Fluorinated Carbohydrates - American Chemical Society

Chapter 8

Sucrose Transport in Plants Using Monofluorinated Sucroses and Glucosides William D. Hitz

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Central Research and Development Department, E. I. du Pont de Nemours and Company, Experimental Station, Wilmington, DE 19898 T h e s u c r o s e carrier in the outer membrane of cells of developing s o y b e a n cotyledons recognizes a n d transports s u c r o s e derivitives which are singly fluorinated at C-1', C-4', a n d C-6'. S o m e a -glucosides are also recognized and transported and may be used a s sucrose analogs in studying carrier-substrate interaction. Phenyl a -D-thioglucopyranosides fluorinated singly at positions C - 3 , C-4, a n d C-6 along with the deoxy-glucosides at these positions indicate that recognition by the plant carrier requires interaction at these three hydroxyls along with hydrophobic interaction with the β -face of the g l u c o s e moiety a n d the a -face of the fructose moiety in sucrose. 1'-Deoxy-1'-fluorosucrose is also recognized a n d transported by the carrier proteins in the vascular tissues of leaves a n d is thus moved in long distance transport in a manner identical to s u c r o s e . At limiting substrate c o n c e n t r a t i o n s , 1'-deoxy-1'-fluorosucrose is metabolized by s u c r o s e synthase at a rate 3.6 times slower than s u c r o s e . Hydrolysis by invertase however occurs at a rate 4200 times slower than sucrose. In vivo rates of metabolism for the 1'-fluoro derivitive a n d for s u c r o s e showed that s u c r o s e is metabolized by both e n z y m e s acting in parallel, a n d that the relative contribution of the individual e n z y m e s varies with tissue development.

In many plant s p e c i e s , including virtually all major field crops and many vegetable crops, s u c r o s e is the carbohydrate utilized 0

0097-6156/88/0374-0138$06.00/0 1988 American Chemical Society

TAYLOR; Fluorinated Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8.

139

Sucrose Transport in Plants

HITZ

for long distance transport a n d for soluble, short term

storage.

T h e carbon which makes up the bulk of the dry weight harvested for economic yield is p r o c e s s e d through the e n z y m e s a n d other proteins in the physical a n d metabolic pathways of s u c r o s e metabolism.

S i n c e much of the increased yield potential

achieved in breeding improved varieties of crop plants h a s c o m e from a more efficient transfer of carbon fixed in photosynthesis to harvestable portions of the plant (1), there is continuing interest in detailing the early steps of s u c r o s e

metabolism in

order to test direct methods of improving yields through or c h e m i c a l

modification

of controlling

steps

genetic

in metabolism.


Two plant e n z y m e s are capable of catalyzing the breakage of the glucose to fructose bond in sucrose. hydrolysis

either

in the intra-cellular

Invertase catalyzes

compartment

or in the

extra-cellular s p a c e , a n d s u c r o s e synthase c a t a l y z e s the transfer of the glucose residue to Uridine diphosphate Since

extra-cellular

membrane

invertase

is present

transport of s u c r o s e

h a s two possibilities

Uptake of the intact molecule occurs a s d o e s followed by uptake of the hexoses produced. fluorinated

carbohydrates

a s alternate

(2.).

in s o m e tissues, the also.

hydrolysis W e have used

substrates

for these

e n z y m e s a n d transport proteins in order to determine both how substrate interaction occurs a n d to determine which of the alternative elements

pathways

is functioning

of substrate

interaction

in vivo . with

T h e important

carrier

determined from the interaction of fluorinated

proteins sucroses and

s u c r o s e analogs w a s used to guide the synthesis of affinity probes

for protein

identification

a n d the fluorinated

sucroses

themselves have been used to determine the relative flux of s u c r o s e through paralell enzymic pathways in vivo. Substrate

Recognition

bv S u c r o s e

Transporters

T r a n s m e m b r a n e movement of sucrose is accomplished by transport proteins in several, but not all tissue types. T h e most obvious example of a specific tissue type is the phloem. T h e s u c r o s e concentration in this tissue c a n approach 0.8 M in contrast to m M concentration in the surrounding tissues a n d probably s u b m M concentration in the extra cellular s p a c e s surrounding the phloem (2). Transport into the cells of the phloem is difficult to study a s they are an integral part of the leaf or stem structure, a n d may comprise only 5 to 10% of the total leaf mass. Another example of s u c r o s e transport is the accumulation of s u c r o s e a n d other nutrients by cells of


140

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

developing embryos or embryo storage tissue.

