Ann. Rev. Plant Physiol. 1986. 37:233-46

FRUCTOSE 2,6-BISPHOSPHATE AS A

REGULATORY METABOLITE IN

PLANTS!

Steven C. Huber

U.S. Department of Agriculture, Agricultural Research Service, North Carolina State

University , Raleigh, North Carolina 27695-7631

CONTENTS

OCCURRENCE IN PLANT TISSUES....... . . . . ....... ...... . . . . . . . . . .... . . . ... . . .... . . . . . . . .... 234

ENZYMES REGULATED BY F26BP ...... . . . . ..... . . ..... . . . . . . . . . . ... . . . . . . . . ...... . . . ... ..... 235

REGULATION OF F26BP CONCENTRATION........... . . . . . . . ....... . . . . . . ...... . . . ... . .... 237 Metabolic Control of F6P, 2KIF26BPase ... .. .. . . . . . . . . ..... . . . . . . . ...... . . . ...... . . . . . . . . . . 237 Possible Coarse Control... . . .. .. . . . . . . .... . . . . . .. ... . . . . . . . . ..... . . . .. . . . ..... . . . . . ..... . . . . . . . . 238

ROLE IN GLyCOLySIS..... . . . . . . ....... . . . . . . ........... . . .. . . . . . . . .. . . . . . . . . .................... 238 Metabolite-Mediated Interconversion of Enzymes............... . . . . . . .. . ... . . . . . . ... ... . . . . 239 Molecular Forms of PFP . . .... . . . . . . . . . . ...... . . . . . . . . . ..... . . . . . . . .... . . . . . . . .... . . . ... . . . ..... 239

ROLE IN GLUCONEOGENESIS.. . . . . . . ...... . . . . . ... . . . ..... . . . . . . ....... . . . . . ..... . . . . ......... 240 Effects on FBPase ..................... ............ .. . . . . .......... .. ............. . . . ...... . . . . . . . 240 Photosynthetic Tissues ........................................................................... 241 Compartmentation in C4 Plants ........ _ _ _ _ ............. . ..... . . .. ... ..... ...... . ..... . . . . . . . .. 242 Nonphotosynthetic Tissues....................................................................... 243

SUMMARy.... . . ... . . . . . . . . . . . . ... . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . ... . . . . . . . . . . 243

Hexose phosphates play a central role in plant carbohydrate metabolism. The degradation of hexose phosphates via glycolysis and the oxidative pentose phosphate pathway is a common characteristic of plant cells, whereas hexose phosphate production, via photosynthesis (reductive pentose phosphate path­way) or gluconeogenesis, occurs in only certain tissues. Some cells, then, have the ability to both degrade and form hexose sugars, and certain enzymes must be highly regulated to prevent futile cycling. Within the past five years, two major discoveries have been made that modify our understanding of plant carbohydrate metabolism: (a) the discovery of fructose 2,6-bisphosphate

IThe us Government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this paper.

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234 HUBER

(F26BP),2 a new regulatory metabolite, and (b) the discovery of a pyrophos­phate-dependent phosphofructokinase in plants.

The aim of this chapter is to review the role of F26BP in the regulation of metabolism in higher plants. Detailed treatment of the overall pathways of hexose metabolism is beyond the scope of this paper. Where necessary or beneficial, reference to animal systems will be made.

OCCURRENCE IN PLANT TISSUES

The occurrence of F26BP in plants was established first in mung bean seedlings by Sabularse & Anderson in 1981 (28). Since that time, F26BP has been identified in a range of tissues, including isolated guard cells (14), leaves (7), and storage tissues (44). With the development of an extremely sensitive bioassay (44, 45), the concentration of F26BP in tissues can be measured accurately, and changes in the metabolite under different conditions can be monitored. In general, the concentration of F26BP in tissues is in the range of 0.1 to 1 nmollg fresh weight (17 ,21, 37,44). Assuming that F26BP is restricted to the cytosol (5), and that the latter is 10% of the tissue volume, the concentra­tion of F26BP would range from 1 to 10 folM. Approximately tenfold higher concentrations of F26BP have been measured in Jerusalem artichoke tubers (44). This does not appear to be a general characteristic of storage tissues because potato tubers contain less than 0.5 nmol F2 6BP/g fresh wt (44). Earlier estimates of extremely high concentrations of F26BP in leaf tissue (up to 30 nmo1!g fresh wt) (7) have not been confirmed. Plant tissues, in general, can

