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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Even though plastid aldolase catalyses a reversible reaction, does not possess properties allowing it to contribute to ‘fine’ regulation, and would therefore be considered unimportant for the control of metabolism and growth, antisense transformants with a 50–70% decrease in aldolase activity showed an inhibition of photosynthesis and growth. We now show that acclimation of photosynthesis to growth conditions includes and requires changes in plastid aldolase activity. Wild-type potato plants and transformants were grown at low irradiance (70 μmol m–2 sec–1), and at high irradiance (390 μmol m–2 sec–1) at 400 or 800 p.p.m. carbon dioxide. (i) Ambient photosynthesis was always inhibited by a 30–40% decrease of aldolase activity, the strongest inhibition being observed when plants were growing in high irradiance and elevated carbon dioxide. (ii) The inhibition was due to a low rate of ribulose-1,5-bisphosphate regeneration in low light, exacerbated by an inadequate rate of starch synthesis in high light and elevated carbon dioxide. Decreased expression of aldolase in antisense transformants was also accompanied by a decrease of fructose-1,6-bisphosphatase protein and activity, and Rubisco activity. Transcript levels for the plastid fructose-1,6-bisphosphatase and the small subunit of Rubisco did not decrease. (iii) In wild-type plants, increasing the growth irradiance from 70 to 390 μmol m–2 sec–1 led to a 15–95% increase of the activity of eight Calvin cycle enzymes, and increasing the carbon dioxide concentration from 400 to 800 p.p.m. led to a 5–35% decrease of these enzyme activities. The largest changes occurred for aldolase, and for transketolase which also catalyses a reversible reaction and is not subject to ‘fine’ regulation.


Abbreviations
A

photosynthesis;

ci

intracellular CO2 concentration;

FBPase

plastid fructose-1,6-bisphosphatase;

Fru6P

fructose-6-phosphate;

FW

fresh weight;

Glc6P

glucose-6-phosphate;

gH2O

stomatal conductance for water;

NADP-GAPDH

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase;

PGK

glycerate-3-phosphate kinase (3-phosphoglycerate kinase);

PRK

ribulose-5-phosphate kinase (5-phosphoribulokinase);

SBPase

sedoheptulose-1,7-bisphosphatase;

WUE

water use efficiency;

pAld

transcript for plastidic aldolase;

pFbp

transcript for plastidic FBPase;

RbcS

transcript for the small subunit of Rubisco;

Tkt

transcript for transketolase;

3PGA

glycerate-3-phosphate;

Ru1

5bisP, ribulose-1,5-bisphosphate.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Metabolic regulation has long distinguished between ‘fine’ and ‘coarse’ regulation ( Newsholme & Start 1973). Fine regulation involves rapid modulation of existing enzyme activity by effectors or reversible post-translational regulation, and coarse regulation involves slower changes in expression and/or protein degradation. It has also been widely assumed that key sites for regulation are located at enzymes that catalyse irreversible reactions, whereas enzymes that catalyse readily reversible reactions are present in excess and, consequently, unimportant for regulation ( Newsholme & Start 1973; Stitt 1995, 1996). Transgenic plants provide an ideal system to test these ideas and deepen our understanding of metabolic regulation.

Recently, Haake et al. (1998) showed that decreased expression of plastid aldolase leads to a dramatic decrease of ribulose-1,5-bisphosphate (Ru1,5bisP), an inhibition of ambient photosynthesis, and decreased growth. The flux control coefficient of aldolase ( Fell 1992; Kacser & Burns 1973; Stitt 1995, 1996) for ambient photosynthesis (Caldolase) in plants growing at 350 p.p.m. carbon dioxide and 250 μmol m–2 sec–1 irradiance was 0.18–0.24. These results were unexpected because aldolase catalyses a reversible reaction and lacks properties that would permit ‘fine’ regulation.

The impact of decreased aldolase expression was comparable to or larger than that found after decreasing the expression of enzymes that catalyse non-reversible reactions and are subject to sophisticated ‘fine’ regulation, including Rubisco ( Stitt & Schulze 1994), plastid fructose-1,6-bisphosphatase (FBPase) ( Kossmann et al. 1994), sedoheptulose-1,7-bisphosphatase (SBPase) ( Harrison et al. 1997), phosphoribulokinase (PRK) ( Paul et al. 1995), and ADP-glucose pyrophosphorylase (AGPase) ( Müller-Röber et al. 1992; Neuhaus & Stitt 1990). Other examples where fluxes in photosynthesis metabolism show surprisingly large responses to decreased expression of a ‘non-regulated’ enzyme include the plastid and cytosolic phosphoglucose isomerase, and plastid phosphoglucomutase ( Neuhaus et al. 1989; Neuhaus & Stitt 1990). Clearly (i) non-regulated enzymes that catalyse reversible reactions are not necessarily expressed in large excess, and (ii) enzymes that are highly regulated are often expressed in considerable excess.

