Regulation of the system is required to balance the autotrophic and the heterotrophic properties of the cell. It is also worth noting that it is not just the rate at which carbon fixation and resource partitioning occurs but the concentrations of metabolites must be kept within a tight range so that they do not upset the kinetic properties of other enzymes in the pathway.
For the purposes of this essay I shall divide regulation of sucrose synthesis into two types, physical separation through the use of a transporter and alteration of enzymes kinetic properties.
Physical Separation.
In order for the triose phosphates to be converted into sucrose they must first be transported out of the chloroplast via the triose phosphate transporter (TPT). The TPT constitutes up to 15% of the total protein in the envelope membrane. Each TPT family protein consists of about 400-450 amino acyl residues with 5-8 putative transmembrane α-helical spanners although the plant chloroplast TPT is thought to have approximately 6 (USCD 2002).
Original in vitro studies on isolated chloroplasts revealed that the TPT has a very high level of activity in an infinite substrate (is that the correct term for a transported molecule?) and in order to obtain adequate levels of triose phosphate in the chloroplast it is necessary to restrict the transport rate by limiting the levels of Pi in the external solution (Stitt 1987). This gave rise to the idea that there was a strict 1:1 ratio of transport between Pi and triose phosphate (also PGA) in and out of the chloroplast.
The strict “one in one out” nature of the TPT provides a good site for regulation. If there is too much sucrose synthesis occurring then there will be a greater than 1/6th triose phosphate export from the chloroplast due to the influx of free Pi and that will inhibit regeneration of Rubisco thus limiting the amount of available triose phosphate and the rate of sucrose synthesis. If there is too much available triose phosphate the sucrose synthesis pathway will become “saturated” with substrate and the Pi release at the end will be at a maximum thus limiting the rate of the TPT. The overflow of triose phosphate in the chloroplast must go somewhere otherwise photodamage will occur and so it is partitioned off into starch synthesis. This is one of the reasons why we see an abrupt cessation in the rate of sucrose synthesis and the change over to starch synthesis.
Enzyme Modulation
The modulation of the kinetic properties of key enzymes in any metabolic pathway can have profound effects on the regulation and control of flux through it. Current opinion divides the field of enzyme modulation into two categories. “Coarse” modulation involves altering the enzymes expression pattern or deactivating it through covalent modification. Such modulation takes time but changes the kinetic activity of an enzyme to a large extent. “Fine” modulation involves the use of allosteric effectors to bring about changes in the enzymes kinetics. Such effectors can be any kind of compound (substrate, steroid etc) that interacts to change the Vmax or km of an enzyme. These tend to bring about rapid small changes in an enzymes activity.
There are two types of reaction that enzymes can catalyse, reversible (near equilibrium) and irreversible (far from equilibrium). General consensus in the 70’s and 80’s was that enzymes that catalysed irreversible reactions would provide good sites for regulating the system as they provided a one-way street to the next step in the pathway and were subject to a lot of allosteric modulation. Today it has been shown that reversible reaction now play a larger part in controlling the flux through a pathway through altering the levels of intermediates (which happen to be allosteric effectors for irreversible reactions). However it is important to note that we are making a distinction between the effects an enzyme has on the flux of a pathway (it’s individual control) and how that pathway is regulated (the model of interactions).
There is a world of difference between the in vitro kinetics of an enzyme and the way in which it behaves in vivo. Merely isolating the enzymes is a hugely difficult task but this is compounded by the huge rates of turnover seen in the intermediates of the pathways investigated. Stitt (1987) discovered that within 2-3 seconds of darkening a leafs metabolites have changed so much that they resemble those found in the leaf at the middle of the night rather than one that is photosynthesising. Initially techniques such as freeze clamping (Stitt 1990) were used but this did not provide the split second cessation of metabolism required to perform studies in vivo. Stitt proceeded to develop techniques involving centrifugation quenching that allowed for metabolism to be studied in protoplasts in very small time steps allowing for analysis of regulation. This was not the answer to everything though since the enzymes in question will undoubtedly be modulated by a wide range of effectors, experiments had to be carefully planned and controlled to obtain a response to only one factor.
