Non - Reducing Saccharides : Floridosides and Sucrose

The none reducing sugar floridosides and sucrose, which are abundant in photosynthetic red algae and green plants, respectively, are recognized as the initial stable neutral saccharides produced in large quantity. In the fifties, when the author started a research program on the assimilation of translocated neutral saccharides in sink organs, live red algae were not readily available and the study on floridosides had to be dropped. Then the assimilation of sucrose in sink organs remained little charted. By using a 14C glucose and a sucrose doubly tagged with 14C and 3H for elucidating the metabolic pathways of sucrose, a cycling of sucrose synthesis-cleavage without net gain of sucrose in many plant systems, and the direct incorporation of glucose moiety of sucrose into starch in the starch filling rice seeds had been brought into foci of research. Sucrose synthase has been identified as the key enzyme in both systems, and thus the genes encoding the enzyme isoforms in the rice have been studied. The active enzyme is consisted of four either identical or mixed protomers, and the three genes encoding individual protomers were cloned and their structures mapped. It has been found that one of the isologous genes is expressed in the endosperm of starch filling rice seed, and the other two were ubiquitously expressed, with one dominating over the other in different tissues. Other sucrose metabolizing enzymes, namely invertase and sucrose phosphate synthase, play distinctly different physiological roles because of the irreversibility in different directions of sucrose metabolic reactions they catalyze. Although being known to the human being as the most important natural sweetener from the time unmemorable, and not many enzymes are directly involved in the metabolic pathways spanning around the sucrose molecule, the importance of sucrose not only as the energy source but also a metabolic regulatory signal in the maintenance of plant life still remains to be studied. Sucrose, the mass-produced crystalline food Sucrose is probably the only organic food material that is massively manufactured and commonly traded in the crystalline form. In Taiwan, the peak production of the “plantation white sugar”, or the granulated sucrose directly recovered from the expressed sugar cane juice in one process, surpassed one million metric tons before the onset of the Pacific War. Being an agricultural chemistry student in Taiwan at the time immediately following the conclusion of the War, the chemistry on cane sugar manufacturing was one of the major courses I had to take. I learned that, the non-reducing sucrose, having both anomeric carbons in α-D-glucopyranose and β-D-fructofuranose linked together, was stable under alkaline conditions but susceptible to acid catalyzed hydrolysis. So, the expressed cane juice could be treated with slaked lime and heating, an apparently drastic chemical condition, to neutralize the juice acidity and flocculate colloidal impurities in the first step of sugar purification. The primary clarified juice goes through carbonatation or sulfitation process to remove excess lime as well as to adsorb impurities on the precipitating calcium salt. The final step of purification is vacuum evaporation-crystallization followed by removal of molasses by centrifugation to recover sugar crystals. Beside the lecture course, we had to spend about a month at a couple of sugar refining factories in the southern Taiwan to learn the whole process on site. Sucrose, the primary neutral saccharide produced in photosynthesis Either the biochemistry or physiology of photosynthesis I learned at the undergraduate level taught me that, based on the transient accumulation of starch granules in chloroplasts, glucose was postulated as the primary stable saccharide produced in the photosynthesis. We were even taught a theory according to which formaldehyde was the intermediate to condense to form a hexose. The synthesis of a product named “formose” from formaldehyde in vitro (Butlerow, 1861) was cited as the evidence to support the theory. It was then a great surprise to learn from an article by Melvin Calvin published in the Journal of Chemical Education (1949) that sucrose was the first detectable neutral saccharide among many acidic compounds, clearly shown in the two-dimensional autoradiograms of radiolabeled photosynthates from green algae. With my knowledge of sucrose chemistry, I fancied that probably the chemical stability conferred by the non-reducing nature of the disaccharide was selected by the