Enzymes of Glucose Oxidation in Leaf Tissues1 - NCBI

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Jan 27, 1986 - The specific activity of P-fructokinase was higher in the epi- ..... and for the synthesis of such secondary metabolites. 506. Plant Physiol. Vol.

Plant Physiol. (1986) 82, 503-5 10 0032-0889/86/82/0503/08/$01.00/0

Enzymes of Glucose Oxidation in Leaf Tissues1 THE DISTRIBUTION OF THE ENZYMES OF GLYCOLYSIS AND THE OXIDATIVE PENTOSE PHOSPHATE PATHWAY BETWEEN EPIDERMAL AND MESOPHYLL TISSUES OF C3-PLANTS AND EPIDERMAL, MESOPHYLL, AND BUNDLE SHEATH TISSUES OF C4-PLANTS Received for publication January 27, 1986 and in revised form June 25, 1986

EVE SYRKIN WURTELE*2 AND BASIL J. NIKOLAU2

Department of Biochemistry and Biophysics, University of California, Davis, California 95616 ABSTRACI The distribution of the glycolytic enzymes, phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, and the oxidative pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was determined in the leaf tissues of two C3-plants, pea and leek, and two C4plants, maize and sorghum. All enzymes examined were found in epidermal tissue. In pea, maize, and sorghum leaves, the specific activities of these enzymes were usually higher in the nonphotosynthetic epidermal tissue than in the photosynthetic tissues of the leaves. In leek leaves, which were etiolated, specific activities were similar in both epidermal and mesophyll tissue. The distribution of the rate limiting enzymes of glycolysis and the oxidative pentose phosphate pathways probably reflects the capacity of each tissue to generate NADH, NADPH, and ATP from the oxidation of glucose. This capacity appears to be greater in leaf tissues unable to generate reducing equivalents and ATP by photosynthesis, that is, in epidermal tissues and etiolated mesophyll tissue.

Microscopic examination of leaves readily demonstrates the diversity of cell types in this organ (9, 16). Studies of the biochemical processes in leaves have often overlooked the intricate role that compartmentation among different cell types may play in the overall metabolism occurring in leaves. Analyses of tissue compartmentation have led, for example, to the discovery of the pathways of photosynthesis, sulfate assimilation, and nitrogen metabolism in C4 plants (4, 20, and references therein). These and other comparisons of primary metabolism among tissues have clearly demonstrated the necessity for cooperation and coordination of biochemical processes in different cell types. The biochemical processes occurring in the epidermis of leaves are poorly understood; however, this tissue is particularly interesting in view of its sequestration and secretion of numerous secondary products. Recently, studies of the biosynthesis of some secondary metabolites have demonstrated portions of the relevant pathways to be localized in epidermal cells (14, 17-19, 23, 28, 30). Glycolysis and the oxidative pentose phosphate pathway are the major routes of glucose oxidation in plants, providing ATP, NADH, and NADPH to nonphotosynthetic cells in the light, 'Supported in part by National Science Foundation Grant PCM7903976 to Dr. P. K. Stumpf, National Science Foundation Grant PCM81-04497 and United States Public Health Service Grant GM-0530 1-25 to Dr. E. E. Conn. 2 Present address: NPI, 417 Wakara Way, Salt Lake City. UT 84108. 503

and to all cells in the dark (1, 27). To gain further insight into primary metabolism in epidermal tissues of leaves, as well as to determine the contribution each cell type may make to the overall rate of glucose oxidation, we studied the distribution of several enzymes involved in glucose oxidation among the different leaf tissues. We report here the activities of five glycolytic enzymes and two enzymes of the oxidative pentose phosphate pathway in the different leaf tissues of the C3-plants, pea and leek, and the C4-plants, sorghum and maize.

