Carbohydrate oxidation during differentiation in roots of Pisum sativum

BIOCHIMICA ET BIOPHYSICA ACTA

33

BBA 2 6 2 5 4

CARBOHYDRATE OXIDATION DURING D I F F E R E N T I A T I O N IN ROOTS OF P I S U M S A T I V U M M. W. F O W L E R AND T. AP REES

Botany School, University of Cambridge, Cambridge (Great Britain) (Received August i s t h , 1969)

SUMMARY

I. The aim of this work was to see whether the relative activities of glycolysis and the pentose phosphate pathway varied during the differentiation of mature cells from apical cells in roots of Pisum sativum. 2. The detailed distribution of 14C from [I-~4CJ- and [6-~4Cjglucose supplied to segments of the apical 26 mm of the root was determined. The distribution in the apical 3 mm indicated a predominance of glycolysis with a relatively small contribution from the pentose phosphate pathway. The distribution in segments from the region 6-26 mm from the apex indicated substantial contributions from both pathways. 3. The activities of four glycolytic enzymes and five enzymes of the pentose phosphate pathway were measured in extracts of root segments at different stages of differentiation. The activities of the glycolytic enzymes predominated in extracts of the apical 5 mm of the root. This predominance was not found in extracts of segments from the region 5-2o mm from the apex. 4. These results were held to support the view that carbohydrate oxidation in the relatively undifferentiated cells of the root apex was mainly via glycolysis and that differentiation involved an increase in the capacity and activity of the pentose phosphate pathway relative to glycolysis.

INTRODUCTION

Although there is good evidence that roots oxidize carbohydrate via glycolysis1 and the pentose phosphate pathway 2, 3, we do not know whether differentiation in the root is accompanied by changes in the relative activities of these pathways. GIBBS AND BEEVERS4 compared 1~C02 production from [I-~CJ- and [6-~4CJglucose by I-cm segments of roots. Their results indicated that glycolysis predominated in the root tips and that the relative activity of the pentose phosphate pathway rose as the cells differentiated. Subsequent appreciation of the general difficulties of interpreting this type of experiment 5, and the particular complication caused by the release of glucose C-6 as CO~ during pentan synthesis s, make these experiments inconclusive. GIBBS AND EARL~ compared the activities of some of the enzymes of the two pathways in extracts of roots of pea plants of different ages. The relative activities of the enzymes Biochim. Biophys. Acta, 2oi (i97 o) 33-44

34

M. W. FOWLER, T, AP REES

of the pentose phosphate pathway were greatest in extracts of roots of the older plants. These results are not directly comparable with the experiments with EleCtglucose a s GIBBS AND EARL 7 used complete roots of different ages whereas GIBBS AND BEEVERS ~ used different segments of roots of the same age. In both the above studies it is difficult to relate the biochemical data to precise stages in root differentiation because the samples of roots used represented m a n y stages of differentiation. The aim of the work in this paper was to see whether the relative activities of glycolysis and the pentose-phosphate pathway varied during the differentiation of mature cells from apical cells in pea roots. We think this problem to be important for two reasons. First, the extent to which differentiation involves changes in the activities of the two pathways is an important aspect of the control of carbohydrate oxidation. Second, the study is related to our understanding of the role of the pentose phosphate pathway. Present evidence strongly indicates that the major function of this pathway is the provision of N A D P H for reductive biosyntheses s. Thus it was important to examine roots as the available evidence indicated that the pentose phosphate pathway was relatively inactive in the root tip, an area normally regarded as being very active in biosynthesis. We cut the root tips into segments, representative of different stages of differentiation, and then used two different methods to assess the relative activities of the two pathways in the segments. First, we determined the detailed distribution of ~C from II-~C~ - and !6-~Clglucose. In short incubations, an increase in the activity of the pentose phosphate pathway, relative to that of glycolysis, would be indicated by the simultaneous occurrence of two changes in the distribution of 14C. The first change is an increase in the proportion of glucose C-I, but not glucose C-6, that is converted to CO~. The second change is an equivalent decrease in the proportion of glucose C-I, but not C-6, that is converted to compounds derived from ribulose 5~phosphate formed by the action of phosphogluconate dehydrogenase. The second approach to the determination of the relative activities of the two pathways was measurement of the activities of their enzymes in extracts of root segments. MATERIALS AND METHODS