nature to serve as the vehicle of transport from the leaf to sink organs, or as the storage material most notably in cane stalks and sugar beets. Non-reducing counterpart of sucrose in the red alga floridosides Reading of papers from the laboratories of Melvin Calvin and Daniel Arnon at Berkeley in the fifties greatly stimulated my interest in the role of sucrose in plant physiology, and also the biochemical pathway that would lead to the biosynthesis of sucrose. By the time I started my job as a teaching assistant in 1950, and was enrolled as a graduate student in the MS program one year later at the department from where I was graduated, I had looked for a research topic that could be done under the prevailing environment. We saw off the Japanese biochemistry chair professor Suguru Miyake, a carbohydrate chemist who had a postdoctoral experience with Haworth of Edinburgh, back to Japan in 1947. His successor Professor Hon-Kai Ho, a graduate of our department who worked at the Manchurian National Institute of Sciences as a lipid chemist, was back from Manchuria, escaping from the siege of the Manchurian capital by the communist, in 1949. Meanwhile, without significant financial inputs for improving teaching and research from the Chinese authorities for almost 10 years since the restoration of Taiwan to China in 1945, the biochemistry laboratory in which I started working had only facilities, equipment and chemicals suitable for carbohydrate chemistry left behind by the Japanese. What I could do then was to read all of the publications from the laboratory in the foregoing 18 years (1928-1945), checked out chemicals and equipment available in the lab, setup a plan to start the biochemistry teaching laboratory as charged to me by Prof. Ho, and find a topic suitable for my own MS dissertation research for Prof. Ho’s approval. The Japanese apparently tried to build a database of carbohydrate resources of Taiwan. Among the materials they studied were rice starches and various plant polysaccharides, especially mucilages and gel forming matters from local plants for food and industrial use, including those from marine algae. A couple of their publications drew my attention. They reported on the galactans from red algae Bangia fuscopurpurea and Porphyra crispata (Hayashi, 1942a,b). Besides isolating crystalline DL-galactose from the acid hydrolysates of water soluble polysaccharides, crystalline D-galactose was obtained from the ethanolic extract of P. crispata. Crystallization of sugars from the concentrated syrup, characterization of various phenylhydrazone and phenylosazone derivatives and oxidation and reduction products were the main techniques of qualitative analysis they employed. They used a dried Porphyra specimen harvested at the Pescadores, a group of islands located between Taiwan and mainland China. So, it was possible that D-galactose was derived from a complex saccharide during the course of sample preparation, shipment and treatment rather than occurred as such in the alga. Besides, the most interesting point was that the L-isomer of galactose was present exclusively in the polysaccharide but none in the low molecular weight fraction. It is well known that, among the naturally occurring aldohexoses, only galactose has D and L forms, and besides, a paper chemistry shows that one form is transformed to the other by simply exchanging their end groups along the carbon chain. And this fact was demonstrated by Neuberg and Wohlgemuth (Neuberg and Wohlgemuth, 1902) by oxidizing dulcitol (or also known as galactitol, obtainable by reducing D-galactose) with a 3% hydrogen peroxide solution in the presence of ferric ion to obtain DL-galactose. My plan was to see whether free D-galactose arose in the alga, and what would be the biochemical mechanism of L-galactose formation. With Prof. Ho’s approval, I obtained some travel funds, setup a heating apparatus to be carried in a wooden case, obtained some knowledge of seaweed taxonomy by studying dry specimens of local marine algae collected by the Japanese, and ventured out to the northern coastal area for sample collection. The transportation available was buses, trains, and on foot. After spending much labor in carrying the equipment to reach a destination, I started setting up an alcohol burner-water bath to heat ethanol containing flasks to fix Porphyra and some other red algae freshly collected from the sea shore. I was suddenly surrounded by several bayonet fixed rifles, and asked questions by soldiers in a language that I could not comprehend well. Only after presenting ID cards to prove