MATERIALS AND METHODS Chemicals and Buffers. Cellulysin and Macerase were purchased from Calbiochem; all other biochemicals were from Sigma. Extracts were prepared with a buffer consisting of 50 mM Tris-HCl (pH 8.0), with 1% (w/v) PVP-40, 10 mM 2-mercaptoethanol, and 0.01 % (v/v) Triton X- 100 (Tris buffer). Plant Material. Seeds of maize (Zea mays, var Golden Hybrid

Blend), pea (Pisum sativum, Argentum mutant) (14), and a sorghum ([Sorghum bicolor (L.) Moench] x sudan grass [S. sudanense (Piper) StapfJ hybrid [cv WAC Forage 99]) were soaked, planted, and allowed to germinate under conditions previously described (30). Sorghum seedlings were harvested 5.5 d after planting and were 3 to 7 cm tall with two expanded leaves. Maize seedlings were 7 d old at harvest. Fully expanded pea leaves were removed from 20 to 30 d old plants. Leeks (Allium porrum) were obtained from a local market; basal sections of etiolated inner leaves were used (22). Preparation of Leaf Tissues. Opposing pea leaves (8-20) were used to prepare the tissue and whole-leaf extracts. One leaf was frozen in liquid N. to be utilized for the whole leaf extract. From the opposing leaf, the upper and lower epidermises were separated from the mesophyll tissue, as previously described (14); all three tissues were weighed and frozen in liquid N2 after separation. Leaves which did not peel well (i.e. the mesophyll tissue adhered to the epidermis) were discarded. The epidermal tissue of leek leaves was separated from the mesophyll tissue (22, 23). The separated tissues were weighed and immediately frozen in liquid N2. All tissues were homogenized in Tris buffer (23), and low mol wt molecules were removed by passage through Sephadex G-25 which was equilibrated with the identical buffer (29). Recovery of proteins was >90%. The integrity and purity of tissues was assessed microscopically. Isolated epidermises from pea leaves were found to have no detectable mesophyll cell contamination when examined under a light microscope. Staining with Neutral Red revealed the majority of the epidermal cells to be intact. A similar examination of the epidermis from leek showed the majority of the epidermal cells to be intact, and to be minimally contaminated

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by adhering mesophyll cells. More than 95% of the RuBP3 carboxylase activity was associated with the mesophyll tissue, indicating minimal contamination of the epidermal tissue by mesophyll cells. Epidermal and Mesophyll Protoplasts, and Bundle Sheath Strands from Sorghum and Maize. Abraded sorghum seedling leaf blades (5-8 g fresh weight) (17), and transversely cut segments of the second leaves of maize seedlings (2-3 g fresh weight) (7) were digested enzymically to yield a mixture of mesophyll and epidermal protoplasts. Epidermal protoplasts were purified by a Ficoll density gradient (23, 29). Epidermal protoplasts were collected from the 5/7% interface. Yields ranged between 0.1 to 1.0 x 106 protoplasts. Mesophyll protoplasts of sorghum were recovered from 10/20% Ficoll interface. Yields ranged between 0.3 and 1.0 x 107 protoplasts. Bundle sheath strands were isolated by a sedimentation/flotation procedure (17). Yields ranged between 1.0 and 5.0 mg, and 0.7 and 6.0 mg of protein for sorghum and maize, respectively. Protoplasts were lysed with Tris buffer (23). Whole leaves and bundle sheath strand were frozen in liquid N2 and homogenized in Tris buffer (23). All samples were immediately passed through Sephadex G-25 columns equilibrated with Tris buffer to remove low mol wt molecules; recovery of proteins was >90% (23). Purity of the protoplast and bundle sheath preparations of maize and sorghum was monitored by microscopic examination and comparisons of marker enzymes and Chl (23, 29). Routinely, epidermal protoplast preparations were 60 to 80% pure, the only contaminant being mesophyll protoplasts. Microscopic examinations of the mesophyll protoplast preparations revealed less than 5% contamination by epidermal protoplasts. Monitoring of the RuBP carboxylase and NADP-malic enzyme activities of the mesophyll protoplasts revealed between 2 and 5% bundle sheath cell contamination. No epidermal or mesophyll cell contamination could be detected by microscopic examination of the bundle sheath strands. However, assays for PEP carboxylase activity revealed between 5 and 7% mesophyll and/or epidermal cell contamination of the bundle sheath strands. Sample Storage. After passage through Sephadex G-25, extracts were either used immediately or were frozen dropwise and stored in liquid N2 for future use. Under these conditions, enzyme activities were stable for at least 2 months. Assays. Prior to assaying, approximately 0.2 ml of each extract was thawed at 4°C and assayed within 4 h. P-glycerate kinase (25), P-fructokinase (10), triose-P isomerase (1 1), pyruvate kinase (15), glucose 6-P dehydrogenase (21), and 6-P-gluconate dehydrogenase (26) were assayed by spectrophotometric assays essentially as described previously. Aldolase was assayed at 24°C as the fructose 1,6-bisP-dependent increase in A340 due to the reduction of NAD+ in the presence of glyceraldehyde-3-P dehydrogenase. The reaction mixture was composed of 15 mm Tris-HCl (pH 8.0), 5 mM 2-mercaptoethanol, 4 mm sodium arsenate, 0.5 mm NADH, 7 mm fructose 1,6bisP, 10 units glyceraldehyde 3-P dehydrogenase, and 10 to 50 Al of enzyme extract, in a total volume of 1 ml. The blank rate of increase in A340 was obtained without the addition of fructose 1,6-bisP. The conditions under which all enzymes were assayed were optimized in relation to enzyme content and substrate concentrations. Furthermore, enzyme activities were proportional to enzyme content, and linear with respect to time. All enzyme activities were dependent on the presence of the substrates. All enzymes were assayed in two to six separate samples of each tissue; the data in the tables represents the mean of these determinations. 3 Abbreviations: RuBP. ribulose pyruvate.

