Carbohydrate Metabolism in the Developing

Plant Cell Physiol. 30(6): 833-839 (1989) JSPP © 1989

Carbohydrate Metabolism in the Developing Endosperm of Rice Grains Yasunori Nakamura, Kazuhiro Yuki1, Shin-Young Park and Toshihide Ohya 2 Laboratory of Environmental Stress Physiology, National Institute of Agrobiological Resources, Tsukuba Science City, Ibaraki, 305 Japan

Key words: ADPglucose pyrophosphorylase — Amyloplast — Endosperm — Q-enzyme — Rice (Oryza sativa L.) — Starch metabolism.

Accumulation of starch in storage tissue is the end process of photosynthesis in some plants. The photosynthetically fixed carbon in green leaves is translocated in the form of sucrose to sink organs, such as the endosperm in cereals, where sucrose is efficiently converted to the end product, starch. Although recently the sites and mechanisms of regulation of carbohydrate metabolism have been elucidated in detail in photosynthetic tissues (Stitt et al. 1987), only limited information has been reported with respect to carbohydrate storage tissues. It is known that enzymes responsible for the biosynthesis of starch are localized in amyloplasts in the endosperm (Echeverria et al. Abbreviations: PP-PFP, pyrophosphate:fructose-6-phosphate 1-phosphotransferase; ATP-PFK, ATP-dependent phosphofructokinase; FBPase, fructose-1,6-bisphosphatase; G1P, glucose-1-phosphate; FBP, fructose-l,6-bisphosphate; G6P, glucose-6-phosphate; RuBP, ribulose-l,5-bisphosphate; PEP, phosphoenolpyruvate; F26BP, fructose-2,6-bisphosphate; DHAP, dihydroxyacetone phosphate. 1 Present address: Yamagata Prefectural Agricultural Experiment Station, Minorigaoka, Yamagata, 990-02 Japan. 2 Present address: Institute of Biological Sciences, The University of Tsukuba, Tsukuba Science City, Ibaraki, 305 Japan.

1988). However, it remains to be determined how sucrose is degraded, what compound is transported from the cytoplasm into amyloplasts through the amyloplast envelopes, how the energy-producing system supports the energy-requiring (ATP-requiring) process of starch synthesis, and how the synthesis of starch is biochemically and enzymically regulated. In this study, carbohydrate metabolism in developing rice endosperm was characterized by a comparison of functionally distinct enzymes in the endosperm with those in green leaves, and by measuring the levels of metabolites involved in carbohydrate metabolism. In addition, localization of key enzymes involved in the metabolism of sucrose and starch was examined with amyloplasts isolated from developing rice endosperm. Materials and Methods Plant material—Rice plants {Oryza sativa cv. Fujihikari) were grown in pots in a greenhouse at 27°C by day and 22°C by night in natural daylight from May to August. Isolation of amyloplasts—Rice kernels were harvested, about six days after pollination, at the early stage of development. The following procedures were perform833

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The metabolism of carbohydrates in developing rice endosperm was characterized by a comparison of levels of activities of 33 major enzymes between the endosperm and green leaves of rice. Activities of ADPglucose pyrophosphorylase, starch synthase and branching enzyme (Q-enzyme), compared on the basis of soluble protein content, were markedly higher in endosperm than in green leaves. The high levels of Q-enzyme may be responsible for the efficient production of starch in the rice endosperm. The measurement of levels of metabolic intermediates and the localization of key enzymes in isolated amyloplasts from rice endosperm support the view that sucrose is metabolized in the cytoplasm via the pathway: sucrose—•UDPglucose-*hexose-P-»-FBP -Hriose-P. Triose-P then enters the amyloplast, where it is converted to G1P via FBP and, finally, G1P is converted to starch by the concerted reactions of ADPglucose pyrophosphorylase, starch synthase and Q-enzyme.