Although this conclusion conflicts with previous notions about the way that metabolic pathways are regulated, it becomes understandable when we consider how a metabolic pathway will respond to decreased expression of an enzyme ( Haake et al. 1998; Stitt & Sonnewald 1995). If the enzyme is subject to ‘fine’ regulation by feedback loops originating within the pathway, changes in the concentrations of several different metabolites (substrates, products, inhibitors, activators) will be able to compensate for decreased expression, by stimulating the residual enzyme. In contrast, enzymes that lack ‘fine’ regulatory properties can only compensate via alterations in the concentrations of their substrate and product and, once these changes start to affect the operation of other enzymes, pathway flux will be inhibited.

This reassessment has interesting implications with respect to the need for ‘coarse’ regulation of enzymes that lack ‘fine’ regulatory properties. Expression of genes is typically modified in response to changes in the environment, for example increased light intensities lead to an increase ( Evans 1989) and elevated carbon dioxide often leads to a decrease of Rubisco activity ( Bowes et al. 1996; Stitt 1991). Investigations of the expression of Calvin cycle enzymes have concentrated on regulated enzymes that catalyse irreversible reactions, in particular Rubisco. However, if non-regulated enzymes like aldolase are not present in a large excess, either (i) they will need to be constitutively expressed at a very high level to avoid such limitations, which is obviously not always the case, or (ii) they will become limiting when the expression of other enzymes in the pathway is increased above a critical level, or (iii) their expression or turnover will need to be regulated to allow co-ordinate changes in the amounts of the regulated and non-regulated enzymes.

In the following experiments we first compared the impact of decreased expression of aldolase on photosynthesis and growth in plants growing in low irradiance, in high irradiance, and in high irradiance plus elevated carbon dioxide, and then compared the magnitude of the changes in aldolase activity with those of other non-regulated and regulated Calvin cycle enzymes during acclimation to altered growth irradiance and carbon dioxide concentration. Aldolase showed larger activity changes than the ‘regulated’ enzymes of the Calvin cycle during acclimation to different growth conditions, and we demonstrate that these large changes are important because aldolase expression is never high enough to avoid a slight, and sometimes marked, limitation of photosynthesis.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Aldolase expression

Wild-type Solanum tuberosum cv. Desirée and four transformant lines A-70, A-3, A-51 and A-2 were grown at low irradiance (70 μmol m–2 sec–1) and ambient carbon dioxide, high irradiance (390 μmol m–2 sec–1) and ambient carbon dioxide, and high irradiance (390 μmol m–2 sec–1) plus elevated carbon dioxide (800 p.p.m.). All experiments were carried out with the youngest fully expanded leaf.

Aldolase activity was inhibited by 40–46%, 69–74%, 78% and 90–96% in A-70, A-3, A-51 and A-2, respectively, when the plants were grown in low irradiance and ambient carbon dioxide ( Fig. 1a) or high irradiance and ambient carbon dioxide ( Fig. 1b). Although the inhibition of aldolase activity in the transformants was apparently smaller in high irradiance plus elevated carbon dioxide ( Fig. 1c), A-70, A-3 and A-51 contained more protein than wild-type plants in high light and elevated carbon dioxide (see below). When aldolase activity was expressed on a protein basis it decreased by 36%, 62%, 75% and 91% in A-70, A-3, A-51 and A-2, respectively (data not shown). In all three growth conditions, the decrease in aldolase activity was accompanied by a decrease in aldolase protein (see below).

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Figure 1. Aldolase activity, protein and photosynthesis in wild-type plants and antisense transformants growing at different irradiance and carbon dioxide concentrations.

Wild-type Solanum tuberosum cv. Desirée (wt) (first column from the left in each set) and antisense transformant lines A-70, A-3, A-51 and A-2 (second, third, fourth and fifth column from the left in each set) were grown at 25°C in a 12-h light/12-h dark photo-regime, at 60% relative humidity at 70 μmol sec–1 m–2 irradiance and 350 p.p.m. carbon dioxide(a, d, g, j), 390 μmol sec–1 m–2 and 400 p.p.m. (425 p.p.m. during measurements) (b, e, h, k), and 390 μmol sec–1 m–2 and 800 p.p.m. (840 p.p.m. during measurements) (c, f, i, l). Samples were harvested from the first fully expanded leaf of 28-day-old plants after 10 h light, and frozen in liquid nitrogen for further analysis. The results are the mean ± SE (n = 4 separate plants). The plant height was adjusted (when necessary) so that the top of all the plants was maintained at the same height and received the same irradiance. (a, b, c) Aldolase activity, (d, e, f) protein content, (g, h, i) photosynthesis measured at the growth irradiance and carbon dioxide concentration related to the remaining aldolase activity. The data points correspond (from right to left) to wild-type plants and the transformants A-70, A-3, A-51 and A-2. All data points are expressed as a percentage of the wild-type value in those growth conditions; the axes are normalized such that the wild-type values for aldolase activity and photosynthetic rate are geometrically identical. A hyperbola fitted through the data points provides an estimate of the flux control coefficient ( Small & Kacser 1993). For absolute values of photosynthesis see (j–l), and for the absolute aldolase activities see (a–c). (j, k, l) Photosynthesis at growth irradiance and carbon dioxide concentration. The results are the mean ± SE (n = 4 separate plants).