1

,S-bisphosphate; PEP, phosphoenol

Plant Physiol. Vol. 82, 1986

Total protein content of the extracts was assayed by the Bradford dye binding method (3), using BSA as a standard. Chl was assayed by the method of Arnon (2). Enzyme activities in this publication are usually discussed on the basis of protein; however, we also present these activities, when appropriate, on the basis of Chl, fresh weight, leaf, and cell to enable the reader to make comparisons with previously published data.

RESULTS Tissue Separation and General Characterization. Epidermal tissue preparations of pea and leek contained both epidermal cells and guard cells; mesophyll tissue of these species contained both the mesophyll cells and vascular strands. The epidermis of pea contained low amounts of Chl in the guard cells. Chl levels in the epidermal tissue homogenates were too low for detection. The protein content of the upper plus lower epidermis comprised 3% (each) of the total leaf protein; 5 and 8%, respectively, of the leaf fresh weight was accounted for by these tissues (Table I). The section of the leek leaf used in these experiments was etiolated and thus contained minimal amounts of Chl. Each gram of epidermal tissue contained over 1.5 times as much protein as a gram of mesophyll tissue. However, the outer and inner epidermis contained only 8 and 5.5%, respectively, of the total leaf protein. The epidermal protoplast preparations from sorghum and maize did not contain Chl. The protein content of these protoplasts were similar for both species (Table II). Mesophyll protoplasts of maize had nearly twice the content of Chl and protein as the mesophyll protoplasts from sorghum. The Chl:protein ratio in the mesophyll cells from both plants was similar; however, the sorghum bundle sheath strands showed a lower Chl:protein ratio than the strands from maize. The distribution of Chl and protein among the leaf tissues of sorghum was calculated (29). The mesophyll protoplasts contained 75% of the Chl and nearly 66% of the total protein of the leaf. The remainder of the Chl was in the bundle sheath cells. Epidermal protoplasts contained no Chl and accounted for only 6% of the total leaf protein. The distribution of protein between the leaf tissues of sorghum was very similar to that found by Kojima et al. (17). Enzymes of Glycolysis in Leaf Tissues of C3-Plants. P-Fructokinase and pyruvate kinase are probably the two regulatory, rate limiting enzymes of glycolysis (27). The specific activities (on the basis of protein or fresh weight) of these enzymes were among the lowest of the five glycolytic enzymes examined in pea and leek leaves (Tables III and IV, respectively). The specific activity of P-fructokinase was higher in the epidermal tissues than in the mesophyll tissue of pea. The difference Table I. Protein, Chl Content and Fresh Weight of Leaf Tissues of Pea and Leek