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Y. Nakamura, K. Yuki, S.-Y. Park and T. Ohya activity was assayed by measuring the increase in absorbance at 340 nm after additon of 1 //I each of P-glucomutase (0.4 unit) and G6P dehydrogenase (0.35 unit). Starch synthase (EC2.4.1.21)—The assay was conducted in 50 mM HEPES-NaOH (pH7.4), 1.6 mM ADPglucose, 0.7 mg amylopectin, 15 mM DTT, and enzyme preparation in a reaction mixture of 280/yl. Twenty min after the start of the reaction, the enzyme was inactivated by placing the mixture in a boiling-water bath for 30 s. Then the mixture was added by 100//I of a solution of 50 mM HEPES-NaOH (pH7.4), 4mM PEP, 200 mM KC1, 10 mM MgCl2, and pyruvate kinase (1.2 unit), and incubated for 30 min at 30°C. The ADP produced by the starch synthase reaction was converted to ATP and the resulting solution was heated in a boiling-water bath for 30 s and then subjected to centrifugation at 10,000xg for 5min. The supernatant (300//I) was mixed with a solution of 50 mM HEPES-NaOH (pH7.4), 10 mM glucose, 20 mM MgCl2, and 2 mM NADP. The enzymic activity was measured as the increase in absorbance of 340 nm after the addition of 1 /A each of hexokinase (1.4 unit) and G6P dehydrogenase (0.35 unit). Branching enzyme (Q-enzyme) (EC 2.4.1.18)—The assay was conducted in 50 mM HEPES-NaOH (pH7.4), 5 mM G1P, 1.25 mM AMP, phosphorylase a (54 unit), and enzyme preparation in a reaction mixture of 200 /A. The reaction was terminated by addition of 50//1 of 1 N HC1. The solution was mixed with "500 /A of dimethylsulfoxide and 700(A of 0.1% I2 and \% KI were added. The enzymic activity was assayed spectrophotometrically at 540 nm. One unit of enzymic activity was defined as the amount causing an increase in absorbance of one unit at 540 nm in one min. FBPase (EC 3.1.3.11)—For the enzyme from rice endosperm, the reaction was carried out in 50 mM HEPESNaOH (pH 7.4), 1 mM FBP, 4 mM DTT, 8 mM MgCl2, 1 mM NADP, P-glucose isomerase (0.7 unit), G6P dehydrogenase (0.35 unit), and enzyme preparation in a volume of 500 /A. The chloroplast and cytoplasmic enzymes from leaves were assayed by the method of Kelly et al. (1982). The other enzymic activities were assayed essentially according to the references cited: debranching enzyme (Renzyme) (EC 3.2.1.70) and amylase (EC 3.2.1.1) (Nelson 1944, Somogyi 1952); sucrose-P synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) (Kerr et al. 1984); Cyt c oxidase (EC 1.9.3.1) and catalase (EC 1.11.1.6) (MacDonald and ap Rees 1983a). The following enzymes were measured by monitoring the change in absorbance of NADPH or NADH at 340 nm in an assay mixture of 500 fA in a Beckman DU-70 spectrophotometer, unless otherwise stated. The enzymic activity was calculated from the linear portion of the curve of changes in absorbance. UDPglucose pyrophosphorylase (EC 2.7.7.9)—The


