This is the normal biological system of DNA transfer and has recently been exploited in antisense inhibition (and sense suppression) of endogenous plant genes. The discovery of these kind of transformations and the development of metabolic control analysis in the 1970’s has meant that the alteration in the expression of endogenous plant genes allows investigations of their contribution to plant growth and thus a much better understanding of the control of metabolic processes. The approach involves the production of a set of plants in which the expression of one enzyme is progressively decreased, in as specific a manner as possible. Following this a flux control coefficient can describe the contribution that an Enzyme, E, makes to the control of flux through a pathway, J;
C= dJ/J
dE/E
The first enzyme from the Calvin cycle to be investigated in this way was Rubisco (Ribulose bisphosphate carboxylase/oxygenase.) Rubisco represents 30-40% of leaf protein. It catalyses the unique step in which carbon dioxide is incorporated into organic compounds in the Calvin cycle and represents a key site in the carbon and nitrogen economy of the plant. Rubisco activity is highly regulated; it is only active when CO2 binds to its lysine201 to form a carbamate to which an MG+ ion is bound. Activase facilities the carbamylation of Rubsico with the consumption of ATP. Rubisco is subject to allosteric regulation and is inhibited by several hexose phosphates and by 3-phosphoglcerate which binds to the active site, CA1P being a very strong inhibitor.
It has been found that the extent to which Rubisco limits photosynthesis depends on the short term conditions under which the flux is measured. When the plants were grown in moderate light, ambient photosynthesis was only slightly inhibited when Rubisco was decreased to about 60% of the wild type activity, and a control coefficient of 0.1-0.3 estimated. When plants were grown in low light and the rate of photosynthesis suddenly increased by increasing light intensity, there was a near proportional relation between the amount of Rubisco and the rate of photosynthesis, C>0.9. Whereas for plants grown in high light only C was about 0.2. A similar result was obtained if CO2 concentration was decreased. However, when photosynthesis was measured in 5% CO2, Rubisco could be decreased by 80% without any effect on the rate of carbon assimilation. Additionally when plants were grown on low nitrogen fertilizer, Rubisco decreased and became more limiting for ambient photosynthesis, C=0.5-0.6, together with the change in Rubisco control when light intensity was suddenly changed this indicates that the extent to which Rubisco limits photosynthesis clearly depends on the short-term conditions under which the flux is measured.
Despite the high levels of regulation mentioned previously however, in ambient conditions the changes in the amount of Rubsico have little effect on the rate of C assimilation.
Sedoheptulose-1,7-bisphosphate (SBPase) and fructose-1,6-bisphsphate (FBPase) are additional enzymes which catalyses irreversible reactions in the Calvin cycle and are found at extremely low levels compared to other enzymes in the Calvin cycle. The activity of SBPase is regulated by a number of different factors; in the light SBPase activity is increased 12-fold by thioredoxin f and is further modulated by pH and Mg2+. A finer level of control is exerted by the products of the SBPase reaction, SBP and Pi and also by glycerate. In common with the other enzymes of the Calvin cycle, the expression of the SBPase genes is regulated by light and development. Antisense plants were constructed to exhibit a range of SBPase activities between 71% and 7% of wild type without detectable changes in the levels of Rubisco, FBPase and PRKase proteins. A reduction in carbon assimilation rates was apparent in plants retaining 57% of wild type SBPase activity. This results in plants with reduced levels of RuBP and 3-phosphoglycerate (PGA) and increased RuBP/PGA ratio. When the levels of SBPase are further reduced the photosynthetic capacity of the plant is greatly reduced as RuBP regeneration further limits carbon fixation. When SBPase activity dropped below 15% the very low flux through the cycle led to a decrease in electron transport most likely due to a build up of ATP and NADPH. When SBPase activity was less than 20% wild type chlorosis was observed. Starch levels dropped in these plants with the maintenance of sucrose levels. Decreased expression of plastid FBPase did not affect photosynthesis until 60% of the wild type activity was removed.
A flux control coefficient of >0.7 has been predicted for SBPase. However, the inhibition of photosynthesis in antisense transformants has been suggested to be mainly because of an inhibition of stromal conductance and the resulting decrease of intercellular Carbon concentration. If photosynthesis is normalized on [Ci], it is inhibited only marginally by a 30-40% decrease of SBPase activity, indicating that SBPase has a fairly small flux control coefficient for the metabolic processes in the mesophyll.