2 Abbreviations used:

DHAP, FBP,

FBPase, F26BP, F26BPase, F6P, F 6P,2K, GIP, GI,6P, G6P, Hexose-P,

P-esters,

PFK, PFP,

PGA, Ru5P,

SPS,

Triose-P, UDPGlc, 6PGlu,

dihydroxyacetone phosphate fructose I,6-bisphosphate

fructose 1,6-bisphosphatase (Ee 3.1.3.11) fructose 2,6-bisphosphate

fructose 2,6-bisphosphatase (EC 3.1.3)

fructose 6-phosphate

fructose 6-phosphate,2-kinase (EC 2.7.1)

glucose I-phosphate glucose 1,6-bisphosphate glucose 6-phosphate hexose phosphates phosphate esters

phosphofructokinase

pyrophosphate: fructose 6-phosphate phosphotrans-ferase (EC 2.7.1.90)

3-phosphoglycerate ribulose 5-phosphate

sucrose phosphate synthase

triose phosphates UDP-glucose 6-phosphogluconate.

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FRUCTOSE 2,6-BISPHOSPHATE IN PLANTS 235

rapidly adjust the concentration of F26BP severalfold, indicating the capacity for F26BP metabolism (synthesis/degradation). This aspect will be discussed in more detail later.

ENZYMES REGULATED BY F26BP

To date, seven enzymes have been identified that are regulated by F26BP; four of these are activated by F26BP and three are inhibited (Table 1). In general, enzymes that may be involved in glycolysis are activated by F26BP, whereas at least one enzyme of gluconeogenesis is inhibited. Of central importance are the opposing effects of F26BP on enzymes involved in the interconversion of F6P and FBP. Soon after the discovery of a PPi-linked PFK (termed PFP) in plant tissues (4), it was recognized that the enzyme from most tissues was activated strongly by F26BP (28, 29). Although the enzyme is freely reversible, it is the only enzyme in addition to A TP-dependent PFK that is capable of converting F6P to FBP. It is also important to note that ATP-PFK from plant tissues is unaffected by F26BP (7). Animal tissues do not contain PPi-PFK, but the ATP-dependent PFK is strongly activated by F26BP (reviewed in 15, 43). It is probable that PFP functions, at least under some conditions, in the glycolytic direction, and this will be discussed in more detail below. Formation of F6P from FBP during gluconeogenesis is catalyzed by a specific FBPase; the cytosolic, but not the plastid enzyme is strongly inhibited by F26BP (7). The differential effect of F26BP on enzymes involved in F6P/FBP interconversion constitutes the central core of a regulatory scheme whereby the flux of carbon through major pathways may be regulated.

In addition, two novel enzymes that may be involved in sucrose utilization have been reported to be activated by F26BP. In tissues that import sucrose and are active in cell wall biosynthesis and/or starch storage, sucrose synthase is thought to play a key role in sucrose breakdown: sucrose + UDP � UDPGlc +