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Leaf protein content

In wild-type plants, total leaf protein was highest in plants growing in high light and ambient carbon dioxide, 25 ± 2% lower in low light-grown plants, and 11 ± 5% lower in plants growing in high irradiance and elevated carbon dioxide ( Fig. 1d–f). There was no consistent change in leaf protein in lines A-70, A-3 and A-51 compared with wild-type plants in high irradiance and ambient carbon dioxide ( Haake et al. 1998 ), a slight downwards trend in low irradiance, and a slight upwards trend (especially in A-70 and A-3) in high irradiance and elevated carbon dioxide. A-2, the most extreme transformant line, always showed a decrease in protein.

Photosynthesis

Photosynthesis was measured at the growth irradiance and carbon dioxide concentration. Decreased aldolase expression inhibited photosynthesis in all three growth conditions ( Fig. 1g–l). The inhibition was weakest in plants growing in low irradiance and ambient carbon dioxide ( Fig. 1g,j), stronger in plants growing in high irradiance and ambient carbon dioxide ( Fig. 1h,k), and strongest in plants growing in high irradiance plus elevated carbon dioxide ( Fig. 1i,l).

The enzyme activity and the rate of ambient photosynthesis in each transformant were normalized by expressing them as a percentage of the corresponding wild-type value in that growth condition, plotted, and a hyperbola fitted through the normalized data points ( Fig. 1g–i). Caldolase for ambient photosynthesis was estimated from the slope in the region corresponding to the wild-type ( Haake et al. 1998 ; Small & Kacser 1993) as 0.15 ± 0.02, 0.21 ± 0.03 and 0.55 ± 0.04 in plants growing in low irradiance ( Fig. 1g), high irradiance ( Fig. 1h), and high irradiance plus elevated carbon dioxide ( Fig. 1i), respectively.

Effect of increased growth irradiance and of elevated carbon dioxide on Calvin cycle enzyme activities in wild-type plants

The effect of the growth conditions on Rubisco ( Fig. 2a), phosphoglycerate kinase (PGK) ( Fig. 2b), NADP-GAPDH ( Fig. 2c), aldolase ( Fig. 2d), plastid FBPase ( Fig. 2e), SBPase ( Fig. 2f), transketolase ( Fig. 2g) and PRK ( Fig. 2h) activity was investigated in wild-type plants. Rubisco, plastid FBPase, SBPase and PRK catalyse irreversible reactions and are subject to regulation by effectors and post-translational modification, NADP-GAPDH catalyses a reversible reaction but is subject to post-translational regulation, and PGK, aldolase and transketolase catalyse readily reversible reactions and are not subject to regulation by effectors or post-translational regulation ( Haake et al. 1998 ; Stitt 1996).

image

Figure 2. Activities of Rubisco and other Calvin cycle enzymes.

Wild-type plants were grown and harvested as in Fig. 1, and assayed for the activity of (a) Rubisco, (b) phosphoglycerate kinase (PGK), (c) NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), (d) aldolase, (e) plastid fructose-1,6-bisphosphatase (FBPase), (f) sedoheptulose-1,7-bisphosphatase (SBPase), (g) transketolase, and (h) phosphoribulokinase (PRK). Growth conditions were as indicated at the bottom of figure: 70 μmol sec–1 m–2 irradiance and 350 p.p.m. carbon dioxide (dark grey bars), 390 μmol sec–1 m–2 and 400 p.p.m. (grey bars), and 390 μmol sec–1 m–2 and 800 p.p.m. (white bars). The results are the mean ± SE (n = 4 separate plants).

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The enzyme activities all increased in high light-grown plants, compared with low light-grown plants, but the extent of the increase varied. Compared with the increase in the leaf protein content (+33 ± 3%; Fig. 1), SBPase (+16 ± 6%) increased subproportionately, PRK (+35 ± 4%) and plastid FBPase (+30 ± 5%) increased proportionately, Rubisco (+45 ± 4%) and PGK (+45 ± 4%) showed a slight and non-significantly larger increase, and aldolase (+63 ± 6%), NADP-GAPDH (+85 ± 7%) and transketolase (+96 ± 4%) showed a significantly larger increase ( Fig. 3, where activities are on a protein basis; the wild-type is the right-hand data point in each plot).

