Chapter 4 in the book "Discoveries in Plant Biology" Volume 3: 69~73 (2000),
published by World Scientific
Non-Reducing Saccharides:
Floridosides and Sucrose
Jong-Ching Su
Department of Agricultural Chemistry
National Taiwan University
Taipei, Taiwan
ABSTRACT
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
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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 D- and 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
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to prepare many biochemicals, including U- C-glucose, ATP, NAD, various
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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
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3
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
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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
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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. DNA- and
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
RSus3.
Recently, we have demonstrated the presence of an SuS activity in the cell wall
fraction of the rice grain. Whether this isozyme is responsible for providing a
precursor to the cellulose synthase, as proposed by Delmer et al. (Delmer and
Amor, 1995) that a SuS constitutes the hypothetical cellulose synthesizing
complex on the plasma membrane, and thus have a parallel function of one of the
maize SuS isozymes, is a problem worth studying.
Sucrose metabolism initiated by invertases
From the facts that the preferential utilization of the glucose over fructose in the
double labeled sucrose feeding is limited to about 6 to 1 even though the tracer
feeding time was shortened to 5 minutes, and that the ratio decreased as the
feeding time is prolonged, we should consider about the invertase initiated
pathway in addition to the SuS pathway. By the enzyme purification and gene
cloning-sequencing, at least three invertase genes and isozymes encoded by them
could be isolated from the rice grain at the laboratory of Hsien-Yi Sung, one of
my colleagues at the same department. The complexity of invertase isozymes is
prevalent among plants, and their physiological significance in the apoplastic and
symplastic transport of sucrose has been demonstrated. One of the isoforms has
been known to be localized in the apoplastic space of many plant species, and its
function in the apoplastic sucrose transport is a commonly accepted view. The
occurrence of a proteinaceous inhibitor of the apoplastic invertase is also common,
and the implication that the inhibitor is a switch to control the apoplastic transport
of sucrose is gaining wider acceptance. Two other forms of invertase are known to
localize in cytoplasm and vacuole, and the roles they may play are symplastic
transport and vacuolar deposition of sucrose.
Conclusion
It is interesting to find that the red marine algae produce a series of non-reducing
floridoside isomers instead of sucrose as the primary neutral photosynthetic
products. Sucrose is produced in all green plants, especially in higher plants as the
major transport of photosynthate from the source (leaf) to sink organs through the
vascular system. The metabolic links between floridosides and
biomacromolecules, including polysaccharides, proteins and others, remain
unknown, although they are supposed to play the counterpart role of sucrose in
green plants (Bean and Hassid, 1955). On the other hand, the biosynthetic
reactions and metabolic fates of sucrose are now well documented. Sucrose is
synthesized in the photosynthetic cell, transported to sink organs via vascular
system, transferred through cell wall and cytoplasmic membrane and metabolized
via invertase or SuS pathways, or further transported into vacuoles as a storage
substance. Externally added sucrose has been known as an osmotic agent, and
now a sucrose carrier is reported to facilitate its membrane transport. The
metabolic pathways of sucrose were established, or deduced, by combining the
textbook knowledge with those derived from rational experiments. The unknown
part of sucrose metabolism resides mainly in the physiological role it may play as
a regulator or a switch of metabolic pathways. We know that sucrose is invariably
the best carbon source to support tissue or cell cultures of higher plants. In the
normal metabolizing plant cells, sucrose may undergo continuous synthesis and
degradation. We may postulate that such metabolic cycling may be a mechanism
of regulating the sucrose level in vivo. Since the discovery of Crabtree effect, the
available sugar level has been known to play pivotal regulatory roles in
carbohydrate catabolism. And with the findings that the sugar level also exerts
effects on the expression level of some genes, the sucrose research now has
entered into the era of molecular genetics-physiology-biochemistry that needs
concerted efforts of plant scientists from multidisciplinary fields. It is hoped that
the physiological significance of the “futile” cycle of sucrose synthesis and
degradation will be deciphered soon.
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