fructose. The UDPGlc formed may be converted to GIP via UDPGlc pyrophos­phorylase or by UDPGlc phosphorylase, the latter of which has been reported to be activated by F26BP and involves Pi rather than PPi as substrate (13). This work needs to be confirmed, as do studies conducted to determine how widely distributed UDPGlc phosphorylase activity is. A source of PPi (for either UDPGlc pyrophosphorylase or the forward direction of PFP) could be the pyrophosphorolytic cleavage of ATP, which is activated by F26BP (10). The occurrence of ATP pyrophosphorylase in tissues other than com scutellum has not been established. Phosphoglucomutase is common to pathways of hexose breakdown and utilization, and it has been determined that F26BP can replace G I ,6P as obligate cofactor (11). The existence of G 1 ,6P in plants has not been firmly established, and thus F26BP may assume this role in plants. It is interesting that 6PGlu dehydrogenase is inhibited strongly by F26BP (27). Hence, F26BP may activate glycolysis but inhibit the oxidative pentose phos-

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Table 1 Enzymes regulated by F26BP

Enzyme Reaction catalyzed

UDPGlc phosphorylase UOPGlc + Pi ==== UOP + GlP

A TP pyrophosphorylase ATP � AMP + PPi

PFP F6P + PPi ==== FBP + Pi

Phosphoglucomutase OIP ==== 06P

Cytosolic FBPase FBP � F6P + Pi

6PGlu DH 6PGlu + NAOP � Ru5P + NADPH + CO2

Trehalose phosphorylase Trehalose + Pi � GlP + glucose

8A, activation; I, inhibition.

Tissue

Potato tuber

Corn scutellum

Many tissues

Mung bean seedlings, animal sources

Spinach leaf, castor bean

Cytosolic and plastid isozymes from

castor bean endosperm

Euglena gracilis

Effect"

A

A

A

A

N Vol 0'1

::I: c::: txl tIl :::e

Reference

13

10

3, 4, 19a, 26, 29, 32

II

16, 20,38

27

27a

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FRUCTOSE 2,6-BISPHOSPHATE IN PLANTS 237

phate pathway. Not only the rate, but also the pathway involved in hexose degradation may be controlled by F26BP. In some algae, trehalose is a major reserve carbohydrate, and the metabolism of trehalose may be regulated by F26BP as a result of inhibition of the reversible enzyme trehalose phosphory­lase (27a).

REGULATION OF F26BP CONCENTRATION

Central to an understanding of the role of F26BP in metabolism is an apprecia­tion of the biochemical control mechanisms that regulate F26BP concentration. F26BP metabolism involves a specific kinase and phosphatase that catalyze the reactions:

ATP ADP Pi

F6P F26BP --4) F6P

F6P,2K F26BPase

In animal tissues, both activities are catalyzed by the same protein (9). Whether this is also true in plants has not been established, but the two activities do co-purify (5, 6, 35).

Metabolic Control of F6P,2KIF26BPase

The concentration of F26BP in vivo will be a function of the relative activities of F6P ,2K and F26BPase, both of which are sensitive to metabolic regulation (fine control). The kinase and phosphatase have been characterized from several tissues, including spinach leaf (5, 6, 35), maize leaf (33) and root (32, 36), castor bean endosperm (21), and several storage or sink tissues (36). In general, similar properties have been identified. F6P ,2K exhibits sigmoidal saturation kinetics with respect to F6P; Pi is a strong activator that increases V max and induces hyperbolic saturation kinetics for F6P. Even in the presence of Pi, the Km for F6P is sufficiently high (0.4 to 0.6 mM) so that changes in F6P concentration in situ would be expected to influence kinase activity. DHAP and PGA are inhibitors, and each interacts differently with the activator Pi. PGA inhibits F6P ,2K even in the absence of Pi, and the two are antagonistic. DHAP, in contrast, has no effect on kinase activity in the absence of Pi, and Pi cannot relieve the inhibition (35). This explains why DHAP was not reported as an effector of F6P,2K in the original study (6). The partially purified F6P,2K preparations studied also catalyze the release of phosphate from the 2-positior. of F26BP to yield F6P. The activity is relatively specific for F26BP and does not require a divalent cation. The Km for F26BP is about 1 J.l.M (38), and the activity is inhibited by Pi and F6P (6, 21, 38). There are no known activators of the phosphatase. Sensitivity to effectors varied greatly when enzymes from different sink tissues were compared (36), suggesting that differences in the regulation of metabolism in various tissues may need to be investigated.

