G6p

Overall: 2 Sucrose + 3 ATP + UTP + 4 H20 2 Sucrose + 3 ADP + UDP + 4Pi, if SuS and IT degrade sucrose equally, or Sucrose + ATP + H20-> Sucrose + ADP + Pi, if only SuS degrades sucrose.

Figure 1. The futile cycle of sucrose synthesis-degradation

2.4. Synthesis of transient and depository starches

A starch synthesizing organelle, either chloroplast or amyloplast, contains two forms of SS; one is soluble and the other starch granule bound. Although both types of SS utilize ADPG as the substrate, the granule-bound one may use UDPG as well, but needing a non-physiologically high concentration. Both enzymes elongate the amylose type a-l,4-D-glucopyranose chains, which undergo a chain transfer reaction catalyzed by a branching enzyme (BE; EC 2.4.1.18), or Q-enzyme, to amylopectin type molecules containing a-1,6-branching residues. The branch chain after being elongated to a considerable length may be debranched (by a debranching enzyme, DE, or R-enzyme, RE, or pullulanase; EC 3.2.1.41) to give an amylose chain, which may be further elongated and branched. The granule bound starch synthase (GBSS) is encoded by the waxy gene (Wx) and is responsible for the formation of amylose. These starch-synthesizing enzymes are present in either the stroma of amyloplast, or in close association with starch granules in insoluble forms. The enzyme distributed in stroma may be released into an extraction buffer simply by breaking the plastid membrane. But those of the latter type may be solubilized only when a detergent, such as sodium dodecylsulfate (SDS) is added to the extraction buffer (4). Rice starch granules isolated from grains incorporate these proteins into the granular structure.

Starch granules, when observed under a microscope, show a shape and structure characteristic to the plant species. Under a polarized microscope, when placed between a pair of crossed polarizers, starch granules show a specific cross-shaped pattern. When observed in an X-ray diffractometer, a diffraction pattern characteristic of partial crystalline structure is revealed. The crystalline structure is due to packed amylopectin layers while the amorphous domains are constituted of amylose chains. These physical methods of observations are useful in characterizing starch, yet elucidation of the mode of layering or packing of gucan chains is not easily achieved.

2.5. Synthesis of starch precursor, ADPG

Since the discovery of ADPG and its higher efficiency as the precursor of starch synthesis by Leloir et al, all starch synthesizing reactions have been recognized to use ADPG as the "natural substrate", or the substrate with the least Km value. Consequently, ADPG pyrophosphorylase (PPase) that catalyzes ADPG synthesis from ATP and a-D-glucose 1-phosphate (G1P) has been regarded as the key enzyme in all starch synthesizing systems, and the rice is not exception. However, there is another enzyme capable of ADPG synthesis and ubiquitously distributed in plants. The enzyme is SuS, and it usually has the same trend of tissue or organ distribution as ADPG-PPase. The main reasons for discriminating SuS but in favor of ADPG-PPase as the sole provider of ADPG in starch synthesis can be that the subcellular localization of the former is in cytosol while the latter in plastids, and also the former uses UDPG as the "natural substrate".

Cardini et al. discovered SuS (5). SuS so far purified from various plants share the same properties as follows (2). It is an enzyme with four protomers in a catalytic entity. It may contain homologous or heterologous protomers, and isoenzymes may be purified. In case of rice plant, a total of five isoenzymes were purified (6), and they all show the highest affinity toward UDP, among other nucleoside diphosphates. However, they also use ADP as the second best substrate, although the Km value for UDP is about 0.1 mM while that for ADP is about 1 mM. The isoenzymes show somewhat different ratios of sucrose synthesis and breakdown reaction rates. It is noteworthy that four SuS isoenzyme species could be purified from maturing rice seed, but only one from leaf which had properties different from those of seed isoenzymes. These facts indicate that the genes encoding these isoenzymes have distinct differences in temporal and spatial expressions. SuS is abundant in starch accumulating grains and catalyzes a sugar nucleotide synthetic reaction energetically more efficient than a PPase. From these reasons, we thought that SuS could be an agent of starch precursor synthesis as well. We designed a couple of radiotracer experiments to explore the possibility.