Tissue

Chl

mg/g tissue

Leek Inner epidermis Outer epidermis

Mesophyll Whole leaf

2.26 1.32

Fresh wt

mg/g mg/ mg/g mg/g mg/g mg tissue leaf leaf leaf protein

Pea Upper epidermis Lower epidermis

Mesophyll Whole leaf

Protein

1.70 1.59

17.3 10.3 0.074 30.9 0.059 27.1 5.15 5.12 3.63 5.19

47 0.82 0.73 70 23.2 750 27.1 1000

0.26 51 0.35 68 3.96 917 5.19 1000

ENZYMES OF GLUCOSE OXIDATION IN LEAVES OF C3 AND C4 PLANTS Table II. Protein and Chl Content in Leaf Tissuies ofSorghum and Maize Protein Chl Tissue ,Ug/lO6 mg/g cells leaf

Sorghuma Epidermal protoplasts Mesophyll protoplasts Bundle sheath strands Whole leaf

0 55.8

1.39 0.39 1.80

mg/ mg

protein 0.175 0.091 0.143

AgI106 mg/g

cells

leaf

236 320

0.85 7.9 4.3 12.6

Maize a 242 0 Epidermal protoplasts 0.185 500 93.3 Mesophyll protoplasts 0.133 Bundle sheath strands 11.7 1.14 0.097 Whole leaf a Distributions between mesophyll and epidermal protoplasts have been corrected for cross-contamination (24).

in the specific activity of P-fructokinase in the pea leaf tissues is also reflected in the tissue distribution of this enzyme; namely, 39% of the total leaf activity is associated with the epidermis. In leek leaves, P-fructokinase specific activity was similar in the three tissues, with 9% of the total leaf activity in the epidermis. In pea, the specific activity of aldolase was slightly greater in the epidermal tissues than in mesophyll tissues (Table III). In leek, aldolase activity was lower than those of any other glycolytic enzymes examined and its specific activity was similar in all tissues (Table IV). The other enzymes of the glycolytic pathway which were examined showed specific activities somewhat greater in the mesophyll tissue than in the epidermal tissues of pea (Table III). In leek leaves, however, the specific activities of P-glycerate kinase and triose-P isomerase were similar in both mesophyll and epidermal tissues (Table IV). Enzymes of the Oxidative Pentose Phosphate Pathway in Leaf Tissues of C3-Plants. Glucose 6-P dehydrogenase and 6-P-gluconate dehydrogenase are two enzymes exclusive to the oxidative pentose phosphate pathway. They catalyse the first two reactions which are rate limiting in this pathway, and their activities are an indication of the maximal capacity of this pathway (27). The specific activities of these two enzymes were similar in the mesophyll tissue of pea (Table V). However, in the epidermis, the specific activity of 6-P-gluconate dehydrogenase was 2- to 3fold higher than that of glucose 6-P dehydrogenase. The epidermis contained up to 12-fold more 6-P-gluconate dehydrogenase activity than the mesophyll tissue. The specific activity ofglucose 6-P dehydrogenase in the epidermis was 2-fold greater than that found in the mesophyll tissue. An examination ofthe distribution of these enzymes over the entire leaf showed 12% of the glucose 6-P dehydrogenase and 36% of the 6-P-gluconate dehydrogenase activities were associated with the epidermis. In leek leaves, the specific activities of glucose 6-P dehydrogenase and 6-P-gluconate dehydrogenase were slightly higher in the epidermis than in the mesophyll tissue (Table VI). The epidermal tissue contained 16% of the total leaf activity of both dehydrogenases.

Enzymes of Glycolysis in Leaf Tissues of C4-Plants. In the leaves and leaf tissues of the C4-plants sorghum (Table VII) and maize (Table VIII), the specific activities of phosphofructokinase and pyruvate kinase were the lowest of all the glycolytic enzymes examined, as would be expected for rate-limiting enzymes. The exception was for mesophyll protoplasts, which contained no detectable aldolase activity. The absence of aldolase from the mesophyll cells would preclude the full operation of glycolysis in