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238 HUBER

Differences also appear to exist in regulatory properties of the enzyme from maize (a C4 plant) and spinach (a C3 plant). With both enzymes, Pi i� a strong activator of the kinase and an inhibitor of the phosphatase. However, with the maize enzyme, DHAP and PGA inhibit F26BPase as well as F6P,2K (33). Furthennore, phosphoenolpyruvate also strongly inhibits both activities, and several metabolites that have no effect on the spinach enzyme selectively inhibit either F6P,2K or F26BPase: oxaloacetate (F6P,2K), pyruvate, and UDPGlc (F26BPase) (33). The metabolic and physiological significance of these differ­ences in properties remains to be established.

Possible Coarse Control As described above, metabolic or "fine" control of F6P ,2K1F26BPase has been finnly established. The enzyme from animal tissues is also highly regulated by metabolic intennediates and is also subject to covalent modification, which constitutes an overriding control mechanism. In liver, a cAMP-dependent protein kinase phosphorylates the protein, which results in inhibition of the kinase and activation of the phosphatase activity (9, 15). The specific protein kinase is subject to honnonal control, and thus F26BP metabolism in animal tissues is regulated at several levels. Preliminary attempts to phosphorylate the partially purified spinach leaf enzyme with cAMP-dependent protein kinase have been unsuccessful (5). However, the possibility that a specific protein kinase is involved, or that some other covalent modification mechanism exists, has not been investigated. It seems likely that a coarse level of control is involved, and this will be an important area for future study.

ROLE IN GLYCOLYSIS

The capacity for hexose degradation via glycolysis and the oxidative pentose phosphate pathway is regarded as a universal characteristic of plant cells (1), including mature photosynthetic tissues (34), One of the central questions regarding the role of F26BP in glycolysis concerns whether PFK and/or PFP function in the fonnation of FBP from F6P. Because PFK is irreversible, it can only function in glycolysis, whereas PFP, being freely reversible, could func­tion in either the glycolytic or gluconeogenic direction. If PFP is to function in the glycolytic direction, an adequate pool of PPi must be present. Recent studies have verified that a substantial PPi pool (5 to 39 nmol/g fresh wt) exists in various tissues of pea and com seedlings (8, 30). In addition, Smyth et al (31) have recently demonstrated a PPi-dependent glycolytic pathway (F6P to pyru­vate) in extracts from pea seeds that was responsive to added F26BP. Ex­periments such as these establish the potential for PPi-linked glycolysis, but the actual flux of carbon through either pathway remains to be established. Two fundamentally different mechanisms have been proposed to explain how the freely reversible PPi-PFK may actually function in glycolysis; these are now considered separately.

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FRUCTOSE 2,6-BISPHOSPHATE IN PLANTS 239

Metabolite-Mediated Interconversion of Enzymes

Recently, Buchanan and coworkers (2, 46, 47) postulated that PFK and PFP can be reversibly interconverted in vitro by incubation with certain metabolites. Specifically, F26BP catalyzed the conversion of PFP to PFK, which being irreversible can only function in the glycolytic direction. The principal evi­dence for the F26BP-mediated conversion of PFP to PFK is measurement of an initial rapid reaction velocity (hyperactive phase) that is not sustained beyond a few minutes. The initial burst of ATP-dependent activity in the presence of F26BP is not catalyzed by PFK, but rather by metabolism of PPi (present as a contaminant in A TP) by PFP (a contaminant of the PFK preparation). Con­sequently, PFK from castor bean endospenn that is prepared absolutely free of contaminating PFP activity does not yield hyperactive kinetics with F26BP (24). In addition, apparent metabolite conversion of PFK to PFP does not result in a decrease in actual PFK activity (12), as might be expected if one fonn was converted to the other.