myself as a student and a university employee, I was allowed to continue the activity under their constant surveillance. Afterward, I requested the university authority to file an application to the Keelung Garrison Command for issuing me a free pass to the coastal area for sample collection. Then, since the fall of the mainland to the communist in 1949 and the massive exodus of military as well as civilians from the mainland that ensued, the whole Taiwan area had been placed under the martial law. Curfews were enforced severely, and the coastal areas were heavily guarded by armed forces to fend off seemingly imminent invasion from the mainland China. My request was turned down by the university for the reason that they did not want me to undertake the dangerous activity. Thus the original project that needed continuous sampling of living algae had to be terminated, and the algae fixed in ethanol obtained by the only successful adventure were kept in a freezer for three years before I could analyze them. By that time, I learned the technique of paper chromatography from the paper by Calvin. Then, recognizing the importance of chemical analysis at the sub-milligram level, I dug into the books of microchemistry to learn how to do chemical operations in capillary tubes and spot plates, and the use of a microscope to enhance the sensitivity of chemical detection and melting point determination. I was then able to couple the paper chromatographic separation with the classical chemical detection techniques to enhance my analytical capability greatly. I was able to show that the ethanol solubles from all of the red algae I collected did not contain reducing sugars with a confidence level of less than one μg per gram fresh weight, but they yielded much D-galactose on acid hydrolysis. From P. crispata, the most abundant specimen available, I isolated and identified floridoside, or α-D-galactopyranosyl 2-glycerol. I could not find other forms of galactoside, and confirmed that DL-galactose could be crystallized from the hydrolysate of its water soluble polysaccharide (Su, 1956). By the time I finished this work, Putman and Hassid (1954) published the isolation and structural determination of floridoside from a red alga Iridophycus flaccidum belonging to the order Florideophyceae. Prof. Hassid further studied the CO2-photosynthesis of the alga and proposed that floridoside would be the counterpart of sucrose in the marine plant (Bean and Hassid, 1955). I wrote to Prof. Hassid to discuss my identification data. This communication brought me to his laboratory as a US-AID trainee in 1958, and also my interest back to sucrose. However, my Ph.D. dissertation that I finished at the Hassid laboratory was not on the plant sugar nucleotide biochemistry that was vigorously pursued in the laboratory at that time. Because I was given by AID only one year of stay in the US, which was later extended to two years, my graduate advisor Prof. H. A. Barker advised me to finish all course and residential requirements for a Ph.D. degree but finish the dissertation work in Taiwan. Professor Baker told me that, as long as I determined to stay in an academic field, a Ph.D. degree would be a necessity. But knowing better than Prof. Barker of the prevailing academic situation in Taiwan, I consulted with Prof. Hassid and he agreed that I continue my line of work on Porphyra so that I might be able to finish a dissertation research within my allowed time of stay in the US. Using P. perforata collected from the rocky beach beneath the Golden Gate Bridge with the help of an old friend from National Taiwan University who had just finished a degree on phycology at Berkeley, Dr. Kung-Chu Fan, the work yielded three papers, one short communication reporting two novel nucleotides (Su and Hassid, 1960), and two full length papers reporting the chemistry of a whole spectrum of nucleotides, two forms of D-galactosylglycerols including floridoside and α-D-galactopyranosyl-1-D-glycerol, two inositols laminitol and scylloinositol, and a DL-galactan sulfate constituting of Dand L-galactoses, 3,6-anhydro-L-galactose, 6-O-methyl-D-galacotse and 6-O-sulfate groups on the unmodified galactose residues in the ratio 1:2:1:1. A possible biosynthetic pathway linking these saccharides together was proposed (Su and Hassid, 1962a,b). After being back in Taiwan, I tried to continue the biochemical aspects of work but was hampered by the fact that the only Porphyra species available to us contained a very active adenylate deaminase (Su et al., 1966) which transformed all ATP required in the in vitro