505

this tissue of C4-leaves. In sorghum, all the enzymes of glycolysis that were examined, apart from aldolase, showed 4- to 20-fold higher activities in the epidermal cells than in the mesophyll cells (Table VII). This was true whether the comparison was made on a cell or protein basis. The specific activities ofthe enzymes in the bundle sheath strands were less than those in the epidermal cells. The epidermal cells of sorghum contain only 6% of the leaf protein, yet over 34% of the leaf activities of phosphofructokinase and pyruvate kinase were located in these cells. Of the total leaf aldolase and Pglycerate kinase activities, over 22% was found in the epidermal cells. Although only three glycolytic enzymes were examined in maize leaves, the differences from sorghum leaves were quite evident (Table VIII). As in sorghum, aldolase activity was absent from mesophyll cells of maize, but, unlike sorghum, bundle sheath cells of maize showed a 17-fold higher activity of this enzyme than epidermal cells. Pyruvate kinase activity was similar in mesophyll and bundle sheath cells, but was 8-fold higher in epidermal cells. Although the distribution of these enzymes among the leaf tissues of maize could not be calculated, comparisons of the specific activities with those found in sorghum suggest there are differences in the distribution of these enzymes between the two leaves. Enzymes of the Oxidative Pentose Phosphate Pathway in Leaf Tissues of C4-Plants. The specific activity of glucose 6-P dehydrogenase in sorghum epidermal cells was 7- and 9-fold higher than in the mesophyll and bundle sheath cells, respectively; the distribution of this enzyme among these three leaf tissues was 34, 45, and 20%, respectively (Table IX). The specific activity of 6-P-gluconate dehydrogenase was also highest in the epidermal cells. The distribution of 6-P-gluconate dehydrogenase among the leaf tissues of sorghum was similar to that found for glucose 6-P dehydrogenase. Glucose 6-P dehydrogenase was the only enzyme of the oxidative pentose phosphate pathway that was examined in the leaves of maize (Table VIII). Like sorghum, the specific activity of this enzyme was highest in the epidermal cells, 3- and 5-fold higher than in the mesophyll and bundle sheath cells, respectively. DISCUSSION Epidermal cells (including epidermal cell modifications such as trichomes, leafhairs, and guard cells) are known to accumulate or secrete high concentrations of a wide variety of natural products and other components. These include, for example, nectars composed of sugars and amino acids, mono- and sesquiterpenes, cellulose, salts (9), cuticular waxes (19), flavonoids (14), and dhurrin (17). The sites of synthesis of most of these natural products have not yet been determined. In some cases, however, there is increasing evidence to indicate that epidermal cells have the ability to synthesize the natural products which they sequester. For example, in leaves of pea, enzymes of flavonoid biosynthesis are exclusively localized in the epidermis ( 14). The biosynthesis of cuticular waxes has also been demonstrated to be in the epidermis of leaves (19). UDP-glucose aldehyde cyanohydrin figlucosyl transferase, the final enzyme of dhurrin biosynthesis, is localized predominantly in the epidermal cells of sorghum (17, 30). Apart from studies to elucidate stomatal function, very little emphasis has been placed on the study of primary metabolism in epidermal tissues and cells. In contrast, many enzymes of primary metabolism, and in particular enzymes of glucose oxidation (4, 13, 20 and references therein), have been compared in detail in mesophyll and bundle sheath tissues. In this study, we have examined the activities of the enzymes involved in glucose oxidation in leaf tissues of pea, leek, sorghum, and maize, with

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Plant Physiol. Vol. 82, 1986