Similarly, the apparent conversion of PFK to PFP in the presence of UDPGlc plus PPi can be explained by utilization of UTP by PFK (22). The UTP is formed from the UDPGlc plus PPi by action of UDPGlc pyrophosphorylase, a contaminant in the auxiliary enzymes used in the PFK assay. In the absence of auxiliary enzymes, PFK from rabbit muscle could not use PPi as substrate (22). To date, there is no convincing evidence to support the notion of "metabolite­mediated catalyst conversion."

These results also emphasize the need for extreme caution about enzyme and reagent purity when studying F26BP effects. Because F26BP is an effective regulator of enzyme activities at low concentrations (""M), but enzyme assays generally involve substrate amounts (mM) , trace contaminants can give erroneous results. New workers who come into this area need to be acutely aware of the need for caution.

Molecular Forms of PFP

A working model to explain how F26BP and PFP may be involved in glycolysis has been forwarded by Black and coworkers (2a). It was originally observed that the activity of PFP and its sensitivity to F26BP changed markedly during gennination and development of pea seedlings (48). Gel filtration experiments resolved two peaks of PFP activity in pea (48) and wheat (50) seedlings: a large form that was relatively insensitive to F26BP and a small form that was activated to a much greater extent. Subsequent work with pea seedlings demon­strated the existence of two interconvertible forms of PFP, with sedimentation coefficients of 6.3 and 12.7S in sucrose density gradient ultracentrifugation (49). The large form has a high ratio of glycolytic to gluconeogenic activity, whereas the reverse is true for the small form. Importantly. F26BP promotes the association of the small form to yield the large form. Thus, the presence of

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240 HUBER

F26BP results in a molecular form of PPi-PFK that favors glycolysis (2a). The wheat enzyme contains two subunits, designated (X and 13; the large form is (X2f32

and the small form is probably 132 (50). PFP has been partially purified and kinetically characterized from castor

bean endosperm (18, 19, 23, 26), mung bean hypocotyl (28, 29), spinach leaf (7), potato tuber (45), and pineapple leaves (3). With the exception of the enzyme from pineapple leaves (3), enzyme activity is virtually dependent upon F26BP. Differences in relative F26BP activation may reflect variable F26BP contamination of some commercial sources of F6P (25); however, species differences noted within the same study cannot be attributed to this (4). Contamination of F6P with F26BP may also explain sigmoidal kinetics; when this problem is eliminated, hyperbolic kinetics for F6P are observed with the enzyme from castor bean endosperm (25), which confirms the earlier results obtained with the mung bean enzyme (28).

The enzyme is confined to the cytosol and has similar activities and responses to effectors in both the forward and reverse directions; F26BP activates and Pi inhibits the reactions. However, stimulation of the reverse '[eaction (F6P production) by F26BP is substantially less than in the forward reaction (FBP production). Activation of PFP by F26BP can be modulated by the con­centrations of F6P and Pi; F6P decreases the Ka for F26BP, whereas Pi

increases the Ka and reduces Vrnax (26). Thus, an increase in the F6P/Pi ratio would increase the activation of PFP at a fixed, limiting F26BP concentration. As described above, an increase in the F6P/Pi ratio might also result in increased F26BP concentration by activation of F6P ,2K. Thus, activity of PFP in vivo may be a function of the concentrations of F6P, Pi, and F26BP.

ROLE IN GLUCONEOGENESIS

It is generally accepted that F26BP plays a role in the regulation of glu­coneogenesis. The basis for this involvement is inhibition of cytoplasmic FBPase, which catalyzes the first irreversible step in the conversion of triose-P to sucrose: Fru 1 ,6-P2 � F6P + Pi.