reactions into ITP. While the mainland China was still in the turmoil of cultural revolution, my friend Dr. Fan, who went back to the mainland soon after helping me collect the seaweed, died at a young age. Professor Pappenfuss, Dr. Fan’s thesis advisor, told me while he visited Taiwan in the late 60’s, that he received a postcard from him with a message of “do not send me any scientific periodicals anymore” in the Chinese. The news made me very sad indeed. Biosynthesis of sucrose: Application of sucrose synthetic enzymes Although the chemical structure of sucrose was established by methylation studies of Haworth school in 1920’s (Avery et al.,1927; Haworth et al.,1927), the feat of its in vitro synthesis was first achieved by Hassid, Doudoroff and Barker (Hassid et al., 1944) from α-D-glucopyranose 1-phosphate (G1-P) and D-fructose under the catalysis of sucrose phosphorylase from Pseudomonas saccharophila. The chemical synthesis was achieved by Lemieux and Huber (Lemieux and Huber,1953,1956) by reacting 3,4,6-tri-O-acetyl-1,2-anhydro-D-glucopyranose and 1,3,4,6-tetra-O-acetyl-D-fructofuranose to yield sucrose octaacetate. Before leaving for the US to join the Hassid laboratory, I tried to find sucrose phosphorylase activity in several plants, but failed. Then the discoveries of the activities of sucrose synthase (SuS) (Cardini et al. 1955) and sucrose phosphate synthase (SPS) (Leloir and Cardini, 1955), both using UDPG as the glucosyl donor, were reported. For the detection of sucrose phosphorylase activity, I could manage to use a G1-P prepared by a potato starch phosphorylase catalyzed reaction, but UDPG needed for the detection of SuS and SPS was beyond my reach then. At the biochemistry department of Berkeley, besides sharpening research techniques by doing dissertation work, I had learned many research methods by taking laboratory courses as well as observing many brilliant postdoctoral fellows, including Elizabeth Neufeld, David Feingold and G. A. Barber, at the Hassid laboratory in action. The technique I learned that benefited me most was the combined application of chemical and biochemical methods for the synthesis of commercially unavailable radiocarbon-tagged sugar nucleotides from commercially available precursors. After returning to Taiwan from the US in 1960, under the conditions that no commercial routes were available for the import of perishable biochemicals, I had to prepare many biochemicals, including U-C-glucose, ATP, NAD, various C-sugar nucleotides, to carry on my biochemical research. Among the synthetic products, a double labeled sucrose prepared by applying SuS from asparagus spears, which had straight forward Michaelis-Menten kinetics in the direction of sucrose synthesis, opened up a new avenue of sucrose research (Lee and Su, 1982). The synthesis started with the preparation of G1-P by the phosphorolysis of sucrose by the P. saccharophila enzyme, coupling the G1-P with UMP by cyclohexylcarbodiimide, and the synthesis of sucrose by the SuS catalysis. By choosing appropriate C and H tagged reactants, sucrose preparations having the two monosaccharide residues independently tagged by the radioisotope of choice were obtained. Many SuS’s exhibit sigmoidal reaction kinetics and could not utilize the substrates at low concentrations, but the asparagus enzyme could and yielded sucrose of high specific radioactivity in good yield. Our later studies on the rice SuS isozymes revealed that they also had the properties of asparagus enzyme, and they are extensively utilized in the synthesis of sugar nucleotides (see for example, Stein et al., 1998). Once Prof. Hassid told me that their sucrose phosphorylase research attracted the interest of the Coca-Cola Company, and obtained some funding from them. Why the enzyme, or the enzyme catalyzed reaction and its products, attracted the soft-drink company appeared to me a mystery. However, the mechanism of the enzyme catalyzed reaction, thoroughly studied by the three discoverers, has provided an excellent model of enzyme mechanism research, and the basic concept developed by them is still of importance to biochemistry (Doudoroff et al. 1947). Besides, the useful enzyme activity could be so easily obtained from the bacterial cells (Doudoroff, 1955) and the reaction catalyzed by the enzyme is so clean and efficient that one can cleave sucrose, even at a “tracer” concentration level, to obtain easily separable G1-P and fructose in quantitative yield (Abraham and Hassid, 1957), and one example of the application is given above. Futile cycle of sucrose in plants Among the research