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Figure 3. Activities of Calvin cycle enzymes in transformants with decreased expression of aldolase, normalized on total leaf protein.

The plants were grown as in Fig. 1 in 70 μmol sec–1 m–2 irradiance and 350 p.p.m. carbon dioxide (dark grey symbols) (a, d, g, j, m, p, s), 390 μmol sec–1 m–2 and 400 p.p.m. (grey symbols) (b, e, h, k, n, q, t), or and 390 μmol sec–1 m–2 and 800 p.p.m. (white symbols) (c, f, i, l, o, r, u). For each genotype, the y-axis shows the various enzyme activities related to the leaf protein content, and the x-axis shows aldolase activity related to the protein content. The original data for enzyme activities in the wild-type plants are shown in Fig. 2, for protein in wild-type plants and transformants in Fig. 1, and for aldolase in the wild-type plants and transformants in Fig. 1. The original data for the activities of the other enzymes in the transformants are not shown. (a, b, c) Rubisco, (d, e, f) phosphoglycerate kinase (PGK), (g, h, i) NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), (j, k, l) plastid fructose-1,6-bisphosphatase (FBPase), (m, n, o) sedoheptulose-1,7-bisphosphatase (SBPase), (p, q, r) transketolase, (s, t, u) phosphoribulokinase (PRK). The data points show (from right to left) the values for wild-type plants and for the antisense transformants A-70, A-3, A-51 and A-2. The results are the mean ± SE (n = 4 separate plants).

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Elevated carbon dioxide led to a decrease of the enzyme activities, whose magnitude again depended on the enzyme in question. Compared with the decrease in leaf protein (–11 ± 5%; Fig. 1), the decrease was small and proportional for NADP-GAPDH (–12 ± 2%), plastid FBPase (–12 ± 4%), PRK (–15 ± 2%) and PGK (–17 ± 2%), somewhat larger for Rubisco (–22 ± 2%) and SBPase (–26 ± 3%), and largest for aldolase (–31 ± 1%) and transketolase (–35 ± 2%) ( Fig. 3).

Activities of the other Calvin cycle enzymes in transformants with decreased expression of aldolase

The experiments were next extended to investigate the reasons for the inhibition of photosynthesis in transformants with decreased expression of aldolase. The effects on the expression and activity of other Calvin cycle enzymes first was investigated.

Because protein changed slightly in the transformants compared with wild-type plants ( Fig. 1), the enzyme activities were related to the leaf protein content and then plotted against aldolase activity ( Fig. 3). Decreased expression of aldolase was accompanied by a clear decrease of FBPase ( Fig. 3j–l) and, to a lesser extent, Rubisco ( Fig. 3a–c) activity in all three growth conditions. The other enzyme activities did not respond so strongly or consistently. PGK activity decreased slightly in high irradiance ( Fig. 3e,f), but not in low irradiance ( Fig. 3d). NADP-GAPDH ( Fig. 3g–i) and transketolase ( Fig. 3p–r) activity showed only small and non-consistent changes. SBPase activity decreased slightly in low irradiance ( Fig. 3m), but not in the other conditions ( Fig. 3n,o). In all three conditions, PRK activity declined slightly but non-significantly ( Fig. 3.–u).

Activation state of Rubisco and the plastid FBPase

Post-translational activation of Rubisco occurs via carbamylation. In high irradiance and ambient ( Fig. 4b) or elevated ( Fig. 4c) carbon dioxide, Rubisco activation showed a marked and progressive increase as aldolase expression was decreased. In low light-grown plants ( Fig. 4a), Rubisco activation was high in wild-type plants and increased slightly and non-significantly in the transformants. Post-translational activation of plastid FBPase involves thioredoxin-mediated reduction. Plastid FBPase activation increased markedly in the antisense transformants in all three conditions ( Fig. 4d–f).

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Figure 4. Rubisco and the plastid fructose-1,6-bisphosphatase.

Plants were grown as in Fig. 1. Growth conditions were as indicated at the bottom of the figure: 70 μmol sec–1 m–2 irradiance and 350 p.p.m. carbon dioxide, 390 μmol sec–1 m–2 and 400 p.p.m. and 390 μmol sec–1 m–2 and 800 p.p.m. (a, b, c) Rubisco activation state, (d, e, f) plastid FBPase activation state. The results are the mean ± SE (n = 4 separate plants). Immunoblots for (g) plastid aldolase protein and (h) plastid FBPase protein. Each lane contains total protein from 0.5 mg FW of leaf tissue. Samples from the different growth conditions were exposed for different times to optimize signal strength. Two replicate experiments are shown for plastid FBPase. Steady-state transcript levels of (i) plastid aldolase (pAld), (j) plastid FBPase (pFbp), (k) small subunit of rubisco (RbcS), and (l) transketolase (Tkt). Steady-state transcript levels of (m) pFbp and (n) RbcS in a separate experiment, in which wild-type, A-3 and A-51 were grown in 70 μmol sec–1 m–2 irradiance and 350 p.p.m. carbon dioxide, and in 390 μmol sec–1 m–2 and 800 p.p.m. carbon dioxide (duplicates are shown). n.a. = not analysed.