Large

accumulations are not obvious in these tissues, but the physiological evidence suggests that s u c r o s e uptake occurs, a n d that in the c a s e of the developing s o y b e a n cotyledon, it has many of the s a m e characteristics a s phloem uptake

(4*5).

S i n c e the soybean cotyledon is quite homogeneous a s to tissue type (S.)

a n d c a n be readily manipulated to remove cell

walls in tissue slices to yield protoplasts (7), we have these protoplasts a s a model system to study transport of s u c r o s e in general.

used

membrane

T h e concentration kinetics of

s u c r o s e influx suggest that at least two p r o c e s s e s are Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: August 2, 1988 | doi: 10.1021/bk-1988-0374.ch008

operating in parallel. O n e of these is saturable with respect to s u c r o s e concentration, of comparatively

low capacity a n d

inhibited by any of several metabolic poisons (7). specificity of this system c a n be studied by using substrates sucrose

a s apparent

inhibitors of the influx of

T h e substrate alternate radiolabled

present at a concentration well below the concentration

required for half-maximal monosaccharides

uptake

(K ). m

Studies in which

a n d natural disaccharides other than

sucrose

were tested a s substrates show the carrier to be quite for s u c r o s e (8.).

singly substituted s u c r o s e s , the

specific

Accordingly, we c h o s e to make a series of using fluorine a s a substitute for

hydroxyl, to systematically

probe the substrate

binding

site

in the s u c r o s e carrier protein in these cells. Fructosvl-Substituted S u c r o s e s . T h e deoxy-fluoro-derivatives of sucrose at C - 1 \ C-4' a n d C-6', along with deoxy a n d deoxyazido-derivatives at C-6' a n d C - V respectively were prepared by the sucrose synthase catalyzed coupling of U D P - g l u c o s e and the substituted fructose (9.10). T h o s e structures a n d their binding constants relative to s u c r o s e (estimated from their respective Kj's) are given in Figure 1a. Substitutions at C - V and C-6' which were more hydrophobic than the hydroxyl they replaced, bound about two fold more tightly than sucrose. T h e amino substitution at C - V (formed by catalytic reduction of the V-azido) bound about one-half a s well a s the native structure. W e reason that this binding pattern could occur if the three dimensional structure of s u c r o s e a s s o c i a t e s with the binding site of the protein such that the relatively hydrophobic surface of s u c r o s e formed by the portions of the two monosaccharide rings which are s h a d e d in Figure 2 a interact with a similarly shaped, hydrophobic site on the protein. T h e hydrophobic substitutions at C - V and C-6' act to increase the size of this



8. HITZ


141

Figure 1a and 1b. T h e relative binding constants for 6 substituted s u c r o s e s (1a) and 10 phenyl

RELATI


25 -

40

O

a X

20 it )

n 1 10

fit 20

a

$

S

A

-

A

-

•

D

1 40

30 TIME (min)

50

i 60

i 70

Figure 6. Translocation of C - s u c r o s e ( x,d) a n d C-1'deoxy-V-fluorosucrose (A, O) into young s o y b e a n leaves. Counts per minute ( C P M ) , measured by Geiger-Mueller counting, were corrected for background a n d expressed a s per cent of C P M at 66 min. C - s u g a r s were supplied to an exporting leaf at time zero by application of a buffered solution containing the sugar to an a b r a d e d surface. (Reproduced with permission from ref. 16. Copyright 1987 T h e A m e r i c a n Society of Plant Physiologists.) 1 4

1 4

1 4


8.

HITZ

Sucrose Transport in Plants 40

151

A - 4% FLL

30 20

O

10 40

B - 7% FLL

30 20 10 CO

z> Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: August 2, 1988 | doi: 10.1021/bk-1988-0374.ch008

-J

o z

50 -

C - 11% FLL

40

id 30 m

3 g o < cc

20 10

50

8

D - 17% FLL O

40

OQ

30 20 10 40 •

E - 40% FLL

30 o

20

„ o o o ° o oS^b o o „^ o

10 • 20

40

60 80 TIME (min.)