Materials Chemicals and enzymes were obtained as follows: D-[I-I~C]- and D-I6-14C] glucose from the Radiochemical Centre, Amersham; glyceraldehyde 3-phosphate, ribose 5-phosphate, and ribose phosphate isomerase from Sigma Chemical Co.; erythrose 4-phosphate from Calbiochem; all other substrates, cofactors, and enzymes from Boehringer, Mannheim.

Preparation of root segments Seeds of pea IPisum sativum L. var. Kelvedon Wonder) were surface sterilized, soaked in aerated distilled water for 6 h and then planted at a depth of 1. 5 cm in sand. After 5-days growth in the dark at 25 ° the seedlings were harvested and the root tips were excised and then sectioned mechanically in an apparatus similar to that described by LOENING9. The freshly-cut segments were kept in 0.03 M KH~PO 4 (pH 5.2) until they were sampled. All operations from the excision of the root tips to the preparation of the experimental samples were carried out at 4 ° . The time between Biochim. Biophys. Acta, 2Ol (197 o) 33-44

CARBOHYDRATE OXIDATION IN PEA ROOTS

35

the cutting of the first segment and the addition of isotope to the experimental samples was 4 h. The time between the start of sectioning and the preparation of extracts for enzyme assays was 30 min.

Metabolism of ~4Clglucose Sufficient roots were sectioned to provide duplicate samples of each type of segment. II-14ClGlucose was supplied to one of the duplicate samples of each type of segment and I6-~Clglucose was supplied to the other. Samples (250 mg fresh weight) were incubated in Warburg flasks in 2.5 ml 0.03 M KH2PO~ (pH 5.2) that contained 2.5 #CI~C~glucose at 0.3 mM. 1~CO2 was collected and assayed as described previously 1°. After 45 or 9 ° rain the incubation medium was removed and each sample was washed for I min in 2.5 ml 0.03 M KH~PO~ (pH 5.2). Glucose uptake was determined by measuring the difference between the amount of ~C supplied to the sample and the amount recovered from the incubation medium and the washings at the end of the incubation. After removal of the incubation medium, the tissue was killed and extracted in boiling 8o % (v/v) aq. ethanol. The residue was then extracted with 80 % /v/v) aq. ethanol for 30 rain at 4 °, homogenized, and extracted successively for periods of 30 rain at 4 ° with 50 % (v/v) aq. ethanol (adiusted to p H 4.0 with glacial acetic acid), and 2 % (v/v) HCIO~ (twice). The extracts were combined, evaporated under reduced pressure at 35 °, and extracted with water to give the water-soluble fraction from which HC104 was removed by precipitation with I.O M K O H at 5 °. The water-soluble fraction was divided into nucleotides, organic acids, a basic fraction (mostly amino acids), and a neutral fraction (mostly sugars) by ion-exchange chromatography. We followed the procedure of HARLEY AND BEEVERS1~ except that the organic acids were eluted from the anion-exchange column with 2.0 M formic acid and then the nucleotides were eluted from the same column with I.O M ammonium formate in 4.0 M formic acid. RNA was extracted from the material that was insoluble in ethanol and HC10~ by incubating this material in I.O M diethylamine at 55 ° for 60 h. The nucleotides derived from the hydrolysis of RNA were separated from the diethylamineinsoluble material by filtration through glass-fibre filter paper. All measurements of I~C were made after it had been converted to Ba~CO.~ as described previously 1°. Radioactivity was assayed with an end-window Geiger counter. The measurements were corrected for self-absorption by the carbonate.