Once the enzymes that made up the sucrose synthesis pathway had been elucidated through in vitro analysis, focus moved over to determining the regulation of the pathway. Studies of the in vitro kinetics of the individual enzymes isolated two enzymes that were good candidates for regulation. Both of them catalysed irreversible reactions and were subject to allosteric modulation by intermediates of the pathway figure 1.
Fructose-1,6-bisphosphatase (FBPase)
Cytosolic FBPase consists of 4 subnits with a total molecular weight of 13,000 resembling FBPase found in mammals or chloroplasts. It is the initial step in the committed sucrose pathway, responsible for the irreversible catalysis of frucotse-1,6-bisphosphate (F-1,6-P2) to Fructose-6-phophate (F6P).
F-1,6-P2 → F6P + Pi
FBPase
In vitro analysis revealed that FBPase is inhibited by frucose-2,6-bisphosphate F-2,6-P2 a signal metabolite found universally in Eukaryotes (Stitt 1990) (I have seen a mention of AMP having an effect is this via the free Pi). This was no easy task as F-2,6-P2 is very difficult to extract as it is acid abile and also sensitive to hydrolysis by phosphatases that are present in plant tissues. Techniques required to extract it involve either heating in alkali or deproteinising with chloroform and subsequent extraction by phase partitioning.
F-2,6-P2 is made from F6P by a bifunctional polypeptide which also catalyses its modification back to F6P.
There also evidence to support its role in regulation in vivo.
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F-2,6-P2 is a potent inhibitor of cytosolic FBPase in vitro that is the first committed step in sucrose synthesis.
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There is an inverse relationship between the level of F-2,6-P2 and the rate of sucrose synthesis and a positive relationship with starch synthesis. (Stitt 1990)
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There is an increase in F-1,6-P2 and triose phosphates in the cytosol indicating that cytosolic FBPase is inhibited. (Stitt 1990)
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The kinase and phosphatase responsible for F-2,6-P2 are also regulated by metabolites of sucrose synthesis, and are present in leaf tissue in much higher concentrations than in other tissues (x10). Implying that finer regulation of F-2,6-P2 production can be obtained.
The problem with all this evidence is that it is correlative and not very quantitative. The introduction of transgenic plants in the late 80’s early 90’s has allowed for some experiments to be performed that further the evidence that FBPase and it’s inhibitor F-2,6-P2 is involved in the regulation of the sucrose pathway. Kruger (1994) directly manipulated the levels of F-2,6-P2 in transgenic tobacco by introducing the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver under the control of the CaMV 35S promoter. The advantages of using an orthologous enzyme especially from a distantly related taxa is that the gene and enzyme itself will not be subject to regulation by the plant and so it is able to express at much higher levels. A range of mutants with differing expression levels was created. Plants were illuminated for 20mins in saturating CO2 and no change on CO2 fixation was seen but a huge change in the partitioning of photosynthate was noted. The amount of 14C entering starch increased 15-28% when F-2,6-P2 was increased 35-85%. This gives good evidence that F-2,6-P2 alters the activity of FBPase and the subsequent partitioning of photosynthate in vitro. (what I don’t understand is how if you over express a BIFUNCTIONAL enzyme do you get an accumulation of one product, where does the equilibrium sit or is it due to the allosteric effectors or the dark arts of transgenics that one function is deactivated).
This study was furthered by removing the bifunctional polypeptide and substituting it for a modified mammalian gene encoding 6-phosphofructo-2-kinase. This only has the capacity to synthesise F-2,6-P2 and hence inhibit FBPase. Mutants were created with a range of expression from 104-230% of wild type levels in the dark. Small levels of 6-phosphofructo-2-kinase showed little change in the partitioning of photosynthate, probably because the plant reacts by deactivating/downregulating its own 6-phosphofructo-2-kinase. But once the plant 6-phosphofructo-2-kinase is nearly completely deactivated then the ectopic expression of modified mammalian 6-phosphofructo-2-kinase comes into play and a change in partitioning is seen (Kruger 1994).
Sucrose phosphate synthase (SPS)
SPS is the enzyme involved in the penultimate step of sucrose synthesis. It is responsible for converting F6P and UDPGlucose (UDPG) into sucrose phosphate.