Effects on FBPase

Cytosolic FBPase has been purified from spinach leaves (16, 38, 41) and castor bean endosperm (20) and characterized with respect to F26BP regulation. 10

general, the properties of the enzyme from these two tissues are very similar. In the absence of effectors,._

�he Km for FBP is quite low (about 2 �M). Micromolar concentrations of F26BP cause marked inhibition of enzyme activity and convert hyperbolic saturation kinetics to sigmoidal (20, 38). Thus, it appears that F26BP is not a simple competitive inhibitor, but it may be an allosteric

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FRUCTOSE 2,6-BISPHOSPHATE IN PLANTS 241

competitive inhibitor that binds to a site other than the catalytic site. Plant FBPase is also inhibited by AMP and Pi, but the inhibition is weak and positive cooperativity is not induced. However, a striking synergism between AMP and F26BP has been observed with both spinach leaf and castor bean endosperm FBPase (20,38,41). Furthermore, the combined effect of AMP and F26BP is further amplified by Pi. The complex regulation of cytosolic FBPase by F26BP and several other effectors appears to explain to a large extent how sucrose formation may be regulated in leaves under different conditions.

Photosynthetic Tissues

In the light, chloroplasts export triose-P to the cytosol where they are converted to sucrose. The rate of triose-P conversion to sucrose must be regulated with respect to the rate ofe02 assimilation (which determines triose-P availability); without such regulat!0!l, stromal metabolites could be dePJ�t�<! rapidly and photosynthesis strongly inhibited under suboptimal conditions. The fact that this does not occur is taken to indicate that a feedforward (39) control of photosynthetic sucrose formation exists. When photosynthetic rates are high, and the rate of sucrose production exceeds the rate of sucrose export, an increased proportion of photosynthate is diverted from sucrose toward starch. This constitutes a feedback (40) type of control that affects partitioning of fixed carbon between starch and sucrose. It is clear that F26BP plays an important role in both feedforward and feedback control mechanisms.

FEEDFORW ARD CONTROL The pioneering experiments of Stitt et al (39)

established that an inverse relationship exists between photosynthetic rate and leaf F26BP concentration. The increases in F26BP concentration would inhibit cytosolic FBPase activity in situ, thereby slowing the rate of sucrose formation under limiting conditions. In this situation, the rise in F26BP concentration can be attributed to lowered concentrations of DHAP, which would increase F6P ,2K activity. The concentration of DHAP changes with the photosynthetic rate to a much greater extent than other metabolites; consequently, Stitt et al (38) have assessed the physiological status of leaf tissue under different con­ditions on the basis of DHAP content of the leaf material. Also, it is likely that as the concentration of P-esters declines as photosynthesis is reduced, the concentration of Pi will be increased. Thus, Pi and DHAP will fluctuate inversely, and for a small change in their absolute concentrations, the Pi/DHAP ratio can change to a greater extent. Under limiting conditions for photosynthe­sis, the large increase in the PilDHAP concentration ratio would activate F6P,2K activity, and the increase in Pi would inhibit F26BPase activity. Another factor contributing to the reduction in FBPase activity would be lower concentrations of the substrate FBP.

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FEEDBACK CONTROL When sucrose production exceeds the rate of assimi­late export, sucrose will accumulate to a higher level than normal and photo­synthate will then be diverted to starch. This shift in partitioning between starch and sucros-e -isasS-ociated with increased concentrations of F26BP (17, 40),

which would inhibit cytosolic FBPase and thereby slow carbon flux into sucrose. This would then restrict export of triose-P out of the chloroplast and thereby stimulate starch synthesis. It is clear that F26BP plays a role in this feedback control mechanism, but all of the details are not understood at present.

Under conditions of excess sucrose accumulation, there is an accumulation of DHAP and hexose-P (17, 40), and this is presumably associated with a reciprocal drop in Pi concentration. The observed increase in F6P concentration

-is greater than that of DHAP. Recall that these P-esters affect F6P,2K in opposite directions; F6P can be considered an activator (as substrate in a reaction with a relatively high Km), whereas DHAP is an inhibitor of the kinase. In addition, F6P directly inhibits F26BPase activity. Collectively, the net result is an increase in F26BP concentration that along with other shifts in metabolites would substantially inhibit FBPase activity in situ (38).