Assay of enzymes Comparisons of activities in different segments were made only with segments derived from the same root tips. Extracts were made in glass homogenizers of samples of 500 mg fresh weight in 3.0 ml 4 ° mM glycylglycine buffer (pH 7.6) at 4 °. The extracts were not assayed until microscopic examination revealed no unbroken cells. The extracts were centrifuged at iooooo × g for 30 rain at 4 ° and the supernatant was used at once for the enzyme assays. Protein in the extracts was determined by the Folin method 12. Standard assays were used for phosphofructokinase (EC 2.7.1.11) 13, fructose1,6-diphosphate aldolase (EC 4.I.2.7)14, glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.I.I2) ~4, pyruvate kinase (EC 2.7.1.41) 15, transketolase (EC 2.2.i.I) ~, and transaldolase (EC 2.2.1.2) 17. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was Biochim. Biophys, Aaa, 2Ol (197o) 33-44

36

M. W. FOWLER, T. AP REES

assayed by comparing NADP + reduction in the presence of glucose 6-phosphate and in the presence of a mixture of glucose 6-phosphate and phosphogluconate according to the method of GLOCK AND McLEAN 18 except that the concentration of NADP + was 0. 3 raM. Phosphogluconate dehydrogenase (EC 1.1.1.44 ) was assayed as described by GLOCK AND McLEAN is except that the concentration of NADP+ was 0.3 mM. Rates of reaction were determined over the first 2 min of the reaction by using a recording spectrophotometer. The activities of the above enzymes are expressed as re#moles of substrate consumed per rain per mg protein. Ribose phosphate isomerase (EC 5.3.1.6) was assayed according to AXELROI~~9. The activity of this enzyme is expressed as change in absorbance per rain per mg protein. RESULTS

Metabolism of I14Clglucose Table I shows the general characteristics of the segments used in these experiments. We found the anatomy of the root tips to be as described by POPHAM~°. Transverse sectioning of pea roots can not yield entirely homogeneous segments. The distinctive features mentioned in Table I do not imply that all the cells in each segment were at the same stage of development nor do they imply that any stage of differentiation was confined to any particular segment. None the less our segments did separate the major aspects of differentiation in the root tips. Extensive changes in metabolism occur when thin slices of m a n y plant tissues are incubated under physiological conditions 22. These changes, which are often apparent within 3 h of cutting, and which include an increase in the rate of respiration, a change in the pattern of 14C02 production from II-I~CI- and E6-~Clglucose 22, and an increase in the activity of at least one enzyme involved in carbohydrate oxidation 23, are prevented by keeping the tissue slices at 2-4 ° . Thus we sectioned and sampled the roots at 4 ° . The success of these precautions m a y be judged from the fact that the O 2 uptake and the pattern of 14CO2 production for all the root segments remained unchanged throughout the period of the experiments. Additional evidence that no marked change in the relative activities of the two pathways of carbohydrate oxidation took place during the experiment is provided by the distribution of label at 45 and 9 ° rain (Tables I I and III). Tables I I and I I I show the distribution of 1~C from II-~4CI- and ~6-~Clglucose supplied to the different segments. We make two general points. First, although glucose uptake varied between different segments, there was very close agreement between replicate samples of the same segment. This is a measure of the reliability of our sampling procedure. Second, we have accounted for nearly all of the label that we supplied to each sample. Thus the amounts of ~4C recovered in the different cell fractions can not have been greatly affected by losses during analysis. Two distinct patterns of distribution of label from II-l~Clglucose relative to that from E6-1~C~glucose were found. One pattern was in the four segments in the apical 3.0 m m and the other was in the segments 6-26 m m from the tip. The pattern in the segments 3-6 m m from the tip was intermediate. In the apical region glucose C-I and C-6 appeared in the different fractions in almost equal proportions. The contribution of C-I to CO~ was slightly greater than that of C-6. This difference was accompanied by a slight excess of label from C-6 over that from C-I in the fractions that contained Biochim. Biophys. Acta, 2 o i (197o) 33-44

segment)

