Plant Physiol. (1992) 100, 560-564 Received for publication February 18, 1992 0032-0889/92/1 00/0560/05/$01 .00/0 Accepted May 15, 1992

Is There an Alternative Pathway for Starch Synthesis?'

Thomas W. Okita Institute of Biological Chemistry, Washington State University, Pullman, Washington

ABSTRACT 15, 17), indicating that the basic enzymology of starch syn- In leaf tissue, carbon enters starch via the gluconeogenesis thesis is the same in amyloplasts as in chloroplasts. The flow pathway where D-glycerate 3-phosphate formed from CO2 fixation of carbon and the exchange of metabolites between the is converted into hexose monophosphates within the chloroplast amyloplast and cytosol in developing sink organs, however, stroma. In starch-containing sink organs, evidence has been ob- are not identical to the processes established for leaves (1, 6, tained indicating that the flow of carbon into starch follows a 7, 9, 17, 19). different pathway whereby hexose monophosphates formed from An alternative pathway of starch synthesis recently has sucrose are transported into the amyloplast, a plastid specialized been proposed based on the capacity of both chloroplasts in starch accumulation. In both chloroplasts and amyloplasts, the and amyloplasts to transport ADPGlc (16 and refs. cited formation of ADPglucose, the substrate for starch synthase, is therein). In this review, I first summarize the latest develop- controlled by the activity of ADPglucose pyrophosphorylase, a key ments regulatory enzyme of starch synthesis localized in the plastid. in starch biosynthesis and then discuss whether the Recently, an alternative pathway of starch synthesis has been operation of this proposed alternative pathway of starch proposed in which ADPglucose is synthesized from sucrose and synthesis is compatible with our present knowledge of the transported directly into the plastid compartment, where it is used biochemistry and genetics of starch biosynthesis. for starch synthesis. On the basis of the biochemical phenotypes exhibited by various plant mutants with defined genetic lesions, it STARCH FORMATION OCCURS VIA ADPGIc is concluded that ADPglucose pyrophosphorylase is essential for PYROPHOSPHORYLASE starch synthesis, whereas the alternative pathway has only a minor role in this process. Extensive evidence indicates that the biochemical events leading to the formation of ADPGlc and its subsequent utilization for starch synthesis are restricted to the chloro- plasts (reviewed in refs. 15 and 17). The more recent isolation and study of mutants defective in carbon metabolism also The events that lead to the flow of carbon into starch and support this view. Caspar et al. (4) obtained a null mutant of their regulation have been well established for chloroplasts Arabidopsis thaliana for the chloroplastic PGM that was (reviewed in ref. 17). In contrast, less is known about the highly defective in starch synthesis, accumulating less than biochemical events that lead to starch synthesis in the amy- 2% of the normal levels. The starchless phenotype of this loplasts of developing sink organs (1). This organelle, con- null plastidic PGM mutant supports the prevailing view that taining one or more starch granules, is delimited by a double the bulk of, if not all, carbon flow into starch occurs via the envelope and is usually devoid of intracellular membranes. gluconeogenesis pathway within the plastid compartment. Because the amyloplast is dependent on the cytoplasm for Kruckeberg et al. (11) examined mutant plants of Clarkia both energy and carbon, the biochemistry of this organelle is xantiana containing reduced activities of the cytosolic PGI likely to be distinct from that exhibited by the autotrophic (64%, 36%, and 18% of wild-type levels) or chloroplastic PGI (ATP-generating, C02-fixing) chloroplasts of leaf tissue. Re- (75% and 50% of wild type) to assess the effect of enzyme cent studies have shown that the allosterically regulated levels on carbon fluxes toward starch and sucrose synthesis ADPGlc2 pyrophosphorylase, as well other enzymes involved under saturating and limiting light conditions. Decreased in starch synthesis, are localized in the amyloplasts (1, 10, levels of the plastid enzyme had very little influence on starch and sucrose synthesis in low light. In saturating light, however, starch synthesis was suppressed, whereas little l Supported in part by Department of Energy grant DE-FG06- effect on sucrose synthesis was observed. Conversely, reduc- 87ER13699, The Rockefeller and Foundation, Project 0590, College tion in the levels of the caused an of Agriculture and Home Economics, Washington State University, cytosolic enzyme increase Pullman, WA 99164. in starch synthesis with a corresponding decrease in sucrose 2 Abbreviations: ADPGlc, ADPglucose; 3-PGA, D-glycerate 3- synthesis; this response was more