ed at 0-4°C. Three hundred dehulled grains were carefully disrupted with forceps with sharp tips and gently agitated in 13 ml of the suspension medium, which contained 30 mM HEPES-KOH (pH7.4), 0.5 M sorbitol, 50 mM KC1, 5mM Na-ascorbate, and 16 mM 2-mercaptoethanol. As a result of this procedure, cells in the milky endosperm were spontaneously but completely disrupted. The suspension was filtered through a nylon net (diameter of pores, 40 fum) and centrifuged on 30 ml of a sorbitol density gradient at 5.3 x g (200 rpm) for 60 min. The linear density gradient consisted of 0.6-1.2 M sorbitol, 50 mM KC1, 16 mM 2-mercaptoethanol, and 5 mM Na-ascorbate and was prepared by using a plastic gradient-maker and pump. The amyloplastrich fraction near the bottom of the centrifugation tube was resuspended in the above mentioned HEPES buffer, with the omission of sorbitol, in a final volume of 0.54 ml. Preparation of enzyme extract—All the procedures were carried out at 0-2°C. For the assays of leaf enzymes, leaf blades (1 g fresh weight) of 20-day-old seedlings were harvested on a clear day, sliced into segments and homogenized with a pestle in an ice-cold mortar that contained 10 ml of 100 mM Tricine-NaOH (pH 8.0), 8mM MgCl2, 2mM EDTA, 50 mM 2-mercaptoethanol, 12.5% (v/v) glycerol, and 5% (w/v) insoluble polyvinylpyrrolidone40. The homogenate was centrifuged at 10,000 xg for 5 min, and the resulting supernatant was used as the preparation of enzymes, unless otherwise stated. For assays of Cyt c oxidase and glycolate oxidase, the supernatant obtained after centrifugation at 1,000 x g for 4 min was used as the preparation of enzymes. For the assays of endosperm enzymes, 25 dehulled grains harvested about 10 days after pollination, at the milky stage, were separated from embryo and pericarp, homogenized with 5 ml of the buffer solution, and the preparations of enzymes were obtained in the same way as described above. Enzyme assays—All assays were carried out at 30° C, in the various reaction mixtures described below. Assays were conducted in the range of concentrations of enzyme where the activity increased linearly with increases in the amount of enzyme preparation and the reaction time. The background values were routinely taken as the activities detected with a reaction time of zero (the enzymes were denatured immediately after their addition to the reaction mixtures). ADPglucose pyrophosphorylase (EC 2.7.7.27)—-The assay was conducted in 100 mM HEPES-NaOH (pH 7.4), 1.2 mM ADPglucose, 3 mM PPj, 5 mM MgCl2, 4mM DTT, and enzyme preparation in a reaction mixture of 650^/1. After 20 min, the reaction was terminated by heating the mixture in boiling water for 30 s. The resulting solution was transferred to an Eppendorf tube and centrifuged at 10,000xg for 10min. A portion (500//I) of the supernatant was taken and mixed with 15 //I of 10 mM NADP. The

Carbohydrate metabolism in rice endosperm

Results Comparison of enzymic activities between developing

endosperm and green leaves in rice—To characterize the metabolism of carbohydrates in non-photosynthetic storage tissue as well as in photosynthetic tissue, the major enzymes were assayed in the developing endosperm and green leaves of rice, as shown in Table 1. On the basis of soluble protein content, activities of ADPglucose pyrophosphorylase and starch synthase, both involved in the predominant biosynthetic pathway to starch (Preiss and Levi 1980), were several-fold higher in endosperm than in leaves. It is noted that the specific activity of the branching enzyme (Q-enzyme), the other key enzyme in starch synthesis, was very much higher in endosperm than that of either of the above two enzymes. The activities of glucan phosphorylase, debanching enzyme (R-enzyme), and aamylase, the major enzymes catalyzing the degradation of starch, were relatively higher in the starch storage tissue. RuBP carboxylase was detected in endosperm although the activity was markedly lower than in green leaves. In contrast, P-glycerate kinase and glyceraldehyde-3-P dehydrogenase, both participants in the 3-P-glycerate reduction pathway in the Calvin cycle, were present at adequate levels in endosperm. The activity of malate dehydrogenase was also high in rice endosperm, while activities of PEP carboxylase, malic enzyme and pyruvate kinase were comparatively low in the storage tissue. Of particular interest is the fact that almost all of the activity of glyceraldehyde-3-P dehydrogenase and malate dehydrogenase was attributable to NAD-type enzymes in the endosperm, while in the green tissue NADP-glyceraldehyde3-P dehydrogenase was detected at the same level as the NAD-type enzyme. In green leaves, the activity of ATP-PEK was higher than that of PP r PFP. In contrast, the P P r P F P activity was markedly higher than the ATP-PFK activity in the storage tissue. P P r P F P was efficiently activated by fructose-2,6-bisphosphate (F26BP) in both tissues, while F26BP had no effect or a slightly inhibitory effect on ATPPFK activities. The specific activity of FBPase in the endosperm was found to be approximately the same as that of the chloroplast enzyme. The activity of the enzyme from rice endosperm was not affected by F26BP (data not shown). This result suggests that the endosperm FBPase was located in the amyloplast, since the cytoplasmic FBPase, but not the chloroplast enzyme, from green leaves has been reported to be inhibited by F26BP (Stitt et al. 1987). The activities of the other enzymes that catalyze hexose-P metabolism were higher in rice endosperm, with the single exception that the activity of G6P dehydrogenase was low as compared to that in leaves. A high level of activity of UDPglucose pyrophosphorylase was observed in endosperm as well as in leaves. Sucrose-P synthase, a key enzyme in the biosynthesis of sucrose in leaves (Huber et al. 1985), was not detected in the