O n u p t a k e (/*l/h p e r

segment

N u m b e r of cells per

(#,g/segment)

Extracted protein

o. 26

65oo

i. 14

Root

cap

anatomical feature

0-0.4

3.5 °

29 500

4.02

initials

Apical

0.4--LO

3.60

28 ooo

5.91

Vacuolation

r.O--L6

Root segment, distance f r o m root tip (ram) :

Distinctive

Characteristic

5.3 °

66 ooo

5.71

Elongation

L6--3.0

6.0--Z6.0

2.60

38 ooo

6.92

4.9 °

98 ooo

34. i

4-6o

99 ooo

3 ° .i

±6.0--26.O

primary phloem and primary xylem

P r o g r e s s i v e m a t u r a t i o n of

3.O 6.0

R o o t s were c u t as d e s c r i b e d in t h e t e x t . P r o t e i n w as e x t r a c t e d in 4 ° m M g l y c y l g l y c i n e buffer (pH 7.6) a n d a s s a y e d b y t h e F o l i n m e t h o d . O n u p t a k e w as d e t e r m i n e d m a n o m e t r i c a l l y w i t h s a m p l e s of 250 m g fresh w e i g h t in 2. 5 ml 0.03 M KH2PO~ (pH 5.2). Cell n u m b e r s ar e c a l c u l a t e d from t h e d a t a of SEXTON AND SUTCLIFFE gl.

CHARACTERISTICS OF SEGMENTS OF PEA ROOT TIPS

TABLEI

© ©

tZ

0 ;~

;~ ,-t

0

(~ ~,

i

O F 14C R E C O V E R E D

FROM SEGMENTS

OF PEA ROOT TIPS SUPPLIED

WITH

[I-14C]GLUCOSE

AND

[6-14C]GLUCOSE

1.o-1.6

o.4-1.o

0-0. 4

Root segment (mm f r o m tip)

C-6

3o

32

9°

C-I

20

21

45

32

C-6

C-I

9°

33

C-6

C-I

15

34

31

13

14

Percentage added laC absorbed

15

45

9o

45

Incubation time (rain)

C-6

C-I

C-6

C-I

C-6

C-i

Position of 14C in glucose

15

17

8

IO

IO

12

9

II

9

ii

7

8

CO 2

23

21

26

21

24

23

28

24

32

31

34

33

Organic acids

9

9

13

13

13

12

14

13

12

i2

18

15

Basic fraction

24

23

25

26

25

25

26

28

29

28

25

25

Neutral fraction

Substances soluble in water

Percentage absorbed 14C per cell fraction

2

2

2

~2

3

2

2

4

3

2

4

4

_

Nucleotides

15

13

Ii

12

io

9

9

9

6

6

4

4

RNA

9

9

8

9

9

9

5

5

6

6

4

5

Residue

~ Insoluble substances

97

94

93

93

94

92

93

94

97

96

96

94

in cell fractions

recovered

29ercentage absorbed laC

R o o t s were g r o w n a n d c u t as d e s c r i b e d i n t h e t e x t . S a m p l e s (25o m g fresh ~veight) were i n c u b a t e d in W a r b u r g flasks i n 2. 5 ml o.o 3 M K H 2 P O 4 (pH 5.2) t h a t c o n t a i n e d 2, 5 pC [ltC]glucose a t 0. 3 raM. I n c u b a t i o n ~vas s t o p p e d b y r e m o v a l of m e d i u m followed b y t h e a d d i t i o n of b o i l i n g 8o % e t h a n o l . Tissue wa s e x t r a c t e d s u c c e s s i v e l y w i t h 80 % e t h a n o l , 50 % e t h a n o l ( m a d e p H 4.0 w i t h g l a c i a l a c e t i c acid), a n d 2 % HCIOa. W a t e r - s o l u b l e c o m p o n e n t s of t h e e x t r a c t were f r a c t i o n a t e d b y i o n - e x c h a n g e c h r o m a t o g r a p h y . N u c l e o t i d e s were e l u t e d from t h e a n i o n - e x c h a n g e c o l u m n s e p a r a t e l y f r o m t h e o th er acidic c o m p o n e n t s of t h e e x t r a c t . R N A w as o b t a i n e d b y e x t r a c t i n g t h e m a t e r i a l i n s o l u b l e in e t h a n o l a n d HC1Oa w i t h i M d i e t h y l a m i n e . Th e m a t e r i a l ins o lu ble in i M d i e t h y l a m i n e is called r e s i d u e a n d is m o s t l y D N A , p r o t e i n , p o l y s a c c h a r i d e , a n d lignin, laCO 2 w as c o l l e c t e d in N O H in t h e cen tr e well.