evident under low light phosphate; PGI, phosphoglucoisomerase; PGM, phosphoglucomu- intensity than under saturating conditions. These observa- tase; SS-ADPGlc, sucrose synthase-dependent synthesis of ADPGlc; tions with the plastid mutant lines reinforce the view that UDPGlc, UDPglucose; Glc 1-P, glucose 1-phosphate; Glc 6-P, glu- the events leading to carbon flow into starch are restricted to cose 6-phosphate. the plastid. Moreover, when sucrose synthesis is depressed 560 THE FLOW OF CARBON INTO STARCH 561 in the cytosolic mutants, more of the carbon is directed away metabolism. Muller-Rober et al. (13) transformed potato with from the cytosol and rerouted toward the synthesis of starch 'antisense' constructs for the small subunit of the heterotet- in the chloroplast. rameric tuber ADPGlc pyrophosphorylase (14). These work- Direct evidence has been obtained showing that most, if ers observed the almost complete absence of starch formation not all, of the ADPGlc formed is controlled by the action of in the developing tubers of these transgenic plants and ADPGlc pyrophosphorylase. Lin et al. (12) isolated two Ar- thereby clearly demonstrated an essential role for ADPGlc abidopsis lines mutated at the Adgl and Adg2 loci that were pyrophosphorylase in starch synthesis. defective in starch synthesis and ADPGlc pyrophosphorylase activity. Plants of the adgl line, which accumulated very little THE FLOW OF CARBON INTO STARCH leaf starch, were devoid of both the large and small subunits of the ADPGlc pyrophosphorylase as viewed by immunoblot In actively photosynthesizing leaves, the events leading to analysis, whereas plants of the line adg2, which accumulated carbon flow into starch are restricted to the chloroplasts 40% as much starch, were deficient in the large subunit of (reviewed in ref. 17). The 3-PGA formed during CO2 fixation ADPGlc pyrophosphorylase. Therefore, the absence or is readily transformed into hexose monophosphates via glu- depression in the levels of starch synthesis can be attributed coneogenesis within the stroma where they can serve as to a direct causal relationship between defects in the expres- substrates for ADPGlc formation (Fig. 1). In young develop- sion of the structural genes for ADPGlc pyrophosphorylase ing leaves that serve as sinks, sucrose is first broken down and the concomitant lower amounts of enzyme activity. by an alkaline invertase or by the successive action of sucrose The rate of CO2 incorporation into starch by isolated chlo- synthase and UDPGlc pyrophosphorylase in the cytoplasm roplasts is directly correlated with 3-PGA levels and inversely correlated with Pi levels (17). This is consistent with the in vitro evidence that ADPGlc synthesis is controlled by the Hexose pathway activation and inhibition of ADPGlc pyrophosphorylase via these metabolites. Recently, a starch-deficient mutant of Glc 6-P do . Gic 6-P Chlamydomonas reinhardtii contained a defective ADPGlc py- that was less to allosteric acti- rophosphorylase responsive Gic 1-P - IT Gic 1-P vation by 3-PGA and inhibition by Pi. This provides direct ATP in vivo evidence that the allosteric activation by 3-PGA of chloroplastic ADPGlc pyrophosphorylase is essential for PP UDPG fructose maximum starch synthesis (2). A direct role for the allosteric ADPG regulation of the amyloplast enzyme in starch synthesis has yet to be resolved. However, Hnilo and Okita (8) have shown that when tuber slices were incubated in the presence of mannose, an effective sequestration agent of intracellular Pi, starch sucrose UDP the incorporation of "4C-sucrose into starch was enhanced by 50%. These results suggest that the activity of the amyloplast amyloplast cytosol enzyme is also modulated by intracellular Pi levels. Genetic studies also indicate a major role for ADPGlc SS-ADPG pathway pyrophosphorylase in starch synthesis of nonphotosynthetic developing sink organs. Maize mutations at two unlinked loci, Shrunken-2 (Sh2) and Brittle-2 (Bt2), result in a 60% reduction in starch levels in the endosperm with correspond- ADPG - -ADPG fructose ing decreases in ADPGlc pyrophosphorylase activities of 66% and 63%, respectively (17). Recent biochemical and molecular studies (3, 15, 17) have shown that Sh2 and Bt2 encode starch ADPGlc pyrophosphorylase subunits of 54 kD and 51 kD, sucrose ADP respectively, and that both subunits are required for maxi- mum enzyme activity and starch synthesis. ADPGlc pyrophosphorylase also seems to have a major role in starch synthesis in pea embryos. Mature seeds reces- sive at the Rb locus have reduced levels of starch and elevated Figure 1. The flow of carbon into starch: two hypotheses. The lipid and sucrose levels and contained less than 8% of the upper panel depicts the transport of hexose monophosphates into normal ADPGlc pyrophosphorylase levels (18). Although the amyloplasts and its