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Plastid aldolase and plastid FBPase protein

The changes in aldolase and FBPase activity in wild-type plants in the three growth conditions ( Fig. 2d,e) were accompanied by similar changes of the corresponding proteins ( Fig. 4g,h). The decrease of FBPase activity ( Fig. 3j–l) in the antisense transformants was always accompanied by a similar decrease of FBPase protein ( Fig. 4h). This decrease was much less marked than the decrease of aldolase protein in the transformants ( Fig. 4g).

Transcript levels for plastid aldolase, plastid FBPase, the small subunit of Rubisco and transketolase

Transcript levels were measured in the first source leaf 10 h into the photoperiod ( Fig. 4i–n). The aldolase transcript level (pAld) did not change markedly in wild-type plants between the three growth conditions ( Fig. 4i). A replicate experiment with shorter exposition time confirmed that aldolase transcript level did not respond to growth irradiance or elevated carbon dioxide (data not shown). The transcript levels for the plastid FBPase (pFbp) ( Fig. 4j,m), the small subunit of Rubisco (RbcS) ( Fig. 4k,n), and transketolase (Tkt) ( Fig. 4l) increased in wild-type plants in response to an increase in the growth irradiance, but did not show a marked response to elevated carbon dioxide.

As expected, the pAld transcript level was strongly decreased in all four antisense lines ( Haake et al. 1998 ). Compared with wild-type plants, the antisense lines had similar or higher levels of the transcripts for pFbp ( Fig. 4j,m), RbcS ( Fig. 4k,n) and Tkt ( Fig. 4l) in all three growth conditions.

Phosphorylated metabolites

To gain more information about the reasons for the inhibition of photosynthesis, the effects on metabolite and carbohydrate levels were investigated. In low light-grown plants, decreased expression of aldolase led to a marked decrease of Ru1,5bisP ( Fig. 5a) and glycerate-3-phosphate (3PGA) ( Fig. 5d), an increase of triose phosphates ( Fig. 5g), and a decrease of hexose phosphates ( Fig. 5j), showing that decreased aldolase expression inhibits Ru1,5bisP regeneration.

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Figure 5. Levels of photosynthesis intermediates.

Plants were grown and harvested as in Fig. 1, in 70 μmol sec–1 m–2 irradiance and 350 p.p.m. carbon dioxide (a, d, g, j, m), 390 μmol sec–1 m–2 and 400 p.p.m. (b, e, h, k, n), and 390 μmol sec–1 m–2 and 800 p.p.m. (c, f, i, l, o). (a, b, c) Ribulose-1,5-bisphosphate, Ru1,5bisP, (d, e, f) glycerate-3-phosphate, 3PGA, (g, h, i) triose phosphates. For (h, i) the triose phosphate content was below the detection limit of 0.4 nmol g FW–1 in all samples. (j, k, l) Total hexose phosphates (the sum of Glc6P, Fru6P and Glc1P) and (m, n, o) the Glc6P/Fru6P quotient. The results are the mean ± SE (n = 4 separate plants).

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A different and unexpected result was found for plants growing at high irradiance. The decrease of Ru1,5bisP was much less marked ( Fig. 5b), 3PGA declined in A-70 (the transformant with the smallest change in aldolase expression) but increased in A-3 and A-51 ( Fig. 5e), the triose phosphates were very low in wild-type plants and the transformants ( Fig. 5h), and the hexose phosphates increased in the transformants ( Fig. 5k). These trends were even clearer in plants growing in high irradiance and elevated carbon dioxide ( Fig. 5c,f,i,l). The increase of hexose phosphates was due to an increase of glucose-6-phosphate (Glc6P), whereas fructose-6-phosphate (Fru6P) remained unaltered or decreased. Consequently, the Glc6P/Fru6P quotient increased dramatically in the transformants in high irradiance ( Fig. 5n) or high irradiance and high carbon dioxide ( Fig. 5o), whereas it remained unaltered in the transformants in low irradiance ( Fig. 5m), indicating that hexose phosphates accumulate in the cytosol of the transformants in high irradiance. The Glc6P/Fru6P ratio is much higher in the cytosol than in the plastid in photosynthesizing leaves ( Gerhardt et al. 1987 ).