strategies that I learned at Berkeley, the mapping of metabolic pathway by the use of radiotracer as the basis of molecular or enzymic studies attracted me most. As I said earlier, buying radiocarbon-labeled glucose was impossible when I wanted to start my research in Taiwan in 1960. I could import C-barium carbonate, though, because it would remain intact during over one year of time needed for the shipment from the US to reach me. So, I, together with my assistant, Mr. Ti-Sheng Lu, who later obtained a Ph.D. degree in plant physiology at the University of California, Davis, used the carbonate as the source of carbon dioxide to obtain starch by the photosynthetic method, devised by Prof. Hassid (in “Isotopic Carbon” , by Calvin, M., Heidelberger, L., Reid, J. C., Tolbert, B. M., and Yankwich, P. F., John Wiley & Sons, New York, and Chapman & Hall, London, 1949, pp. 263-268; Abraham, S. and Hassid, W. Z., The synthesis and degradation of isotopically labeled carbohydrates and carbohydrate intermediates, Methods in Enzymol. III, Academic Press, New York, 1957, pp. 489 560). The radioactive starch was recovered from the tobacco leaf that was exposed to CO2 under light, hydrolyzed with acid and the recovered glucose was used in a feeding study using bamboo shoot slices. From the kinetic analysis of radioactivity levels of ethanol soluble saccharides from the tissue slices, we could show that the radioactivity level of free glucose depleted rapidly but that of sucrose increased in proportion, but the free fructose gained practically none of the radioactivity. The radioactive sucrose was hydrolyzed and the recovered hexoses were analyzed. We found that they gained radioactivity in a similar fashion, but with the increase of glucose level leading that of fructose in about 2 to 1. Since the levels of all soluble sugars in the tissue slices remained practically constant in the short duration of tracer feeding, these results suggested that the metabolism of free fructose was segregated from the sucrose synthesis pathway by either not sharing the same intermediate or by compartmentation of metabolic sites. We may further see that, if UDPG is used as the glucosyl donor in the sucrose synthesis, the glucosyl acceptor is generated at the site of UDPG synthesis, or both of them share the same precursor, and a continuous cycling of sucrose synthesis and degradation takes place in the same cellular compartment because the total sucrose remained constant (Su, 1982). Years later, we did the same type of experiment on banana fruit slices and found the same. An interesting finding was that, when the banana fruit was subjected to a chilling injury treatment, the cycle was disrupted, as indicated by the finding that no radio-labeled fructose was incorporated into sucrose (Niu and Su, 1969). We may write such cycling pathway of synthesis and degradation by incorporating known enzyme activities, including SuS, SPS, sucrose phosphate phosphatase (SPase), invertase, hexokinase, phosphohexoisomerase, phosphoglucomutase and UDPG pyrophosphorylase, but these enzyme activities were not affected by the chilling injury. Thus we had proposed that the injury was incurred on the membrane transport of substrates. We may see that the overall balance of the cycle constructed from the enzyme activities listed above is “futile” in the sense that pyrophosphate bond energy is lost at no gain of sucrose if the sucrose synthesis in the cycle is catalyzed by SPS and SPase while that of sucrose degradation is catalyzed by either invertase or SuS, especially by invertase, because invertase catalyzed hydrolysis is as irreversible as the synthetic reaction catalyzed by SPS and SPase. As we know, the SuS catalyzed reaction is fully reversible and also may utilize nucleoside diphosphates other than UDP, and if only this enzyme is involved in the degradation and synthesis of sucrose, and if the change in direction of reaction is governed by a signal, the response to the signal will be more rapid than the futile cycling, and the sucrose cleavage reaction may be coupled to different requirements of sugar nucleotides. If the cycling is the common phenomenon in plants, then we may see that the one catalyzed by SuS single-handedly would be more versatile than the futile cycle operated by the invertase/SPS-SPase system. However, as we have shown by the radio-labeled glucose feeding done on bamboo shoots and banana fruits, the fructose residue in the sucrose molecule is in equilibrium with the precursor of the glucose residue, not in favor of the cycling catalyzed by SuS only. Then, what is the