P. and Sanwal, G. G. (1969). Colorimetric determination of protein and orthophosphate. Indian J. Biochem., 6 : 21-25.

Khanna, S. K., Sanwal, G. G. and Krishnan, P. S. (1971). Glucan phosphorylase in the leaves ot

Dendrophthoe faleata: Purification and characterization of enzyme. Phytochemistry, 10: 551-60.

Krisman, C. R. (1962). A method for the colorimetric estimation of glycogen with iodine. Anal. Biochem.,

4: 17-23 .

PLANT STARCH PHOSPHORYLASB

₃₂₃

Kumar, A. and Sanwal. G. G. (1977). Multiple forms of starch phosphorylase from Musa paradisiaca leaves. Phytochemistry, 16: 327-28.

Kumar, A. and Sanwal, G. G. (1931). Immobilized starch phosphorylase from mature banana (Musa

p;uadisiaca) leaves. Indian J. Biochem. Biophys., 18: 114-19.

lowry, O. H., Rosebrough, N. J., Farrh, A. l. and Randall, R. J. (1951). Protein measurement with lolin phenol reagent. J. BioI. Chem" 193 : 265-75.

Peavey, D. G., Steup, M. and Gibbs, M. (1977). Characterization of starch breakdown in isolated spinach chloroplast. Plant Physio{., 60 : 305-08.

Shivaram, K. N. (1976). Purification and properties of potato phosphorylase isoenzymes. Z. NaturForsch,

31e : 424-32.

Singh, S. and Sanwal, G. G. (1975). CharacteriZ3tion of mUltiple forms of a-glucan phosphorylase from

Musa paradisiaca leaves. Phytochemistry, 14 : 113-18.