In addition to reducing SPBase activity, cyanobacterial FBP/SBPase has been introduced into tobacco chloroplasts and it has been shown that the overexpression of FBP/SBPase in the chloroplasts of transgenic plants leads to increased photosynthetic capacity in source leaves, carbohydrate accumulation and accelerated growth rate. FBP/SBPase has no homology with FBPase and SBPase genes derived from higher plants and thus is useful in this respect as is less likely to be prone to transgene silencing. Why the increase in photosynthetic activity occurs has been postulated to be due to increased levels of RuBP. Rubisco activase may only bind Rubisco when RuBP occupies the active site, therefore the increased levels of RuBP induced may be followed by the activation of Rubisco and the increase in photosynthesis.
It has been proposed that starch accumulation directly inhibits photosynthesis, that the rate of sucrose and starch synthesis limits photosynthesis, and that sugar accumulation in the source leaves triggers changes in gene expression, resulting in a decrease in Rubisco and photosynthetic activity under high CO2 conditions. However in the aforementioned transgenic plants, increased levels of starch and sucrose did not show any significant down regulation of photosynthesis and the enzyme activities involved in the Calvin cycle in source leaves. This gives insights into possible integration of metabolism that was not previously possible. In general these experiments showed that FBPase and/or SBPase involved in the regeneration of ribulose-1,5-bisphosphate are one of the limiting factors that participate in the regulation of the carbon flow through the Calvin cycle and the determination of the partitioning of carbon to end products. As was shown with the experiments reducing their activity.
The final irreversible enzyme to be discussed is Ribulose-5-phosphate kinase/ phosphoribulokinase (PFK), which synthesizes RuBP and constitutes less than 1% of leaf protein. It is subject to various regulation: It is rapidly activated in the light as a result of reduction of thiol groups via thioredoxin and ferredoxin-thioredoxin reductase, the kinetics of which is influenced by stromal concentrations of H+, MG2+ and metabolites. Light activated PRK is competitively inhibited with respect to Ru5P by 6-phosphogluconate, RuBP, 3-phosphoglyceric acid (s-PGA) and phosphate; fructose-1,6-bisphosphate (FBP) is also inhibitory. Tobacco was transformed with antisense gene constructs to reduce the activity of PRK.
In plants grown in low light 85% of the enzyme can be removed before there is any effect on the rate of photosynthesis. Therefore PRK has a flux control coefficient of zero for carbon assimilation in wild type tobacco growing under these conditions, with only about 6% of maximal activity required to sustain the rates of CO2 assimilation observed in the wild type. When PRK activity was reduced to between 20 and 5μmol m-2sec-1 (reduced 20 fold to the wild type) there was a reduction in assimilation rate with a flux-control coefficient of about 0.6. It was calculated that measured alteration in metabolites would have maintained sufficient carbon flux through PRK to realize the CO2 assimilation rates observed. This is a good example of an enzyme that defies traditional metabolic biochemistry.
Plants have also been investigated with decreased expression of NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH). Although this enzyme catalsyes a near-equilibrium reaction, it is regulated by thioredoxin. Photosynthesis was inhibited when NADP-GAPDH activity was decreased by 65% or more. The results so far show little correlation with the view point that points of control lie only with those enzymes that catalyse irreversible reactions and/or are highly regulated.
Aldolase catalyses the readily reversibe conversion of glyceraldehydes-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) to fructose-1,6-BP, and erythrose-4-phosphate (Ery4P) and DHAP to SBP and has no regulatory properties. This made it a good candidate to investigate whether fluxes around and out of the Calvin cycle are sensitive to decreased expression of an enzyme that catalyses a non-regulated and reversible step and again antisense transformants were examined. Decreased expression of plastid aldolase was accompanied by a selective decline of pFBPase, SBPase and PRK activity – this is the first example where decreased expression of one enzyme leads to selective changes in other Calvin cycle enzymes emphasizing metabolic intergration. The antisense transformants showed marked inhibition of photosynthesis in ambient conditions (C=0.15-0.24) and saturating light and CO2 (C=0.26-0.32) and a weaker inhibition in saturating CO2 and limiting light. Comparison with studies of transformants with altered expression of pFBPase, SBPase and PKA shows that changes in the activities of the individual enzymes are not responsible for the inhibition of photosynthesis in the antisense aldolase transformants. When aldolase activity fell below 50% of wild type activity, there was a near linear relation between photosynthesis and aldolase activity, showing that photosynthesis is strongly limited by aldolase. This shows that the Calvin cycle is remarkably sensitive to a small decrease of plastid aldolase, even though it is a non-regulated enzyme that catalyses a readily reversible reaction.