In the first experiment (7) we fractionated the milk-ripe rice seeds into soluble and insoluble fractions, and titrated the enzyme activities in one fraction with the other. The soluble fraction contained UDPG- and ADPG-PPases and SuS, and the insoluble starch granules had SS activity. Different proportions of the two fractions were combined, to which were added substrates for either ADPG-PPase (G1P plus ATP) or SuS (sucrose plus ADP) at an equimolar level. The insoluble product was characterized as starch by an enzymic hydrolysis technique. From the yield of starch, it was estimated that the soluble fraction contained 4 times more ADPG synthesizing activity derived from SuS than ADPG-PPase. It was further shown that the SuS activity could synthesize either ADPG or UDPG, and the former was more efficiently incorporated into starch in the presence of starch granules.

To further elucidate the role played by SuS in starch synthesis, we designed an experiment using a radioactive sucrose in which the two hexosyl residues were labeled with different radioisotopes (8). The rationale of the experimental design is as follows. As shown in Fig. 1, SuS is the only enzyme that may synthesize a sugar nucleotide directly from sucrose by transferring the glucose residue to a nucleoside diphosphate acceptor. If the sugar nucleotide so synthesized is used directly in starch synthesis, in the pool of hexose phosphates, the glucose moiety of sucrose will not be in equilibrium with the fructose moiety derived from sucrose. Thus the starch synthesized will have more radiotracer derived from the glucose moiety than that from the fructose moiety of sucrose. On the other hand, if sucrose is metabolized via the IT pathway, the two hexose moieties will be metabolized all the way in equilibrium, and starch so synthesized will be derived from equimolar amounts of the two hexoses.

We synthesized a radioactive sucrose in which glucose and fructose residues were labeled with 14C and 3H, respectively. A sample of the sucrose solution was hydrolyzed with a yeast invertase to be used as an equimolar mixture of glucose and fructose. Rice seeds with pedicels were sampled at different DAP. Feeding of the radioactive sugars was done by dipping the pedicel of each seed into 10 |il of the radioactive sugar solution. In an air stream, a seed absorbed the solution in about 5 min, and 10 of water was used to chase the residual sugar into the seed. After being incubated at room temperature further, the reaction was terminated at 80 to 120 min from the start of sugar feeding. The seed was crushed in 80 % ethanol and the mixture was boiled. Starch in the ethanol insoluble fraction was hydrolyzed to glucose with a mixture of a-amylase, glucamylase and pullulanase, and glucose was recovered by paper chromatography. The phosphate compounds in the ethanolic extract were separated by paper electrophoresis. The separated compounds were quantitated for 14C and3H. As shown in Table 1, the glucose moiety of sucrose was the better precursor of starch when sucrose was administered. This tendency is weakened in more mature seeds and when the time for incubation after tracer administration is extended. When the hydrolysate of sucrose was fed, the two monosaccharides were assimilated into starch at about an equal rate. These results verified the rationale of the experimental design as described above. However, as mentioned,

ADPG is the only acceptable substrate of SS while the "natural substrate" of SuS is UDPG. So, many investigators postulated that, if SuS contributes to starch synthesis, UDPG derived from the reaction between UDP and sucrose should be transformed into ADPG by coupling UDPG- and ADPG-PPase catalyzed reactions in which G1P was the intermediate. The presence of both PPase activities in rice grains seemed to support the postulation.

Table 1

Mole ratios of 14C- and 3H-glucose residues in starch. Rice seeds were fed with a doublelabeled sucrose, a-D-14C-glucopyranosyl-(3- JH-fructofuranoside, or its hydrolysate.

Incubation Fed with Days after pollination

Hydrolysate 1.16 1.04 0.98 0.95 0.97

Hydrolysate 1.09 1.07 1.07 1.01 1.02

Hydrolysate 1.11 1.02 1.10 0.98 1.06

Hydrolysate 1.09 1.06 1.08 1.04 1.03

Table 2

Mole ratios of 14C- and 3H-hexose in starch and sugar phosphate compounds isolated from rice seeds fed with a double-labeled sucrose or its hydrolysate.

Table 2

Mole ratios of 14C- and 3H-hexose in starch and sugar phosphate compounds isolated from rice seeds fed with a double-labeled sucrose or its hydrolysate.

Fed with

0 0

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