Table III. Activities ofthe Enzymes of Glycolysis in Extracts from Leaf Tissues of Pea Recoveries of the enzyme activities in the separated tissues relative to the whole leaf were as follows: Pfructokinase, 82%; pyruvate kinase, 119%; aldolase, 102%; P-glycerate kinase, 74%; triose-P isomerase, 100%. Leaf Tissue Enzyme Units Units Lower Enzyme eUpper Leaf Mesophyll Mspyl La epidermis epidermis P-fructokinase nmol/min * mg 335 487 42.3 72.9 protein nmol/min-g 5800 5020 1310 2000 tissue % of total leaf 17.0 22.0 60.9 activity

~~~~~~~~~~~Whole

Pyruvate kinase

nmol/min-mg protein

nmol/min.g tissue % of total leaf activity

Aldolase

P-glycerate kinase

144

357

2490

3670

4.6

10.2

93.5

2890

78.5 2130

85.2

nmol/min * mg protein nmol/min.g tissue % oftotal leaf activity

255

224

177

162

4400

2310

5460

4380

nmol/min.mg

1880

1970

3310

3980

protein nmol/min-g tissue % of total leaf

32500

20300

1.02 x I0

1.80 x 105

4.7

1.9

3.6

1.8

91.7

96.0

activity Triose-P isomerase

nmol/min.mg protein nmol/min.g tissue % of total leaf activity

14100

11800

13400

12200

2.44 x 105

1.21 x 105

4.15 x 105

3.31 x 105

3.5

particular emphasis on the activities of these enzymes in the epidermis. The activities of the rate limiting enzymes of glycolysis and of the oxidative pentose phosphate pathway are interpreted as indicating the maximal capacity of carbon flux through these pathways. However, this interpretation is to some extent a simplification as these enzymes exist as isozymes (8, 13, 27) which are located in different subcellular compartments. Furthermore, a substantial proportion of the activities of aldolase, triose-P isomerase, and phosphoglycerate kinase in leaf extracts originate from chloroplasts where they are preferentially utilized in the Calvin-Benson cycle of photosynthesis (1, 27). All enzymes of primary metabolism examined were present in the epidermal tissues. Comparisons of activities in the separated tissues and in the whole leaf were made to check for possible loss of activity associated with tissue preparation. In pea and leek, the sums of the activities in each tissue were similar to the activities found in the whole leaf extract, and we are confident that the observed specific activities of these enzymes are not distorted by the preferential inactivation of enzyme activity in one or more of the leaf tissues. For sorghum, the sum of the activities in the tissue extracts were between 35 and 80% of those for the total leaf extract. This discrepancy is probably attributable to a loss of activity during protoplast preparation. Thus, the results may be an underestimation of the in vivo specific activity

2.6

93.9

in each cell type. We could not quantitate the recovery of activities for maize; however, the specific activities in the separated tissues were similar to those in the whole leaf extracts, suggesting a high recovery. In the two C4-plants examined, mesophyll cells did not contain detectable aldolase; similarly, only very low levels of adolase have been previously demonstrated in two other C4-plants (4). A lack of aldolase precludes the operation of glycolysis in mesophyll cells of these C4 species. Possibly to compensate for the lack of glycolysis in mesophyll tissue, the epidermal and bundle sheath tissues of sorghum have a higher capacity for glycolysis compared to the oxidative pentose phosphate pathway, as judged by the specific activities of the rate limiting enzymes in these tissues. The limited data for maize similarly suggests that epidermal and bundle sheath tissue have the capacity for greater glucose oxidation via glycolysis than the oxidative pentose phosphate pathway. In pea, sorghum, and maize, the specific activities of glucose 6-P dehydrogenase, P-fructokinase, and pyruvate kinase were 2to 10-fold higher than the corresponding activities in cells of the other tissues. This may reflect the requirements by epidermal cells for reducing equivalents and ATP which cannot be obtained by photosynthesis in these cells. The NADPH, NADH, and ATP generated by oxidation of glucose could be for general cell maintenance, and for the synthesis of such secondary metabolites