physiological significance of the cycle, regardless of either “futile” or “non-futile”? This is the question also raised by Pontis (Pontis, 1978) based on the biochemical properties of SuS, SPS and SPase. Unfortunately, besides knowing that the sucrose level will determine the growth, differentiation, etc., of plant tissue culture and plants, clear metabolic pathways have not yet been charted. What is the physiological role played by sucrose synthase? When Leloir and Cardini discovered sucrose synthase, they undoubtedly considered it as the enzyme for catalyzing sucrose synthesis. Then, on studying the metabolism of UDPG, they further found the presence of SPS, and later sucrose phosphate phosphatase in green plants. The sucrose synthesis catalyzed by SPS is energetically more favorable than that of SuS, probably because that fructose 6-phosphate (F6-P) has only the furanose form to readily condense with glucose to form sucrose, while fructose has the more abundant pyranose form in addition. Furthermore, the phosphatase catalyzed reaction remove sucrose phosphate from the reaction system to render the overall reaction irreversible. With such development, it is natural to look for the physiological significance of SuS catalyzed reaction in the direction of sucrose degradation. We know that the rate of SuS catalyzed reversible reaction is rapid in kinetics, and it may be very important in the “sucrose cycle” to regulate the sucrose concentration level to meet the requirement of sucrose as a metabolic regulator. Besides being a possible regulator of metabolism, the SuS catalyzed sucrose degradation reaction has an advantage over the invertase catalyzed one because it directly gives UDPG, a precursor for complex saccharide synthesis by itself, as well as the starting substrate for a series of saccharide transformation reactions in the formation of uridyl nucleotides of D-galactose, D-glucuronic acid, D-galacturonic acid, D-xylose and L-arabinose, all needed for the biosyntheses of plant cell wall polysaccharides, as elegantly demonstrated by the Hassid school in the sixties. Here I have to mention about the substrate specificities of SuS catalyzed reaction. From our results obtained from nearly ten plant sources (Su, 1982) and also many reported by others, we can say that sucrose is the only disaccharide substrate SuS uses. However, although UDP is invariably the nucleotide substrate with the least Km value, usually ADP or TDP comes to the next best, and other nucleoside diphosphates are also reactive, though at more reduced rates. It is therefore highly possible, with the adenylate nucleotides usually predominating in the soluble nucleotide pool, in vivo formation of ADPG, in addition to UDPG, by the SuS catalyzed reaction is possible (Chen et al., 1982). Besides the multiplicity of sugar nucleotide specificity, we have found that all of the SuS we studied have a quaternary structure constituting four either identical or not identical protomers. Recent studies on the SuS isozymes have revealed that the wide spectrum of sugar nucleotide specificity is not due to the heterotetrameric structures (Yen et al., 1994; Huang and Wang, 1998). My radiotracer study on bamboo shoot slice indicated the importance of the SuS catalyzed reaction in the cell wall polysaccharides syntheses. However, trials in solubilizing the enzyme activities identified by the tracer study were a failure, although the catalytic activities were all found in a particulate fraction sedimented at 105 x g (Sung et al., 1971). With no means of purifying the enzymes, I had to divert my effort to another class of plant polysaccharide, the starch. My choice of the system was the rice. By noting that the starch synthesizing activity of the rice seed resided almost exclusively associated with the insoluble starch granules while the precursor nucleotides synthesizing activity was extractable into a buffer solution, we titrated the two activities in the formation of starch by reconstituting them in different proportions in test tubes. It was found that sucrose plus ADP were not only better substrates than G1-P plus ATP in the starch synthesis, but also used less soluble fraction for a fixed amount of starch granule to achieve a higher level of starch synthesis, indicating that the activity of sucrose synthase to synthesize ADPG was much more adequate than the ADPG pyrophosphorylase activity in the rice seed (Chen et al., 1981). Then we used rice seeds at different maturing stages to feed a double labeled sucrose for 5 to 60 minutes. We were excited to find that, when the feeding time