DISTRIBUTION

TABLE II

~

~ t~

~

:-I

.~

~

~

fi

III

C-I

c-6

~o

~

C-I

~

C-6

C-6

p

~"

C-I

16.o-26.o

C-6

C-I

,~."

~

C-6

~

~

C-I

~

6.o-16.o

C-6

C-I

C-6

90

45

9°

45

90

45

28

31

14

16

56

5°

20

23

73

72

27

28

49

53

90

C-I

21

23

45

Incubation Percentage time added x4C (min) absorbed

C-6

C-I

Position of 14C in glucose

~.

3.o-6.o

1.6-3,o

Root segment (mm from tip)

9

22

4

14

9

23

4

II

8

II

3

7

13

15

5

6

CO~ Nucleotides

29

20

35

20

28

22

44

27

32

27

35

3°

24

22

38

29

IO

7

9

7

12

9

II

13

13

II

18

16

12

IO

18

I6

14

27

27

27

32

19

17

18

19

21

19

12

12

21

23

II

2

2

5

7

I

I

4

4

I

I

3

3

2

2

2

2

12

IO

8

9

8

Io

IO

8

9

7

9

9

9

13

12

II

II

IO

7

5

IO

13

IO

II

14

13

14

15

12

12

13

17

Residue

RNA

Neutral fraction

Organic acids

Basic fraction

Insoluble substances

Substances soluble in water

Percentage absorbed 14C per cell fraction

E x p e r i m e n t a l p r o c e d u r e is d e s c r i b e d i n l e g e n d of T a b l e I I .

DISTRIBUTION OF 14C RECOVERED FROM SEGMENTS OF PEA ROOT TIPS S U P P L I E D WITH [I-14C]GLUCOSE AND [6-14C]GLUCOSE

TABLE

98

96

96

93

89

95

99

94

96

91

94

92

97

96

98

96

Percentage absorbed 14C recovered in cell fractions

(~

© ©

>

1>

©

40

M . W . FOWLER, T. AP REES

the organic and amino acids. In the more differentiated tissue C-I made an appreciably greater contribution to CO2 than did C-6. This difference coincided with a contribution from C-I to the organic and amino acid fractions that was substantially smaller than that from C-6. Other fractions were labelled equally.

Activities of enzymes of carbohydrate metabolism in extracts of root segments Because of the similarity of the labelling patterns in the four apical segments of the roots we decided not to subdivide this region for measurements of enzyme activities. Thus the segments taken for these assays were 0-5, 5-1o, Io-15, and 15-2o m m from the root tip. These segments contained I544oo, 44100, 40000, 43000, cells and 28. 7, 17. 5, 17. 4, 16.6, #g extractable protein, respectively. Evidence that the extracts did not contain inhibitors or activators of the enzymes assayed was obtained by examining the effects of the extracts on the activities of the purified enzymes where these were available. Thus we compared the activities of purified enzymes, of untreated extracts, and of extracts to which measured amounts of the purified enzymes had been added. The activities of purified phosphofructokinase, fructose-I,6diphosphate aldolase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase were not affected by extracts of any of the segments used. The activity of purified glyceraldehyde-3-phosphate dehydrogenase was inhibited b y up to 15 % by extracts of each of the segments. The activities of the enzymes assayed are given in Table IV. Four features of these results are emphasized. Firstly, we stress the consistency with which we found this pattern of enzyme activities. Each value depends upon results obtained from at least five separate batches of plants. Secondly, there was no qualitative difference between the extracts of the four different segments. Thirdly, the activities of the enzymes of the pentose phosphate pathway, in relation to protein content, did not change appreciably from segment to segment. Finally, when expressed on a protein basis, the activities of the glycolytic enzymes in extracts of the segments from 5 to 20 m m from the tip were very similar but were about half the activities found in extracts of the apical 5 m m of the roots. DISCUSSION