conversion into starch; the bottom panel depicts nature of this genetic defect has not been determined at the the SS-ADPGlc pathway. In the upper panel, Glc 1-P is the preferred metabolite transported in wheat endosperm amyloplasts (1), molecular level, it is likely that the reduction of starch in the whereas Glc 6-P is specifically transported in pea embryo (7) and rb mutant pea lines is the result of a defect in ADPGlc root amyloplasts (6). In both instances, ADPGlc pyrophosphorylase pyrophosphorylase activity and not in any other major en- is required for the formation of ADPGlc, the substrate for starch zyme activity involved in starch synthesis. synthase. In contrast, the SS-ADPGlc pathway utilizes the cytosolic- In potato tuber, a 'reverse genetics' approach was used to localized sucrose synthase for the formation of ADPGlc, which is evaluate the role of ADPGlc pyrophosphorylase in starch subsequently transported into the plastid for starch synthesis. 562 OKITA Plant Physiol. Vol. 100, 1992

(1). The hexose sugars are then metabolized via glycolysis ages of sucrose and UDPGlc (1) are directly transported into into three carbon intermediates that are transported via the the amyloplasts, where they are converted into starch via the Pi translocator (6) into the chloroplasts where they can be combined action of ADPGlc pyrophosphorylase and starch metabolized further for starch synthesis. synthase. Although several studies (reviewed in refs. 15 and 17) have suggested the transport of C3 intermediates into isolated THE ALTERNATIVE PATHWAY OF STARCH SYNTHESIS amyloplasts, this does not appear to be the main pathway by which carbon enters starch synthesis. Keeling et al. (9) ex- An alternative pathway of carbon flow into starch has been amined the extent of redistribution of 13C between carbons 1 proposed by Akazawa et al. (16 and refs. cited therein). These and 6 in the starch glucosyl moiety when developing wheat authors (16) have shown that both chloroplasts and amylo- endosperm were fed [1_-3C]- or [6-13C]glucose or -fructose plasts contain an ADP/ATP translocator that also appeared and found that there was only partial redistribution (12-20%) to be capable of transporting radiolabeled ADPGlc, but not of '3C into starch between the Cl and C6 atoms (9). Because UDPGlc, with a percentage of the radioactivity incorporated the redistribution of label in the glucose moiety of starch into starch. The transport of ADPGlc and the subsequent would be expected to be more extensive if carbon flow into incorporation of the glucosyl moiety into starch led these starch occurred by the C3 pathway via triosephosphate iso- authors to suggest the direct synthesis of ADPGlc from merase, Keeling et al. (9) suggested that hexose monophos- sucrose and subsequent incorporation into starch. In this phates, and not triosephosphates, are the most likely candi- hypothesis, ADPGlc is synthesized directly from sucrose via dates for entry into the amyloplast. The observation of direct the action of a cytosolic sucrose synthase and then trans- import of hexose units into amyloplasts of potato tubers, fava ported across the amyloplast membranes where it could be beans (19), maize endosperm, and suspension cells of Chen- utilized by starch synthase, a process labeled as the SS- opodium rubrum (5) led to similar conclusions. Several studies ADPGlc pathway (Fig. 1). (1, 6, 7) have demonstrated the direct transport of hexose The proposed SS-ADPGlc pathway has the added advan- monophosphates as well as other metabolites into isolated tage over the commonly accepted routes of carbon flow into amyloplasts and plastids from nonphotosynthetic tissue. starch in being more energy efficient and is consistent with Tyson and ap Rees (reviewed in ref. 1) observed that only three lines of evidence. First, sucrose synthase can use ADP Glc 1-P served as an effective precursor for starch synthesis as the glucosyl acceptor to form ADPGlc (16). Second, a by intact wheat endosperm amyloplasts and that this labeling mutation in the major maize endosperm-specific form of of starch was dependent on the degree of intactness of this sucrose synthase, Sh1, results in a decrease in overall starch organelle. A somewhat different specificity of transport was levels. Third, the SS-ADPGlc pathway is consistent with the exhibited by pea amyloplasts (7), which readily took up and asymmetric distribution of carbon in the glucose moiety of incorporated Glc 6-P, but not Glc 1-P, into starch at rates sucrose, starch, and phosphate esters when green leaves are comparable with those measured in vivo (7). The incorpora- exposed to "4CO2 under photosynthetic conditions or are fed tion of 14C label from Glc 6-P was dependent on the presence [Cl-11C]glucose, or when rice grains are radiolabeled with of ATP and on the integrity of the amyloplasts. In