Sugar and starch levels

In wild-type plants, starch was low in low light-grown plants ( Fig. 6a, note the y-axis scale), 25-fold higher in high irradiance and ambient carbon dioxide ( Fig. 6b), and another 2.5-fold higher in high irradiance plus elevated carbon dioxide ( Fig. 6c). Sucrose was lowest in low light-grown plants ( Fig. 6d), and 2.8-fold higher in high irradiance ( Fig. 6e) and high irradiance plus elevated carbon dioxide ( Fig. 6f). Reducing sugars were highest in low light-grown plants ( Fig. 6g,j), and slightly lower in high irradiance ( Fig. 6h,k) and in high irradiance plus elevated carbon dioxide ( Fig. 6i,l).

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Figure 6. Levels of carbohydrates.

Plants were grown and harvested as in Fig. 1, in 70 μmol sec–1 m–2 irradiance and 350 p.p.m. carbon dioxide (a, d, g, j), 390 μmol sec–1 m–2 and 400 p.p.m. (b, e, h, k), and 390 μmol sec–1 m–2 and 800 p.p.m. (c, f, i, l) and assayed for the content of (a, b, c) starch, (d, e, f) sucrose, (g, h, i) glucose (j, k, l) fructose and (m, n, o) total sugars (sum of glucose, fructose and sucrose given in hexose equivalents). Note the different scale of the y-axis for (a). The results are the mean ± SE (n = 4 separate plants).

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Decreased aldolase expression led to a decrease of starch ( Fig. 6a), sucrose ( Fig. 6d) and reducing sugars ( Fig. 6g,j) in low light-grown plants. Starch decreased more than sugars, as seen earlier for plants growing at 250 μmol sec–1 m–2 ( Haake et al. 1998 ). In high irradiance, the decrease of starch in A-3 and A-51 was more marked ( Fig. 6b), whereas sucrose decreased only slightly ( Fig. 6e) and reducing sugars increased threefold ( Fig. 6h,k) compared with wild-type plants. In high irradiance plus elevated carbon dioxide, there was an even more dramatic decrease of starch in A-3 and A-51 ( Fig. 6c), sucrose remained high ( Fig. 6f), and reducing sugars increased fivefold ( Fig. 6i,l). A severe decrease in aldolase expression in transformant line A-2 resulted in a marked decrease of starch and sugar in all three growth conditions ( Fig. 6a–l).

Whole plant biomass and biomass allocation

Plants were harvested after 28 days, when tuber growth had just commenced. Total wild-type biomass increased 2.2-fold when the growth irradiance was increased from 70 to 390 μmol sec–1 m–2 ( Fig. 7a,b), due to a 2.1-fold increase in leaf weight ( Fig. 7d,e; note the different scale of the y-axis), a slight increase in stem weight ( Fig. 7g,h) and a 3.1-fold increase in root weight ( Fig. 7m,n). Side shoots ( Fig. 7j,k) and tubers ( Fig. 7p,q) were initiated by this time in high irradiance, but not in low irradiance. Total wild-type biomass increased by another 36% when the carbon dioxide concentration was increased from 400 p.p.m. to 800 p.p.m. ( Fig. 7b,c), due to a slight non-significant increase of leaf weight ( Fig. 7e,f), a more marked increase of stem ( Fig. 7h,i) and root ( Fig. 7n,o) weight, and an increase of side shoots ( Fig. 7k,l) and tuber weight ( Fig. 7q,r).

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Figure 7. Plant biomass.

Plants were grown and harvested as in Fig. 1, in 70 μmol sec–1 m–2 irradiance and 350 p.p.m. carbon dioxide (a, d, g, j, m, p), 390 μmol sec–1 m–2 and 400 p.p.m. (b, e, h, k, n, q), and 390 μmol sec–1 m–2 and 800 p.p.m. (c, f, i, l, o, r), and the fresh weight (FW) was determined. (a, b, c) total plant biomass, (d, e, f) leaves including midribs, (g, h, i) main stem and leaf stalks, (j, k, l) side shoots, (m, n, o) roots, (p, q, r) tubers. Note the different scale of the y-axis for the low-light-grown plants (a, d, g, j, m, p). The results are the mean ± SE (n = 4 separate plants).

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Biomass was not decreased in A-70, with the smallest change in aldolase expression. There was a 41% and 49% decrease in total biomass in A-3 and A-51 in low irradiance ( Fig. 7a), and a smaller (28% and 27%) decrease in high irradiance ( Fig. 7b) or in high irradiance and elevated carbon dioxide (22% and 34%; Fig. 7c). Biomass allocation was altered in a similar manner in all conditions ( Fig. 7). The decrease in leaf biomass was relatively small ( Fig. 7d–f), and the decrease in the stem ( Fig. 7g–i), side shoot ( Fig. 7j–l), root ( Fig. 7m–o) and tuber ( Fig. 7p–r) biomass disproportionately large.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Decreased expression of plastid aldolase is accompanied by a selective decrease in plastid FBPase activity, and sometimes by small changes in the activities of other Calvin cycle enzymes