100

120

Figure 7. The time course of percent incorporation into ethanol insolubles of phloem translocated sucrose (o) and 1 '-deoxy-1 'fluorosucrose (•) in soybeanfirsttrifoliolate leaves of various ages (%FLL is the percent offinalleaf length of a comparable leaf at maturity). Radiolabled sucrose and/or 1'-deoxy-1'-fluorosucrose were supplied as described in Figure 3 and importing leaves were either removedfromsampling at time points or parts of the leaf were removed at time points and extracted as described in the text. (Reproduced with permissionfromref. 16. Copyright 1987 The American Society of Plant Physiologists.)


152

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS Quantitative

determination

of the relative

contributions

of s u c r o s e synthase and invertase to the breakdown of phloemsupplied s u c r o s e c a n b e determined from the total

metabolism

rate for e a c h sugar a n d the discrimination factor for the two sugars a s substrates for invertase and sucrose synthase.

The

total rate of breakdown of either sucrose or V - F S is the s u m of the fluxes through the two e n z y m e s so that: R


and

= Is + S S

s

Rf

s

(3)

S

= Ifs + S S f

(4)

s

where R is the total rate of metabolism for either sugar, I is the contribution of invertase to that rate, a n d S S is the contribution of s u c r o s e synthase. At the tracer concentrations arriving in the importing leaf, the ratio of s u c r o s e to V - F S metabolism by s u c r o s e synthase is 3.6 times the ratio of substrate concentration, a n d the s a m e ratio for invertase is 4200 times the ratio of substrate concentration. Thus:

SS

S

= 3.6 S S f s

[

1

4

C

-

S

U

C

r

°

S

e

l

[14 c - V - F S ]

and

l

s

= 4200 l f

[

1 4

(5)

C-sucrose]

s

[14c- V-FS]

(6)

S i n c e the two substrates are transported identically, a n d supplied at the s a m e concentration to the mature leaves, their initial concentrations in their respective importing l e a v e s will be very similar a n d the substrate ratios in equations 5 a n d 6 will be essentially o n e . S i n c e s u c r o s e is always metabolized faster than V - F S , the ratio will b e c o m e less than o n e with time and the apparent relative metabolism rates for the two sugars will c h a n g e a s c a n be s e e n in figure 7. During the initial, linear period of label incorporation into insolubles however the ratio can be a s s u m e d to be o n e . Assuming this simplification, dividing equation 3 by equation 4 and substituting for S S f s and Ifs from equations 5 and 6 respectively, then solving for I s / S S s , gives


8. HITZ

153


,

s

/

S

S

s

m

1-(Rs/Rfs)/3.6 (Rs/Rfs)4200-1

T h e ratios Rs/Rfs from the data of figure 7 and the corresponding contributions of invertase and s u c r o s e

synthase

calculated from those ratios are given in T a b l e I for leaves of different


T a b l e I.

developmental

ages.

T h e relative contribution of invertase and

sucrose

synthase to s u c r o s e metabolism in developing s o y b e a n Leaf A g e

Rate of Sugar Metabolism

(% F L L i a

_Rs_.

% of Total. Metabolism

b _R /Rfs_ _ I / S S

_Rfs_

S

S

C S

_. Js_

S

0.25

0.074

3.4

0

0

100

7.6

0.64

0.095

6.7

0.86

46

54

1.5

0.19

7.9

1.20

54

46 58 57

17.0

1.0

0.16

6.2

0.72

42

40.0

0.37

0.059

6.5

0.75

43

L e a f age as determined by the % of length attained by a

comparable leaf b

_SS _

4.0 11.0

a

leaves

at full development.

R a t e of sucrose or 1'-FS

metabolism in % of total radiolabel in

the leaf converted to insolubles per minute. c

C a l c u l a t e d using equation 7.

T h e rate of carbon import and s u c r o s e metabolism increased very rapidly during early development. Most of the increased rate of sucrose metabolism could be accounted for by a sharp rise in the flux through invertase. A s the leaf b e c a m e photosynthetically competent, the rate of utilization of c a r b o n imported from mature leaves declined, however about one-half of s u c r o s e

metabolism

remained through

invertase.