Even though F26BP plays a key role in the feedback mechanism, it may not

be the primary or initial factor. As sucrose accumulates and the rate of sucrose formation declines, there is an increase in the concentrations of UDPG1c and F6P, the substrates of SPS (40). This indicates that SPS activity is regulated and has been inhibited in situ by some mechanism. Thus, in feedback control, F26BP concentration may be adjusted in response to the regulation of SPS activity (17a). This is an important area for future study.

Compartmentation in C4 Plants

A new concept in the area of C4 photosynthesis is that sucrose formation may be strictly compartmented within the mesophyll cells of maize. Usuda & Edwards (42) initially concluded that at least some of the enzymes involved in sucrose biosynthesis are enriched in mesophyU cells, and recently SolI et al (33)

established that F6P,2K and F26BPase were primarily, if not exclusively, localized in the mesophyll tissue. Hence, it appears that the enzymatic capacity and associated regulatory systems of sucrose and starch metabolism are strictly compartmented between the mesophyll and bundle sh,eath cells. Future studies will undoubtedly examine the intercellular distribution of other key enzymes, namely, PFP and PFK. It should be stressed that the localization of sucrose synthesis in the mesophyll cell has only been suggested for maize; earlier work with other C4 species conclusively established that SPS activity is distributed between the two cell types (for review see 7a). More extensive studies with a range of species from the three C4 subgroups will be required to resolve this issue.

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FRUCTOSE 2,6-BISPHOSPHATE IN PLANTS 243

Nonphotosynthetic Tissues

Leaves are not the only tissues active in gluconeogenesis. During germination and early growth of certain plants such as castor bean, there is a high rate of conversion of fat to sucrose in the endosperm. A cytoplasmic FBPase that is displaced from eqUilibrium in vivo (1) has been long recognized as an important regulated step in the gluconeogenic pathway. Castor bean endosperm contains F26BP, the enzymes involved in its metabolism (21), and an F26BP-inhibited cytosolic FBPase (20). Various treatments that are known to inhibit glu­coneogenesis result in a rapid increase in F26BP concentration in this tissue (21). Hence, F26BP appears to play an important role in the regulation of gluconeogenesis in nonphotosynthetic as well as photosynthetic tissues.

SUMMARY

F26BP plays a central role in the regulation of carbon metabolism in plants. It appears to function as an activator of glycolysis and inhibitor of glu­coneogenesis. F26BP and the enzymes involved in its synthesis and degrada­tion have been identified in a range of tissues, and it is likely the occurrence of this regulatory system will be universal in plant tissues. Metabolic fine control of the activity of F6P ,2K1F26BPase is well established, but the significance of differences in properties of the enzyme from various sources needs to be established. Another question concerns whether some coarse control mech­anism(s) exists along with the metabolic control of F26BP metabolism.

A number of enzymes have been identified that are regulated by F26BP. The cytosolic FBPase and PFP occur widely in plants, but the distribution of some of the other regulated enzymes has not been explored. Even though PFP occurs widely, can it operate in both directions in situ? Many specific questions remain to be answered, but a general scheme is now available that can be used as a working model to study the regulation of carbon metabolism. The next few years should reveal new insights in this area.

ACKNOWLEDGMENTS

I am grateful to the numerous colleagues who shared their preprints and unpublished data with me. I also thank Drs. C. C. Black and P. S. Kerr, and Ms. W. Kalt-Torres for reading the manuscript and making helpful sugges­tions, and Ms. Adina Homer for her expert assistance in preparing the manu­script. This has been part of the cooperative investigations of the USDA-ARS and NCARS. This is paper No. 10136 of the Journal Series of the NCARS, Raleigh, North Carolina.

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