assay was conducted in 100 mM HEPES-NaOH (pH7.4), 1 mM UDPglucose, 1 mM Na-PPj, 4.8 mM MgCl2> 0.8 mM NADP, P-glucomutase (0.4 unit), G6P dehydrogenase (0.35 mM), and enzyme preparation. For measurement of the enzymic activities with preparations of amyloplasts, a two-step assay method was used. The enzymic reaction was run for 20min in a reaction volume of 650 /A, but NADP, P-glucomutase and G6P dehydrogenase were omitted from the above reaction mixture. Then the activity was assayed by measuring the amount of G1P formed by the same procedure as in the assay for ADPglucose pyrophosphorylase. The other enzymic activities were assayed by the methods in the references cited: phosphorylase (EC 2.4.1.1) (Nakamura and Imamura 1983); RuBP carboxylase (EC 4.1.1.39) (Usuda 1985); P-glycerate kinase (EC 2.7.2.3), NAD-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12), NAD-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13), PEP carboxylase (EC 4.1.1.31), NAD-malate dehydrogenase (EC 1.1.1.37), NADP-malate dehydrogenase (EC 1.1.1.82), NAD-malic enzyme (EC 1.1.1.38), NADP-malic enzyme (EC 1.1.1.38) and pyruvate kinase (EC 2.7.1.40) (Winter et al. 1982); ATP-PFK (EC 2.7.1.11) and P P r P F P (EC 2.7.1.90) (Kombrink et al. 1984); FBP aldolase (EC 4.1.2.13), P-glucose isomerase (EC 5.3.1.9), P-glucomutase (EC 2.7.5.1), G6P dehydrogenase (EC 1.1.1.49), glucokinase (EC 2.7.1.1), fructokinase (EC 2.7.1.4) and invertase (EC 3.2.1.26) (MacDonald and ap Rees 1983a). Analysis of metabolites—A sample of 110 rice kernels, about 10 days after pollination, were harvested during the daytime on a clear day and quickly frozen in liquid nitrogen. The endosperm was dehulled, removed from the embryo and homogenized with a mortar and pestle in 10 ml of 3% perchloric acid on ice. The homogenate was mixed with 0.2 g of activated charcoal and allowed to stand for 15 min. The mixture was neutralized with 0.98 ml of 2.5 M K2CO3 to approximately pH7.0 and centrifuged at 10,000 x g for 5 min. The supernatant was immediately used for the measurements of metabolites by the coupledenzyme assay method of Usuda (1985) in a Shimadzu UV150-02 spectrophotometer. Protein determination—An aliquot of the sample to be assayed was mixed with trichloracetic acid at a final concentration of 10%. The mixture was centrifuged at 10,000 x g for 10 min and the pellet was washed with 1 ml of 10% trichloracetic acid. The protein was redissolved in 1 ml of 2% sodium dodecyl sulfate in 0.1 N NaOH at 40°C for 1 h and the protein content was measured by the method of Lowry et al. (1951).