In leaves at early stages of development, extractable invertase activity (measured at its p H optima) c a n e x c e e d extractable s u c r o s e synthase activity by 10 to 20 fold (15L), yet compartmentalization of e n z y m e and substrate may make the relative flux through these parallel paths quite different from


154

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

the proportionate activities.

T h e method described above is o n e

of the few that c a n discern in vivo flux in this way.

As a

general method it could be applied to any system for which differentially

metabolizable

substrates c a n be produced.

In the

c a s e of 1'-FS used in this way, a problem arises d u e to the extreme discrimination against 1'-FS by invertase.

A very

small change in the ratio Rs/Rfs corresponds to a large change in the flux through invertase, sensitive to errors

making the calculation

very

in the ratio.


Conclusions a n d Future Studies T h e tenative identification of a s u c r o s e carrier protein o p e n s many a v e n u e s of research including the identification of s u c r o s e binding proteins in other tissues using the s u c r o s e photoprobe a n d utilizing antibody c r o s s reactivity. Ultimately the function of the 62 kD membrane protein from cotyledons needs to be proven either by the biochemical methods of reconstitution into artificial m e m b r a n e v e s i c l e s , by c o m p a r i s o n of the implied protein structure (obtained from the structural g e n e sequence) with other, known transporters, or by genetically manipulating transport. T h e use of fluorinated s u c r o s e s a s tracers of s u c r o s e metabolism in order to differentiate between glycolysis started by invertase a n d glycolysis started by s u c r o s e synthase should be quite useful in determining the in vivo flux of these paths and the large differences in phosphate metabolism which a c c o m p a n y the two routes of carbon metabolism.

Acknowledgments T h e active collaborations of Dr. Peter C a r d in the synthesis of fluorinated carbohydrates, Mr. Kevin Ripp in protein purification and preparation of antibodies, Dr. Vincent Francheschi in immunohistochemistry a n d Dr. Judy Schmalstig in the m e t a b o l i s m of 1 '-deoxy-1 '-flurosucrose are greatfully a c k n o w l e d g e d .

Literature 1.

Cited

Gifford, R. M.; Thorne, J . ; Hitz, W.; Giaquinta, R. 1984, 225, 801-8.

Science


8. IIITZ 2.

3. 4. 5. 6. 7.


8. 9. 10. 11. 12. 13. 14. 15. 16.


155

Avigad, G. In Plant Carbohydrates I. Encyclopedia of Plant Physiology. New Series: Springer-Verlag: Berlin, 1982; vol. 13A; pp. 217-347. Giaquinta, R. T. In The Biochemistry of Plants: Academic: New York, 1980; vol. 3; pp. 271-320. Lichtner, F. T.; Spanswick, R. Plant Physiol. 1981, 68, 693-8. Thome, J. H. Plant Physiol. 1982, 70,953-8. Thome, J. H. Plant Physiol. 1981, 67, 1016-25. Lin, W.; Schmitt, M.; Hitz, W.; Giaquinta, R. Plant Physiol. 1984, 75, 936-40. Schmitt, M. R.; Hitz, W.; Lin, W.; Giaquinta, R. Plant Physiol. 1984, 75, 941-6 Card, P. J.; Hitz, W. J. Am. Chem. Soc. 1984, 106, 5348-50. Card, P. J.; Hitz, W. J. Am. Chem. Soc. 1986, 108, 158-61. Hitz, W. D.; Card, P.; Ripp, K. J. Biol. Chem. 1986, 261, 11986-91. Mathlouthi, M.; Cholli, A.; Koenig, J. Carbo. Res. 1986, 147, 1-9. Hindsgual, O.; Norberg, T.; Lependu, J.; Lemeiux, R. Carbo. Res. 1982, 109, 109-142. Guthrie, R. D.; Jenkins, I.; Yamasaki, R. Aust. J. Chem. 1982, 35, 1003-8. Giaquinta, R. T. Plant Physiol. 1978, 61, 380-5. Schmalstig, J. G.; Hitz, W. Plant Physiol. 1987, 85, 40712.

RECEIVED January 15, 1988


Fluorinated Carbohydrates - American Chemical Society

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