contrast, [(U-'4C)glucose, [6-3H-fructose]-sucrose (see 16 and refs. cited an amyloplast preparation from developing maize endosperm for a more complete discussion). preferred dihydroxyacetone phosphate for uptake and incor- It should be pointed out, however, that these studies only poration into starch, although Glc 6-P and fructose 1,6- indirectly support the SS-ADPGlc pathway; moreover, they bisphosphate were also transported, albeit at lower levels cannot discriminate between the various hypotheses pro- (reviewed in ref. 10). posed for the flow of carbon into starch, i.e. they are also not Heldt et al. (6) have proposed that amyloplasts and other inconsistent with the consensus pathways of starch synthesis. plastids of nonphotosynthetic tissue contain a modified Pi An important distinction between the SS-ADPGlc hypothesis translocator with a relatively broad metabolite specificity as and the consensus pathways of starch synthesis is the sub- inferred from the transport capabilities displayed by plastids cellular location of ADPGlc formation that is required for from pea root. These plastids contain a Pi translocator similar starch synthesis. Evidence obtained from more recent studies, to the type observed in chloroplasts in their ability to trans- discussed above and elaborated below with regard to this port Pi, dihydroxyacetone phosphate, and 3-PGA in a coun- alternative pathway, demonstrate that the sugar nucleotide ter-exchange mode, but the root-specific Pi translocator is utilized by starch synthase is synthesized in the plastid. also able to transport Glc 6-P but not Glc 1-P. Because pea If the SS-ADPGlc pathway were a major route, starch root plastids also appear to lack a plastid fructose 1,6-bis- synthesis would be dependent on sucrose synthesis and phosphatase, Heldt et al. (6) reasoned that the transport of ADPGlc pyrophosphorylase would have a minor role in Glc 6-P would not only allow entry of carbon into starch but ADPGlc formation and, hence, in starch production (Fig. 1). would also provide a source of reducing power via the These predictions are not supported by the results obtained oxidative pentose phosphate pathway required for nitrite from the biochemical-genetic analyses of plant mutants. As reduction. discussed earlier, Clarkia mutants defective in the cytosolic In light of the above discussion, it appears that the flow of PGI accumulated less sucrose under limiting light conditions carbon from the cytoplasm into starch accumulated by the (11). If ADPGlc were formed from sucrose via the action of amyloplasts does not involve C3 intermediates; instead, car- sucrose synthase (Fig. 1), then starch levels should also be bon is directed into starch in a more direct route (Fig. 1). depressed. Instead, increased levels of starch were observed, Hexose monophosphates formed from the successive cleav- indicating that the restriction of carbon into sucrose in the THE FLOW OF CARBON INTO STARCH 563

cytosol caused a rerouting of this carbon into starch in the hexose monophosphates across the amyloplast membranes? plastid (11). Results of the study of mutants defective in With regard to the latter, the permeability properties of the chloroplast PGI are also inconsistent with the SS-ADPGlc isolated amyloplast are completely dependent on the degree pathway, which predicts that a defect in plastid PGI would of intactness, but present methods to assess amyloplast integ- have no significant effect on starch synthesis. Instead, re- rity rely on metabolic activities and enzyme latency studies duced starch but not sucrose levels were observed in the (1) and on cytological examination (16). These criteria, how- plastid PGI mutants, indicating that the ADPGlc formation ever, are insufficient to assess the extent of membrane dam- required for starch synthesis was restricted to the chloroplasts age and cytoplasmic contamination that can occur during (11). amyloplast isolation (see ref. 1 for more discussion). In many The most compelling evidence for a direct role of ADPGlc respects, the current definition of amyloplast intactness is at pyrophosphorylase in ADPGlc formation is also derived from a stage comparable with the time preceding the development mutant analysis. The Arabidopsis mutants adgl and adg2 (12, of cellular fractionation techniques for the successful isolation 17), the maize endosperm mutants sh2 and bt2 (3, 15, 17), of intact functional chloroplasts (20). Once it was recognized the transgenic potato plants harboring antisense DNA con- that the ability to fix CO2 was dependent on chloroplast structs to an ADPGlc pyrophosphorylase gene (13), and a intactness, 'Class A' chloroplasts were routinely obtained Chlamydomonas mutant (2) have depressed levels of starch (20). A similar biochemical test to assess the intactness of the and deficient levels of ADPGlc pyrophosphorylase activities amyloplast