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Y. Nakamura, K. Yuki, S.-Y. Park and T. Ohya

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Table 1 Activities of enzymes in endosperm and leaf of rice (cv. Fujihikari) Enzyme

325

58.2

17.8 4,540° 56.1 462° 303° 21.1

5.5

351 502 1,120 1,564

321 912

26.5 53.2

PEP carboxylase NAD-malate dehydrogenase NADP-malate dehydrogenase NAD-malic enzyme NADP-malic enzyme Pyruvate kinase ATP-PFK ( + 20/UM F26BP) PPrPFP ( + 20//M F26BP) Aldolase P-glucose isomerase P-glucomutase Glucose-6-P dehydrogenase Glukokinase Fructokinase UDPglucose pyrophosphorylase Sucrose-P synthase Sucrose synthase Invertase Cyt c oxidase Glycolate oxidase Catalase

222° 10.8 75.3° 57.7°

31.5 1,534

(Chloroplast) (Cytoplasmic)

88.3 12.2 108 448

7.4

15.4

3.3 4.5

1.7 5.3

4.6 4.4

29.3 19.5 15.7

3.8 77.4 186 166

4.2

9.6 47.0

842

193

360 39.6 55.1 31.7 8,937 trace

132 68.8 13.3 21.8 2,929 20.0

387

8.7

25.0 8.0

0.15 nd

12.8 59.1 265 40,000

Endosperm/Leaf 5.58 3.24 20.5 5.19 6.14 5.25 0.060 0.64 0.81 0.017 0.60 —

0.29 3.42 0.48 1.94 0.85 0.16 0.23 0.24 18.4 19.4 3.53 4.36 2.73 0.58 4.14 1.45 3.05 0.00 44.5 1.95 0.14 0.0006 0.00

The each value is the mean of results of at least three replicate incubations. " The activity is expressed in absorbance units as described in "Materials and Methods" See "Materials and Methods" for details. nd, not detected.

starch storage tissue. By contrast, the activity of sucrose synthase in the endosperm was about 45-fold higher, on the basis of soluble protein content, than that in leaves, where the activity of sucrose synthase was about 45% of the activity of sucrose-P synthase. Alkaline invertase activity was found in the endosperm. In the endosperm, the activity of Cyt c oxidase was de-

tected, but it was lower, on the basis of protein content, than that in green leaves. Glycolate oxidase activity was extremely low in the endosperm and no catalase activity was detected. Levels of metabolites in developing rice endosperm— The amount of sucrose, the starting material for starch biosynthesis, was high in developing rice grains, the concentra-


ADPglucose pyrophosphorylase Starch synthase Q-enzyme (branching enzyme) Phosphorylase R-enzyme (debranching enzyme) Amylase RuBP carboxylase P-glycerate kinase NAD-glyceraldehyde-3-P dehydrogenase NADP-glyceraldehyde-3-P dehydrogenase FBPase

Enzymic activity [nmol • (mg protein)""'•min-'] Endosperm Leaf


Table 2 Levels of metabolites in developing rice grains (cv. Fujihikari) Metabolite

/miol/g grain

44.9 1.52 8.97 3.27 2.11

1.80 0.061 0.36 0.13 0.085 nd 0.82 0.18 3.79 0.031 64.4

nd

20.5 4.49 94.5 0.76 1,610 741

29.7 19.6

488

The values are the means of results from three replicate samples. See "Materials and Methods" for details.

tion being 64fimo\ per gram fresh weight of rice grain (Table 2). The molar concentrations of glucose and fructose were about 46% and 30% of the concentration of sucrose, respectively. It should be noted that FBP, as well as hexose mono-P, was present in substantial amounts in the developing endosperm, suggesting that FBP is involved in the pathway from sucrose to starch in rice endosperm. The level of 3-P-glycerate was about ten-fold higher than that of DHAP. In rice endosperm, a larger amount of malate was found to have accumulated, as was also found in the developing endosperm of maize (Liu and Shannon 1981). The other compounds involved in the C4-metabolism, P-enolpyruvate and pyruvate, were also detected. Localization of enzymes in amyloplasts isolated from developing rice endosperm—In an attempt to isolate