ENZYMES OF GLUCOSE OXIDATION IN LEAVES OF C3 AND C4 PLANTS Table IV. Activities of the Enzymes ofGlycolysis in Extracts from Leaf Tissues of Leek Recoveries of the enzyme activities in the separated tissues relative to the whole leaf were as follows: Pfructokinase, 122%; pyruvate kinase 89%; aldolase, 98%; P-glycerate kinase, 110%; triose-P isomerase, 80%. Leaf Tissue Whole Enzyme Units Inner Outer leaf Mesophyll epidermis epidermis 602 591 547 874 P-fructokinase nmol/min * mg protein nmol/min .g tissue % of total leaf activity

Pyruvate kinase

Aldolase

P-glycerate kinase

Triose-P isomerase

nmol/min * mg protein nmol/min .g tissue % of total leaf activity

nmol/min * mg protein nmol/min *g tissue % of total leaf activity nmol/min * mg protein nmol/min .g tissue % of total leaf activity nmol/min * mg protein nmol/min *g tissue % of total leaf activity

5.1

4.0 90.0 464

3310

3170

2800

3040

90.9

126

123

120

645

446

660

4.2

8.1

87.8

34.5

33.6

47.6

173

172

178 4.2

5.7

40.9

225

90.0

3170

1790

3350

2580

16300

9160

12200

14200

4.3

5.5

90.2

3760

4690

4350

4760

19400

24000

15800

26200

4.8

8.4

86.8

Table V. Activities of the Enzymes of the Oxidative Pentose Phosphate Pathway in Extracts from Leaf Tissues of Pea Recoveries of the enzyme activities in the separated tissues relative to the whole leaf were as follows: glucose 6-P dehydrogenase, 93%; 6-P-gluconate dehydrogenase, 89%. Leaf Tissue Whole Units Enzyme Upper Lower leaf epidermis epidermis Mesophyll Glucose 6-P dehydrogenase 95.1 100 44.8 47.3 nmol/min-mg protein 1650 nmol/min.g 1030 1380 1280 tissue % of total leaf 6.5 6.1 87.4 activity 6-P-gluconate dehydrogenase

nmol/min-mg protein nmol/min.g tissue % of total leaf activity

160

366

2770

3770

11.8

24.0

30.6

945 64.1

45.8 1240

507

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Plant Physiol. Vol. 82, 1986

Table VI. Activities of the Enzvmes of the Oxidative Pentose Phosphate Pathway in Extracts from Leaf Tissues of Leek Recoveries of the enzyme activities in the separated tissues relative to the whole leaf were as follows: glucose 6-P dehydrogenase, 148%: 6-P-gluconate dehydrogenase, 85%. Enzyme Enzvme Units

nmol/min-mg protein nmol/min g tissue % of total leaf activity

Glucose 6-P dehydrogenase

-

6-P-gluconate dehydrogenase

nmol/min - mg protein nmol/min-g tissue % of total leaf activity

Inner epidermis 364

1880 6.9

Leaf Tissue Outer epidermis 335 1710 8.7

Mesophyll

leaf ~~~~~~~~~~Whole

M

288

175

1050

965

84.4

266

376

262

280

1370

1930

951

1540

5.5

10.8

83.7

Table VII. Activities of t/e Enzymes ofGlycolvsis in Extracts from Leaf Tissues ofSorghum Recoveries of enzyme activities in the separated tissues relative to the whole leaf were are as follows: phosphofructokinase. 61%: pyruvate kinase, 49%; aldolase, 35%; P-glycerate kinase, 57%; triose-P isomerase, 72%. Leaf Tissue Enzyme

Units

P-fructokinase

Pyruvate kinase

nmol/min. 106 cells nmol/min -mg protein

26.4 112

nmol/min * mg Chl % of total leaf activity

39.0

nmol/min- 106 cells nmol/min-mg protein

26.4 112

nmol/min mg Chl % of total leaf activity

34.0

-

Aldolase

nmol/min. 106 cells nmol/min-mg protein nmol/min mg Chl % of total leaf activity

Bundle sheath strands

Whole

1.24 3.87 22.1 12.0

27.1 297 48.0

31.8 222

3.61 11.3 64.4 32.0

21.4 236 33.0

44.9 314

70.4 493

Mesophyll Epidermal Epideral Mesphyll

protoplastsa

protoplastsa

22.7 96.3

N.D.b N.D.

26.0

0

52.7 580 74.0

27.7 86.7 494 13.0

752 8280 63.0

-

P-glycerate kinase

nmol/min- 106 cells nmol/min-mg protein nmol/min * mg Chi % of total leaf activity

316 1340 22.0

leaf

703 4920

624 2520 nmol/min- 106 cells 314 2860 1950 10700 nmol/min-mg protein 20000 3460 11100 nmol/min-mg Chl 5.2 59.0 35.0 % of total leaf activity a Distributions between mesophyll and epidermal protoplasts have been corrected for cross-contamination b Not detected. (24).

Triose-P isomerase

flavonoid, cuticular waxes, and dhurrin which are known to be synthesized in this tissue. Few previous studies of the distribution of the enzymes of glucose oxidation in leaf tissues have included epidermal tissues. Wurtele et al. (30) have previously established a higher specific activity of triose-P isomerase in epidermal protoplasts than mesophyll protoplasts from sorghum, and have evidence for both a cytoplasmic and plastid localization for this enzyme in epideras