F6P + UDPG → Sucrose-P
SPS
During the mid 80’s experiments were performed on the activity of SPS with respect to light. SPS showed a distinct cycling of high activity during the day (light) and a low activity during the night (dark). The first evidence that this was a diurnal fluctuation was obtained from soybean plants where highs were seen at the beginning and the end of the photoperiod. However this was not strictly tied to light dark transitions as cycles persisted in plants for few days that were grown in either full light or complete darkness. The diurnal rhythm is controlled by some endogenous clock mechanism (do we know what it is? Is it the build-up of sucrose mentioned in you lectures?). The mechanism for the activation of SPS was isolated to a member of a family of plant phosphatases called PP2A (Stitt 1990). This was shown by inhibiting the other known (a bit dodgy?) plant phosphatases using toxins and showing the SPS’s diurnal pattern still remained. The protein responsible for the deactivation (SPS kinase) has also been discovered.
More correlative evidence for SPS being involved in regulation of the sucrose synthesis pathway was found in vitro studies of the allosteric effectors of the enzyme and its synthesis enzymes. Much like the evidence for F-2,6-P2 the allosteric effectors are present in appropriate levels and are intermediates of sucrose synthesis. All this information combined produces this model.
This is a good example of the “fine” modulation of SPS via its allosteric effectors and the “coarse” modulation achieved by protein modification. There is a degree of amplification seen in the system where not only are the allosteric effectors acting on the “fine” modulation of SPS but also they are allosteric effectors of the “coarse” control components. This allows the system to respond very sensitively to the changes in metabolic intermediates.
To my knowledge no transgenic experiments have been performed using SPS modification and so the information here is still only model and has yet been proved conclusively.
Discussion.
This essay has only briefly described some of the known control components of sucrose synthesis and how it is coordinated with photosynthesis and the levels of photosynthate. But from the information given two major concepts of regulation have arisen. Firstly that there is a “feed forward” regulation in the system where the intermediates of one step act as the allosteric activators of enzymes further downstream. This coordinates sucrose synthesis with the rate of photosynthesis. The next component is a “feed back” regulation which responds to the levels of free Pi in the system. Due to the kinetics of the TPT the levels of triose phosphates in the chloroplast will increase if the sucrose synthesis pathway is not able to liberate enough Pi and this cross-talk between the sucrose synthesis pathway and the Calvin cycle allows for coordinated repartitioning of triose phosphates into starch. The system provides for a lot of very fine control and is able to respond to a wide range of changes in the environment and its use of amplification in certain scenarios allows for a rapid and sensitive response.
Bibliography
Huber, S.C. and Huber, J.L. (1992) Plant Physiol. 99: 1275-1284.
Siegel, G., MacKintosh, C. and Stitt, M. (1990) FEBS Lett. 270: 198-202. Elegant little paper on phosphorylation of SPS. A classic set of expts.
Stitt, M., Huber, S.C. and Kerr, P. (1987) In The Biochemistry of Plants, vol. 10, pp. 327-409. (only in Plant Sci library, NOT RSI)
Stitt, M. (1990) Ann. Rev. Plant Physiol. Plant Mol Biol 41: 153-185.
Kruger, N.J. and Scott, P. (1994) Manipulation of fructose 2,6-bisphosphate levels in transgenic plants. Biochem. Soc. Transactions 22. 904-909.
Scott, P., Lange, A.J., Pilkis, S.J. and Kruger, N.J. (1995) Carbon metabolism in leaves of transgenic tobacco containing elevated fructose 2,6-bisphosphate levels. The Plant, Journal 7: 461-469.
Stitt M (1997) The flux of carbon between the chloroplast and cytoplasm. In Plant Metabolism. (Dennis DT, Turpin DH, Lefebvre DD, Layzell DB, eds.) pp.382-400. This is a good one with which to begin.
Stitt M (1996) Plasmodesmata play an essential role in sucrose export from leaves. The Plant Cell 8: 567-571 (Only the first half of this editorial comment article is directly relevant to the question)
Huber, S.C. & Huber J.L. (1996) Ann Rev Plant Physiol. Plant Mol. Biol.47: (sorry, forgotten the page numbers)