amyloplasts efficiently from developing rice endosperm, several procedures and reagents were tested. It was difficult to prepare protoplasts, probably because the plasma membrane as well as the cell wall of the developing endosperm was exceptionally fragile and sensitive to physical and chemical shock; no endosperm cells could be observed after agitation of the tissue in the suspension medium. As judged from observations of amyloplasts under the light microscope, sorbitol was the best, among the agents tested, at maintaining the integrity of the amyloplasts. The addition of percoll or Ficoll to the sorbitol mixture did not increase the percentage of intact amyloplasts. The use of sucrose resulted in low recovery of intact amyloplasts during centrifugation, possibly as a result of its higher viscosity. Note that the speed of centrifugation should be as low as or below 200 rpm. It is also important to note that the endosperm at an early developmental stage gave the highest percentage of intact amyloplasts. All attempts to increase the yield of intact amyloplasts were unsuccessful. Resuspension of the precipitated amyloplasts in the suspension buffer markedly decreased the number of intact amyloplasts. Table 3 shows that the total activity of ADPglucose pyrophosphorylase, a marker enzyme for amyloplasts, in the rice amyloplast fraction was about 0.46% of the total original level in the suspension of cells. However, it should be noted that the specific enzymic activity, on the basis of protein content, was higher in the amyloplast fraction. By contrast, the recovery of the activity of UDPglucose pyrophosphorylase, a marker enzyme for the cytoplasm, in the amyloplast fraction was as low as 0.018%, the value being one order of magnitude lower than in the case of ADPglucose pyrophosphorylase. The relative activity of FBPase in the amyloplast fraction was of about the same order of magnitude as that of ADPglucose pyrophosphorylase, while the activity of sucrose synthase was equivalent to that of UDPglucose pyrophosphorylase.

Table 3 Distribution of enzymes in amyloplasts isolated from developing rice endosperm

Enzyme

ADPglucose pyrophosphorylase FBPase UGPglucose pyrophosphorylase Sucrose synthase

Crude Suspension" Amyloplast Function" Activity Activity Total Total activity per mg of protein activity per mg of protein (nmol/min) (nmol • mg • min" 1 ) (nmol/min) (nmolmg~'min~') 957 85.7

108 9.66

4.43 0.18

16,800

1,894

2.97

5,500

620

0.42

168

6.82 112

15.9

Yield*

(%) 0.46 0.21 0.018 0.0076

" The volume and protein content of the crude suspension were 13 ml and 8.87 mg, respectively, and those of the amyloplast fraction were 0.54 ml and 0.0264 mg, respectively. * The yield was estimated from the total activity in the amyloplast fraction divided by that in the crude suspension. See "Materials and Methods" for further details.


Glucose-6-P Glucose-1-P Fructose-6-P Fructose-1,6-P2 Dihydroxyacetone-P Glyceraldehyde-3 -P 3-P-glycerate P-enolpyruvate Malate Pyruvate Sucrose Glucose Fructose

nmol/grain

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These results suggest that ADPglucose pyrophosphorylase and FBPase are localized in the amyloplast, while UDPglucose pyrophosphorylase and sucrose synthase are localized in the cytoplasm in developing endosperm cells. Discussion

We thank Dr. H. Usuda (Teikyo University) and Dr. K. Wakasa (National Institute of Agrobiological Resources) for help in assays of metabolites and preparation of amyloplasts, respecitvely. We are also grateful to Ms. R. Fujisaki and Ms. S. Morimoto for their skillful technical assistance. The work was supported by grants from the Ministry of Education, Science and Culture, and from the Ministry of Agriculture, Forestry and Fisheries, Japan.

References Boyer, C. D. and Preiss, J. (1979) Properties of citratestimulated starch synthesis catalyzed by starch synthase I of developing maize kernels. Plant Physiol. 64: 1039-1042. Echeverria, E., Boyer, C D . , Thomas, P. A., Liu, K.-C. and Shannon, J. C. (1988) Enzyme activities associated with maize kernel amyloplasts. Plant Physiol. 86: 786-792. Hawker, J. S., Ozbun, J. L., Ozaki, H., Greenberg, E. and Preiss, J. (1974) Interaction of spinach leaf adenosine diphosphate glucose a-l,4-glucan a-4-glucosyl transferase and a-1,4glucan, a-1,4-glucan-6-glycosyl transferase in synthesis of branched a-glucan. Arch. Biochem. Biophys. 160: 530-551. Huber, S.C., Doehlert, D. C , Kerr, P. S. and Kalt-Torres, W. (1985) Regulation of photosynthetic sucrose formation in