The use of transgenic plants to investigate metabolic regulation requires accurate quantification of the change in expression of the targeted enzyme ( Stitt 1995), and evidence that the expression of other enzymes is not significantly altered ( Ap Rees & Hill 1994). The inhibition of aldolase expression resembles that in Haake et al. (1998 ), where the accuracy of the quantification was examined critically. Decreased expression of aldolase actually has interesting effects on the expression of other Calvin cycle enzymes. The transformants show a consistent decrease of plastid FBPase activity in all growth conditions examined so far, and decreases of Rubisco, SBPase and PRK activity in some conditions ( Fig. 3; Haake et al. 1998 ). The activities of NADP-GAPDH, PGK and transketolase remain unaltered or only change slightly in plants with a five- to 10-fold decrease in aldolase expression, provided the activities are related to protein ( Fig. 3). The inhibition of photosynthesis, starch synthesis and growth can nevertheless be ascribed to the changes in aldolase expression because these secondary changes in FBPase, SBPase or Rubisco activity are too small to explain the observed changes in photosynthetic rate, and because decreased expression of these proteins anyway leads to very different changes in metabolite levels and allocation ( Harrison et al. 1997 ; Kossmann et al. 1994 ; Paul et al. 1995 ; Stitt & Schulze 1994).

Decreased expression of plastid aldolase leads to changes in photosynthesis across a range of growth conditions

A 30–50% decrease in aldolase expression leads to a significant inhibition of ambient photosynthesis and growth when plants are growing in low irradiance, in high irradiance, and in high irradiance plus elevated carbon dioxide. In all cases aldolase exerted significant control over the rate of photosynthesis. The inhibition of ambient photosynthesis was surprisingly marked in high irradiance and elevated carbon dioxide (Caldolase = 0.55), and smaller but still substantial (Caldolase = 0.15; Haake et al. 1998 ) in low irradiance.

The impact of decreased aldolase expression on leaf carbohydrate levels and growth depends on growth conditions

Decreased expression of aldolase also inhibits starch synthesis, leading to a change in photosynthate partitioning. In low light, decreased aldolase expression led to a larger decrease of the starch than the sugar content ( Haake et al. 1998 ). This shift is accentuated in growth conditions that allow high rates of photosynthesis. In high irradiance there was a dramatic collapse of the starch content when aldolase activity was decreased below about half of that in wild-type plant, whereas leaf sugar levels remained high or, in elevated carbon dioxide, increased.

The inhibition of plant growth in the transformants was largest in low irradiance, and smallest in high irradiance and elevated carbon dioxide. This is opposite to the effect on photosynthesis, but can be explained as follows. First, due to the low levels of carbohydrates at low irradiance ( Fig. 6), a relatively small shortfall in photosynthate production will have a relatively large effect on growth. Indeed, growth is not inhibited in plants at high irradiance until carbohydrates decline to levels resembling those found at low irradiance. Second, in low irradiance, the decrease in biomass in the antisense aldolase transformants includes a near-proportional decrease of leaf biomass, which will amplify the inhibition of photosynthesis on a whole plant basis. In high irradiance and, even more so, in elevated carbon dioxide, leaf biomass is maintained in the transformants because the decrease in whole plant biomass is largely due to decreased stem, root and tuber growth.

The mechanism whereby decreased expression of plastid aldolase inhibits photosynthesis depends on the ambient conditions

Haake et al. (1998 ) reported that decreased expression of aldolase led to an accumulation of triose phosphates and a depletion of Ru1,5bisP and 3PGA, showing that photosynthesis is inhibited by a low rate of Ru1,5bisP regeneration. Similar results were obtained in the present experiments when the potato plants were grown in low irradiance (70 μmol sec–1 m–2).

Different, and unexpected, results were obtained for plants growing at high irradiance, especially with elevated carbon dioxide ( Figs 5 and 6). Although line A-70, with the smallest decrease in aldolase expression, showed a slight (6–17%) inhibition of photosynthesis, a slight decrease of Ru1,5bisP and a marked decrease of 3PGA, when aldolase expression was reduced further (A-3 and A-51) there was a further 31–68% inhibition of photosynthesis, even though Ru1,5bisP did not decrease further and 3PGA rose 2.2- to 3.5-fold. This unexpected increase of 3PGA was accompanied by an increase of hexose phosphates, especially glucose-6-phosphate which is preferentially located in the cytosol, an increase of sugars, and a dramatic decrease of starch, indicating that photosynthesis has become limited by the rate of starch synthesis. This interpretation is supported by the high flux control coefficient (0.42; Haake et al. 1998 ) of plastid aldolase for starch synthesis in high irradiance and saturating carbon dioxide. The accumulation of 3PGA may be a consequence of a phosphate limitation, and a restriction of 3PGA reduction ( Stitt 1996). The low triose phosphate levels will further exacerbate the effect of low aldolase on Ru1,5bisP regeneration and on starch synthesis. The immediate reason for the inhibition of carbon fixation might be the rise of 3PGA in the absence of a compensating rise of Ru1,5bisP ( Stitt 1996; Woodrow & Berry 1988).