mal cells. Hampp et al. (12), reported an approximately 2-fold higher specific activity of 6-P-gluconate dehydrogenase in epidermal cells compared to mesophyll cells of Viciafaba. Kojima and Conn (18) reported a distribution of glucose 6-P dehydrogenase among the leaf tissues of sorghum similar to that found in this study. Both glucose 6-P dehydrogenase (6) and phosphofructokinase (5) were detected in epidermal cells from young leaves of Dianthus; the activities of these two enzymes were

ENZYMES OF GLUCOSE OXIDATION IN LEAVES OF C3 AND C4 PLANTS Table VIII. Activities of the Enzymes of Glutcose Oxidation in Extracts from Leaf Tissues ofMaize Leaf Tissue Enzyme

Pyruvate kinase

Units

nmol/min- 106

Epidermal

Mesophyll

protoplastsa

protoplastsa

64.1

Bundle strands

Whole leaf

32.0

51.9

15.9

cells

nmol/min*mg protein nmol/min*mg Chl

nmol/min. 106

Aldolase

cells nmol/min*mg protein nmol/min-mg Chl

Triose-P isomerase

nmol/min. 106 cells nmol/min * mg protein nmol/min-mg Chi

31.8

265

172

5.83 24.1

535

240

N.D.b N.D.

1860

3670

7690

7340

412

102

3090

1050

6450

9060

4.84 x 105

39600

1.06 x 105

7.04 4.83 nmol/min. 106 cells 6.12 29.1 9.66 9.33 nmol/min*mg protein 52.2 45.9 96.1 nmol/min*mg Chl a Distributions between mesophyll and epidermal protoplasts have been corrected for cross-contamination b Not detected. (24).

Glucose 6-P dehydrogenase

Table IX. Activities ofthe Enzymes of the Oxidative Pentose Phosphate Pathway in Extracts from Leaf Tissues of Sorghum Recoveries of the enzyme activities in the separated tissues relative to the whole leaf were as follows: glucose 6-P dehydrogenase, 40%; 6-P-gluconate dehydrogenase, 80%. Leaf Tissue Enzyme

Units

Epidermal Epiderma

Mesophyll protophlass

protoplasts'protoplastsa Glucose 6-P dehydrogenase

nmol/min. 106 cells nmol/min * mg protein nmol/min * mg Chl % of total leaf activity

6-P-gluconate dehydrogenase

nmol/min- 106 cells

11.8

2.27

50.0

7.09

34.0 25.3

Bundle sheath strands

Whole

5.87

24.6

40.4

64.6

45.0

20.0

Leaf

172

8.23

107.2 46.8 25.7 42.0 nmol/min*mg protein 147 327 462 nmol/min * mg Chl % of total leaf 19.0 43.0 38.0 activity a Distributions between mesophyll and epidermal protoplasts have been corrected for cross-contamination (24).

509

510

WURTELE AND NIKOLAU

similar in epidermal and mesophyll cells. This study and others demonstrate that there is tremendous variation in the distribution of the enzymes of glucose oxidation among the leaf tissues. This could be due to variation between species, environmental conditions of growth, or the developmental stage of the leaf. Examples of such variations are the studies by Croxdale (5) and Croxdale and Outlaw (6) of the distribution of phosphofructokinase and glucose 6-P dehydrogenase in apical meristem and early primordia of leaves of the C3dicot Dianthus chinensis L. Whereas the capacity to oxidize glucose via glycolysis was greater in the apical meristem and youngest leaf primordia, the capacity of the pentose phosphate pathway exceeded that of glycolysis in the young leaves. Similarly, an elevated capacity of the pentose phosphate pathway is seen in developing leek leaves; in the epidermal and mesophyll tissues of these leaves, the potential to oxidize carbohydrates via the pentose phosphate pathway is greater than for glycolysis (Tables IV and VI). It is becoming apparent that the biosynthesis of secondary metabolites may occur only in certain specialized cell types. The high capacity of the epidermal cells for carbohydrate oxidation via glycolysis and the oxidative pentose phosphate pathway may in part be necessary to provide an immediate source of energy for the biosynthesis of natural products in the epidermal cells. Acknowiledgments-We

are

grateful for the support of Dr. Eric E. Conn and Dr.

Paul K. Stumpf, in whose laboratories this work was carried out. We thank Dr. G. A. Marx, New York State Agricultural Experiment Station, New York, for the P. sativuin argentum mutant seeds.

LITERATURE CITED

APREES

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jB-cyanoal-

,3-glucosyl