It is generally accepted that the predominant pathway for the biosynthesis of starch in both photosynthetic and non-photosynthetic tissues of higher plants involves reactions catalized by ADPglucose pyrophosphorylase, starch synthase and Q-enzyme (Preiss and Levi 1980). The ratios of ADPglucose pyrophosphorylase activity to starch synthase activity were similar in the endosperm and leaves of rice plants (Table 1), but the specific activity of both enzymes, on the basis of soluble protein content, was much (3- to 6-fold) higher in endosperm than in leaves. Q-enzyme is considered to activate starch synthase by providing it with the non-reducing end needed for acceptance of a newly synthesized glucan unit (Hawker et al. 1974, Pollock and Preiss 1980, Preiss 1988). Therefore, the result that the relative activity of Q-enzyme was much higher than the activities of ADPglucose pyrophosphorylase and starch synthase in endosperm as compared in leaves might explain the potential for efficient production of starch production in rice endosperm. However, the mechanism of the concerted actions of these three enzymes remains to be elucidated (Preiss 1988). The activities of sucrose synthase, PP r PFK and UDPglucose pyrophosphorylase were very much higher in rice endosperm than in leaves (Table 1), suggesting that these enzymes play a key role in the breakdown of sucrose in rice endosperm. The occurrence of FBP and DHAP in developing rice endosperm (Table 2) also suggests the involvement of these compounds in the conversion of sucrose to starch in the endosperm. The same extent of recovery of FBPase activity as that of ADPglucose pyrophosphorylase activity in isolated amyloplasts from developing rice endosperm indicates that most, if not all, of the enzymic activity was located in the amyloplast. In summary, the following pathway is the most likely, given the present data, for the metabolic conversion of sucrose to starch in developing rice endosperm. The process is composed of three steps. First, in the cytoplasm, sucrose is degraded to form triose-P (DHAP) via reactions catalized by sucrose synthase, UDPglucose pyrophosphorylase, P-glucomutase, P-glucose isomerase, fructokinase, PPj-PFP (and ATP-PFK), aldolase and triose-P isomerase. Next, DHAP is translocated into the amyloplast. Finally, DHAP is converted to starch in the amyloplast via reactions catalized by triose-P isomerase, aldolase, FGPase, Phexose isomerase, P-glucomutase, ADPglucose pyrophosphorylase, starch synthase and Q-enzyme. Involvement of triose-P in the biosynthesis of starch in

non-photosynthetic tissues has been proposed by MacDonald and ap Rees (1983a, 1983b) and by Echeverria et al. (1988) who studied this problem using suspension cultures of soybean cells and amyloplasts from developing maize endosperm, respectively. Such involvement is further supported by the results of Ngernprasirtsiri et al. (1988) who found that, in the amyloplast envelope-membrane from cultured sycamore cells, the Pj translocator-like protein with molecular weight of 31 kDa was immunoreactive with an antibody raised against the pea chloroplast P-, translocator. In contrast, Keeling et al. (1988) recently reported that hexose-mono-P, such as G1P, G6P or F6P, but not triose-P, is the most likely candidate for the predominant substrate in the synthesis of starch in amyloplasts of developing wheat grains. To test these hypotheses further with developing rice grains, a method must be developed for efficient isolation of intact amyloplasts which can be used for experiments in vitro. The implications of the presence of high concentrations of 3-P-glycerate and malate are unclear at present. These compounds might paticipate in the process of production of ATP described above. It is also possible that they play a direct and key role in the synthesis of starch, since it has been reported that 3-P-glycerate is a potent allosteric activator of plant ADPglucose pyrophosphorylase (Preiss and Levi 1980), and malate can enhance the unprimed synthesis of glucan that is catalyzed by starch synthase (Boyer and Preiss 1979, Pollock and Preiss 1980).


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