was shorter, or the seed growth stage was earlier, the incorporation of the glucosyl residue into starch was higher than that of the fructosyl residue. The segregation ratios of the two hexoses as the precursor of starch were from 6 to 4, the higher when the feeding time was shorter. When the radio-labels were analyzed on ADPG, UDPG, hexose 6-phosphates and G1-P isolated from the seed, the results indicated that glucosyl residues in the two nucleotides were derived mainly from the glucose part of sucrose, while that in all of the hexose phosphates were from the fructose part, showing that the pyrophosphorylase catalyzed reactions were not responsible for the formation of sugar nucleotides if sucrose was fed, and that the SuS catalyzed reaction directly provided the precursor of starch synthesis in the rice seed (Lee and Su, 1982). Same kind of experiment done on other plants, such as pea seedlings, asparagus spears, bamboo shoot, etc., yielded results not as distinct as that of the rice seed but showed the same trend. However, the result obtained from sweet potato root slices was entirely different; it indicated that G1-P was a more direct substrate than a sugar nucleotide at the initial phase of starch synthesis. These results lead us to concentrate on the studies of SuS in the rice grains and the starch phosphorylase in the sweet potato tuberous root (Chang et al., 1987). Genes encoding rice sucrose synthase isozymes With its richness in genetic backgrounds, the Sus mutants and their direct relevance to the carbohydrate biochemistry were first described for the maize system. The maize shrunken-1 and sugary mutants are the best documented examples in which the SuS deficiency is attributed to the poorer biosynthesis of endosperm glucans. Analogous to the maize, many plants are known to have two isoforms of Sus genes. From the rice, we cloned and established the cDNA and genomic structures of three genes encoding sucrose synthase polypeptides, and their primary structures were deduced (Yu et al., 1992; Wang et al., 1992; Huang et al., 1996). From the homology comparison analysis, we could see that two of the three genes are highly homologous and correspond to the maize Sus, while the third one, named as RSus2, is closely related to the maize Sh1. DNAand immuno-probes specific to the three genes and polypeptides, respectively, are now available. By applying these identification tools in the mapping of spatial and temporal expression of these genes at the transcription and translation levels, we could conclude that RSus2 is a house keeping gene. Of the two genes similar to maize Sus, the one we named RSus3 is unique in having its expression almost exclusively in the endosperm of milk-ripe stage seeds. The close cousin of RSus3, RSus1, has its temporal and spatial expression patterns compensating those of RSus2. Besides the homotetrameric quaternary structures which are prevalent when only one gene is expressed at a time in a confined tissue, the presence of heterotetrameric structures could be found if two or more genes are expressed simultaneously in a same compartment. The gene products of RSus1 and RSus2 catalyzed reactions show different initial rates in the directions of sucrose synthesis and breakdown, and the rice leaves with earlier and later order of emergence have different activity ratios of sucrose synthesis over breakdown, implicating that the expression patterns of the two genes change as the leaf tissue ages. Besides the temporal and spatial changes, availability of sugar and oxygen have been reported to affect the expression of Sus genes as well (Yen, 1998). Many plants are known to have two functional isologous Sus genes, and the occurrence of three has been reported in the rice only. Whether such diversification is due to the adaptation to the semi-anoxic condition by the rice root system merits further studies. In this respect, it is noteworthy that, we found that the vascular tissues of rice root and leaf have only one type of Sus expressed at the translational level . The importance of SuS in the polysaccharide biosynthesis has been demonstrated in the maize grains. It has been shown that one of the maize genes contribute to the formation of cell wall in the endosperm tissue, while the other enhances the starch synthesis. We specifically inhibited the expression of RSus3 in the rice grain by the antisense technique and obtained the shrunken phenotype of the grain, demonstrating that the conclusion we had drawn from our earlier radiotracer study using double-labeled sucrose was right, and the responsible SuS is encoded by