The distribution of glucose C-I and C-6 in segments of the apical 3 m m of the root is entirely consistent with the view that in these segments carbohydrate was oxidized predominantly via glycolysis with a relatively small contribution from the pentose phosphate pathway 1°. We think that it is unlikely that the pentose phosphate pathway made a major contribution to carbohydrate oxidation in these segments. This is because of the similar yields of CO s from glucose C-I and C-6 and because this similarity does not appear to have been due to the release of C-6 as CO~ via some pathway other than glycolysis. Our present understanding of carbohydrate metabolism in higher plants indicates two possible routes for the release of glucose C-6 as CO~ via non-glycolytic pathways. These are the synthesis of UDP-xylose from glucose ~4, and the recycling of ribulose 5-phosphate through the pentose phosphate pathway. Even if appreciable amounts of glucose C-6 were released as CO~ during the formation of UDP-xylose, the simultaneous operation of an active pentose phosphate pathway would still have yielded pentose phosphate labelled by C-6, but not b y C-I, of the Biochim. Biophys. Acta, 2Ol (197o) 33-44

OF GLYCOLYSIS

PHOSPHATE

PATHWAY

5 (7)

3 (7)

1. 5 :~ 0.2 (5) I ' 2 4 4 - ° ' ° 4 ( 5 )

35 4-

I9 4-

Ribose-phosphate isomerase

6 (7)

3o ~= 5 (7)

5 (7)

5 (7)

5 (7)

49 4-

Transketolase

P ~

5I ±

57 2_ 7 (8)

39 ~

loo ± 13 (5)

44 ~

8 (7)

5 (7)

19o ± 13 (7)

55 4-

7 (8)

55 ~

51 ~

245 ± 53 (5)

II 5 -10

Transaldolase

Phosphogluconate dehydrogenase

P y r u v a t e kinase

dehydrogenase

Glyceraldehyde-3-ph°sphate

3 (7)

31I ± 23 (7)

95 ±

I

0-5

N

I 4~ 4~

PENTOSE

Activity

(ram f r o m tip)

Glucose-6-phosphate dehydrogenase

~ & ~

AND

Root segment

Fructose-i,6-diphosphatealdolase

Phosphofructokinase

Enzyme

4 ~

~.

~"

~

OF ENZYMES

IN EXTRACTS

OE SEGMENTS

OF

PEA

ROOT

TIPS

III

7 (7)

2 (7)

5 (7)

7 (8)

I (8)

6 (7)

I'66~-°'I5(5)

33 ±

25 -t-

51 4-

39 4-

34 4-

88 4- 13 (5)

175 4- 14 (7)

55 4-

lO-I5

IV

6 (7)

3 (7)

4 (7)

5 (7)

2 (7)

5 (7)

I'36 -4- o.I (5)

36 4-

24 4-

59 ±

47 ±

54 ~

I13 ± Io (5)

167 4- 21 (7)

59 ±

~5 2 0



N.S.


<0.o5

N.S.

N.S,

N.S.

N.S.


N.S.

N.S.

N.S.

<0.05





III

II

I vs.

vs.

I

N.S.


N.S.

N.S.

N,S.

N.S.




IV

vs.

I

Fisher's P values

N.S.

N.S.

N.S.

N.S.

N,S.

N.S.

N.S.

N.S.

N.S.

III

vs.

I[

N.S.