Transketolase (TK) additionally catalyses a reversible reaction; transferring a carbohydrate residue with two carbon atoms from fructose 6-phosphate to glyceraldehydes 3-phosphate, yielding xylulose 5-phosphate and erythrose 4-phosphate in a reversible reaction. A 20%-40% reduction of TK activity inhibits RuBP regeneration and photosynthesis. The inhibition of photosynthesis becomes greater as irradiance increases across the range experienced in growth conditions. TK almost completely limits the maximum rate of photosynthesis in saturating light and saturating. TK expression resulted in the activities of the other Calvin cycle enzymes remaining high or increasing slightly. In ambient conditions the flux control for TK was 0.07-0.32 resembling Rubisco and aldolase.
The discussed genetic manipulation of the Calvin cycle shows there is no connection between the impact of changes of the expression of a gene on pathway flux and the role of the gene product on metabolic regulation. Genes whose level of expression is near critical do not necessarily play a key role in the short-term metabolic regulation of the pathway. Conversely, changes in the expression of enzymes with complex regulatory properties such as PRK do not necessarily lead to a change in pathway flux. Enzymes that are subject to regulation, in particular via feedback loops originating from within the pathway, can compensate for decreased expression because changes in the concentrations of substrates, products, inhibitors and activators will stimulate the activity of the residual enzyme. In contrast, enzymes that lack regulatory properties can only compensate for decreased expression by altering the concentrations of their substrates and products, and flux will be inhibited when these changes affect the operation of other enzymes in the pathway.
The genetic manipulation of the Calvin cycle reinforces what metabolic control analysis now emphasizes. That;
-Single rate limiting steps are probably rare in metabolic sequences.
-Control of pathway fluxes is usually shared among several steps.
-Their relative contribution to overall control will vary with flux.
The antisense experiments described above with TK exhibits a good example of how plants have been used to reveal information about the integration of metabolism. The reactions TK catalyses in the Calvin cycle and the oxidative pentose phosphate pathway (OPPP) produce erythrose-4-phosphate, which is a precursor for the shikimate pathway – this pathway generates amino acids for photosynthesis and provides precursosrs for a large variety of other substances including phenylpropanoids such as flavonoids and lignin. Therefore a decrease in TK in its primary pathway will influence secondary pathways. Alteration in gene expression enables this interaction to be further understood. Along with the inhibition of photosynthesis mentioned previously as TK was reduced there was additionally a decrease in levels of sugars and of aromatic acids and so decreased phenylpropanoid intermediates and lignin. These results demonstrate that there are no major alternative pathways that are able to substitute for plastid TK, and as metabolism and growth are inhibited in response to an unexpectedly small decrease of activity it indicates that TK is co limiting or near rate limiting for several important metabolic pathways. Why this enzyme simply is not expressed in excess has been suggested to be because greater TK activity might lead to an increase of Ery4P, which would be unfavorable to starch synthesis.
In addition it was found that decreased Rubsico activity led to a fall in sugars and starch and decreased activity of aldolase, plastid FBPase and SBPase led to a marked decrease of starch but not sugar levels giving examples of integration.
Transgenic plants have therefore shown how a change in an enzyme in one pathway can affect another, and particularly the importance of TK as a key determinant of plant metabolism. There have been found no alternative routes to substitute for a decrease in this enzymes level in contrast to many other processes in primary carbon metabolism, which show a high degree of redundancy. Transgenic plants have also revealed the traditional understandings of control in metabolic processes to be false in that a protein does not have to catalyse an irreversible reaction of be highly regulated to exhibit a high level of control over a pathway.