must be implemented before the transport prop- due to specific losses of one or both of the ADPGlc pyro- erties of this organelle can be accurately studied. In the case phosphorylase subunits or reduction in allosteric activation of pea embryo amyloplasts (7) and pea root plastids (6), the by 3-PGA. Likewise, a direct correlation was also observed uptake of metabolites and their incorporation into starch and between the levels of starch and ADPGlc pyrophosphorylase as a source of reducing power for nitrite reduction, respec- enzyme activity and not between starch and sucrose synthase tively, may be useful criteria to establish the degree of in- activity in the pea embryo mutant rb (18). These studies tactness and, hence, facilitate efforts that will lead to a better demonstrate direct causal relationships between decreased understanding of the biochemistry of this specialized, starch- levels in gene products, of enzyme activities, and of starch containing organelle. levels, and therefore substantiate the view that ADPGlc pyrophosphorylase is absolutely required for starch synthesis. ACKNOWLEDGMENTS Therefore, one can conclude that, on the basis of the results I thank Professors Frank Loewus and Gerald Edwards and Dr. of these studies (2, 3, 12, 13, 15, 17), the ADPGlc used for Mirta Sivak for their helpful discussions and editorial suggestions on starch synthesis is formed in the plastid via the activity of this minireview. I apologize to the many workers in the area of ADPGlc pyrophosphorylase and that the SS-ADPGlc path- carbon metabolism whose publications were not referenced in the way plays little, if any, role in this process. manuscript. The omission was not due to an oversight but rather dictated by space limitations. CONCLUSIONS LITERATURE CITED Overwhelming biochemical evidence gathered in the past 1. ap Rees T, Entwistle G (1989) Entry into the amyloplasts of years has documented the events leading to starch production carbon for starch synthesis. In CD Boyer, JC Shannon, and in chloroplasts. Carbon flow into starch is mediated by glu- RC Hardison, eds, Physiology, Biochemistry, and Genetics of coneogenesis, and one of the pivotal reactions, ADPGlc for- Nongreen Plastids. American Society of Plant Physiologists, is controlled the allosteric of ADPGlc Rockville, MD, pp 49-62 mation, by activity 2. Ball S, Marianne T, Dirick L, Fresnoy M, Delrue B, Decq A pyrophosphorylase (15, 17). Although several studies have (1991) A Chlamydomonas reinhardtii low-starch mutant is de- suggested the presence of a similar pathway in developing fective for 3-phosphglycerate activation and orthophosphate sink organs, sufficient evidence has been gathered indicating inhibition of ADP-glucose pyrophosphorylase. Planta 185: that most of the carbon is routed into starch by the direct 17-26 3. Bhave MR, Lawrence S, Barton C, Hannah LC (1990) Identi- transport and utilization of hexose monophosphates (1, 6, 7). fication and molecular characterization of Shrunken-2 cDNA Likewise in several developing sink organs, evidence has clones of maize. Plant Cell 2: 581-588 been obtained from biochemical and genetic studies (1, 2-4, 4. Caspar T, Huber SC, Somerville C (1986) Alterations in growth, 8, 11-13, 17, 18) that conclusively demonstrates that ADPGlc photosynthesis and respiration in a starch mutant of Arabidop- pyrophosphorylase activity is absolutely essential for starch sis thaliana (L.) Heynh deficient in chloroplast phosphogluco- mutase activity. Plant Physiol 79: 11-17 synthesis. Although the alternative SS-ADPGlc pathway is a 5. Hatzfeld W-D, Stitt M (1990) A study of the rate of recycling potential source of ADPGlc, there is no evidence that directly of triose phosphates in heterotrophic Chenopodium rubrum supports the formation of ADPGlc in the cytoplasm and its cells, potato tubers, and maize endosperm. Planta 180: incorporation into starch in leaf tissue and in developing sink 198-204 6. Heldt HW, Flugee U-I, Borchert S (1991) Diversity of specificity organs. On the contrary, results of biochemical-genetic stud- and function of phosphate translocators in various plastids. ies (4, 11, and refs. cited in 15, 17) discount the utilization of Plant Physiol 95: 341-343 the SS-ADPGlc pathway as a major route leading to starch 7. Hill LM, Smith AM (1991) Evidence that glucose 6-phosphate synthesis. is imported as the substrate for starch synthesis by the plastids of developing embryos. Planta 185: 91-96 Questions that remain unresolved include to what extent 8. Hnilo J, Okita TW (1989) Mannose feeding and its effect on does ADPGlc synthesis occur in the cytoplasm and what are starch synthesis in developing potato tuber discs. Plant Cell the rates of transport of ADPGlc, triosephosphates, and Physiol 30: 1007-1010 564 OKITA Plant Physiol. Vol. 100, 1992

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