N.S.

N.S.

N.S.

N.S.

<0.05

N.S.

N.S.

N.S.

IV

vs.

II

N.S.

N.S.

N.S.

N.S.

N,S,

<0.02

N.S.

N.S.

N.S.

IV

vs.

III

T h e roots were c u t a n d t h e e x t r a c t s were p r e p a r e d a n d a s s a y e d as described in t h e t e x t . T h e a c t i v i t y of ribose p h o s p h a t e i s o m e r a s e is g i v e n as zlA per m i n per m g protein. T h e activities of all o t h e r e n z y m e s are g i v e n as n m o l e s of s u b s t r a t e c o n s u m e d per m i n per m g protein. V a l u e s are g i v e n as m e a n s 4- S.E. T h e n u m b e r of e x t r a c t s a s s a y e d is g i v e n in p a r e n t h e s e s . F i s h e r ' s P v a l u e s are g i v e n for c o m p a r i s o n of t h e activities in different s e g m e n t s . Values of o.o 5 or less are considered significant. Values g r e a t e r t h a n o.o 5 are g i v e n as N,S. (not significant).

ACTIVITIES

T A B L E IV

H

>

©

H b~

o

42

M . W . FOWLER, T. AP REES

added glucose. Thus the activity of the pentose phosphate pathway, though concealed in the labelling of CO~, would be characterized by a much greater contribution from glucose C-6 than from glucose C-~ to compounds derived from pentose phosphate produced in the pathway. Our data show that this disparity of labelling did not occur after either 45 or 9 ° min. There is general evidence against the occurrence in higher plants of the form of recycling that brings C-6 of any added glucose into position ~ in glucose 6-phosphate formed from ribulose 5-phosphate produced b y the pentose phosphate pathway ~°,aS. Such recycling in a tissue with an active pentose phosphate pathway would probably be marked by a ratio of glucose C-6 to C-~ in CO~ that was initially very low and which then rose rapidly. Our results show that in the apical segments the ratio was between o.8 and ~ even after only 45 rain and did not change markedly during the next 45 min. In the more mature regions of the root, 6-26 m m from the tip, the distribution of label is that which we would expect if both glycolysis and the pentose phosphate pathway had made appreciable contributions to carbohydrate oxidation. The labelling of these segments is very similar to that found in carrot roots ~0. The significance of this labelling pattern has already been discussed ~. Our interpretation of the labelling patterns is supported by our measurements of enzyme activities. Firstly, although the activities of the enzymes of the pentose phosphate pathway in extracts of the apical 5 m m of the root were appreciable, the activities of the glycolytic enzymes were much higher. Secondly, the differences in the labelling pattern between the apical and mature segments were accompanied by corresponding differences in the relative activities of the enzymes of the two pathways. Thirdly, the similar labelling pattern in segments 6-~6 tnm and i6-~6 m m correlates well with the constancy of enzyme activities in extracts of the three segments taken from the region 5-2o m m from the root tip. In view of the general agreement between these two very different experimental approaches we conclude that in the apical 5 m m of the pea root, carbohydrate is oxidized mainly via glycolysis with a relatively small contribution from the pentose phosphate pathway. In the more mature segments (6-26 m m from the tip) both pathways make appreciable contributions to carbohydrate oxidation. Study of the enzyme patterns reveals that, of the glycolytic enzymes, phosphofructokinase and pyruvate kinase had the lowest activities. This correlates with independent evidence that glycolysis in peas m a y be controlled at these steps ~. In the pentose phosphate pathway transketolase, as in rat tissues ~v and yeast ~a, appeared to be the limiting enzyme when measurements were made under optimum conditons i n vitro. The relatively low activity of transketolase m a y be significant in relation to the control of the concentration of erythrose 4-phosphate. This compound, by its action on glucose phosphate isomerase, could affect the relative activities of the two pathways of carbohydrate oxidation in the manner suggested previously ~. The changes in enzyme pattern indicate that variation in enzyme content exerts what U~A~GER ~ has called coarse control over the relative activities of the two pathways of carbohydrate oxidation in pea roots. The relationship between this coarse control and differentiation in pea roots is perhaps best seen by considering our data in relation to individual cells. Comparison of a cell in the apical 5 m m with a cell in the segment ~5-2o m m from the apex strongly indicates that differentiation of the apical cell was accompanied by a 3-fold increase in protein and the enzymes of the pentose phosphate pathway but only a doubling of the enzymes of glycolysis. Study of Table IV shows Biochim. Biophys. Acta, ~o~ (~97o) 33-44

43

CARBOHYDRATE OXIDATION IN PEA ROOTS

that during these changes in enzyme pattern the relative activities of the enzymes of the pentose phosphate pathway did not vary very widely. Thus the synthesis of these enzymes m a y be co-ordinated by a common control mechanism. A similar case can be made for the glycolytic enzymes. We think that differentiation in pea root tips m a y involve the independent regulation of the synthesis of the enzymes of glycolysis and the pentose phosphate pathway to give cells in which the capacity of the latter pathway, in relation to that of glycolysis, is significantly greater than in undifferentiated cells. The significance of the predominance of glycolysis in the apex and the relative increase in the pentose phosphate pathway during differentiation in the root is not obvious. The available evidence argues strongly against any specific role for the pentose phosphate pathway in the provision of carbon compounds for biosyntheses. From Tables II and I I I it can be seen that in each segment C-I and C-6 of glucose contributed almost identically to the insoluble fraction that contained the pentans, and to nucleotides and RNA. Thus it is unlikely that the pathway served specifically to produce pentose for the synthesis of either pentans or the pentose moieties of nucleic acids. Data from other organisms supports this view in respect of both pentans ~° and nucleic acids al. If we accept that the role of the pathway is production of N A D P H for biosyntheses then we are faced with the evidence that the activity of the pathway is minimal in a region normally regarded as being active in biosynthesis. A possible explanation of this anomaly is suggested by the work of OAKS32 who has presented evidence that amino acids used for protein synthesis in the apical cells of maize roots are synthesized in more differentiated cells and then translocated to the tip. If this occurs in pea roots then it would greatly reduce the demand for N A D P H in the apex and increase the demand in mature tissue. This difference in demand would be increased further if the reduction and assimilation of nitrate a*, as well as the formation of aromatic amino acids for lignin biosynthesis, were all preferentially localised in the more differentiated cells of the root tip. ACKNOWLEDGEMENT

M.W.F. thanks the Science Research Council for a research studentship. REFERENCES H. BEEVERS AND M. GIBBS, Plant Physiol., 29 (1954) 318. M. GIBBS AND 13. L. HORECKER, J. Biol. Chem., 2o8 (1954) 813. H. BEEVERS, Plant Physiol., 31 (1956) 339M. GIBBS A~I:0 H. BEEVERS, Plant Physiol., 3° (1955) 343. J- I~ATZ AND H. G. WOOD, J. Biol. Chem., 238 (1963) 517 . W. G. SLATER AND H. BEEVERS, Plant Physiol., 33 (1958) 146. M. GIBBS AND J. M. EARL, Plant Physiol., 34 (1959) 529. B. L. HORECKER, Harvey Lectures, Series 57, Academic Press, New York, 1962, p. 35. U. E. LOENING, Biochem. J., 81 (1961) 254. T. AP REES AND H. BEEVERS, Plant Physiol., 35 (196o) 830. J. L. HARLEY AND H. BEEVERS, Plant Physiol., 38 (1963) 117. E. LAyNE, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 447. 13 K. J. SCOTT, J. S. CRAIGIE AND R. M. SMILLIE, Plant Physiol., 39 (1964) 323 • 14 1R. W u AND E. RACKER, J. Biol. Chem., 234 (1959) lO29. 15 Z. BOCHER AND G. PFLEIDERER, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p- 435I 2 3 4 5 6 7 8 9 IO II 12

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