Changes in the irradiance and carbon dioxide concentration lead to a co-ordinate ‘coarse’ regulation of all the Calvin cycle enzymes, with especially large changes in the activity of ‘non-regulated’ enzymes like aldolase

An exquisite system of ‘fine’ control involving thioredoxin-mediated regulation of several enzyme steps, changes in pH and magnesium, and changes in the levels of Calvin cycle intermediates allows rapid and co-ordinated changes in the in vivo activities of the four enzymes that catalyse irreversible reactions in response to rapid changes in the environment ( Stitt 1996). During acclimation to sustained changes in the growth conditions, these rapid changes are complemented by a slower ‘coarse’ regulation of Calvin cycle enzyme activity.

Our results show that acclimation involves a change in the activity of each individual Calvin cycle enzyme. Strikingly, the largest ‘coarse’ changes in enzyme activity occurred for aldolase and transketolase, which lack ‘fine’ regulatory properties and catalyse readily reversible reactions ( Fig. 2). Aldolase and transketolase showed a clear increase in activity on a protein basis in response to the growth irradiance, whereas the activities of Rubisco, the plastid FBPase, SBPase and PRK were unaltered or even decreased on a protein basis. In elevated carbon dioxide, aldolase and transketolase activity decreased more than total protein and the activity of the other Calvin cycle enzymes.

Several mechanisms could contribute to the co-ordinated changes in the Calvin cycle enzyme activities

‘Coarse’ control can involve transcriptional or post-transcriptional regulation. The role of light in regulating the expression of key genes encoding photosynthesis proteins, including components of the light harvesting complexes, the photosynthetic electron transport chain, Rubisco, and the other ‘regulated’ Calvin cycle enzymes, has been researched intensively. Strikingly, an increase in growth irradiance leads to a similar increase of the transcripts for ‘unregulated’ enzymes like transketolase and ‘regulated’ enzymes Rubisco and plastid FBPase ( Fig. 4). Although the level of the plastid aldolase transcript did not show a marked response to irradiance, it is possible that there are subtle changes or a shift in the diurnal regulation that were not addressed in the present experiments.

Some aspects of our results indicate that further novel mechanisms also contribute to the co-ordination of Calvin cycle expression. Decreased expression of aldolase resulted in a marked although non-proportional decrease of plastid FBPase activity in all of the growth conditions used in the present study ( Fig. 3j–l; Haake et al. 1998 ). There was a slight but consistent decline of PRK activity ( Fig. 3.–u; Haake et al. 1998 ), a decrease of SBPase activity in low irradiance ( Fig. 3m; Haake et al. 1998 ) but not at high irradiance ( Fig. 3n,o), and a decrease of Rubisco activity in all three conditions used in the present study ( Fig. 3a–c) but not in Haake et al. (1998 ). The latter discrepancy might be due to differences in the nitrogen fertilization regime. In the present study plants were watered daily with nutrient solution and contained higher wild-type Rubisco activity (9.4–10.3 μmol sec–1 g–1 protein) than in Haake et al. (1998 ) (6.7 μmol sec–1 g–1 protein), where nitrogen was supplied via slow release fertilizer. Rubisco represents a larger proportion of the total protein in nitrogen replete plants than in nitrogen-limited plants ( Evans 1989; Fichtner et al. 1993 ). The slower growth rates in the transformants relieved their nitrogen deficiency (data not shown), and the resulting shift in protein allocation might have masked the effect of decreased aldolase expression on Rubisco activity.

These results provide indirect evidence for ‘cross talk’ between aldolase expression and the expression of other Calvin cycle enzymes, especially plastid FBPase. Further experiments are needed to investigate the mechanisms involved. The decrease in plastid FBPase activity is not due to reversal of the thioredoxin-mediated light activation of plastid FBPase ( Fig. 4). It involves a decrease in FBPase protein but not of the pFbp transcript level, indicating that plastid aldolase might modulate the translation and/or the stability of plastid FBPase. This might, speculatively, involve protein–protein interactions or altered translation or degradation of FBPase as a result of the lower levels of Fru1,6bisP, which is the product of plastid aldolase and the substrate for the FBPase. The decrease in Rubisco activity was also not due to deactivation by decarbamylation, and was not accompanied by a decrease of the RbcS transcript level ( Fig. 4), again indicating that the interaction may involve a novel post-transcriptional mechanism.

The contribution of ‘fine’ and ‘coarse’ control to regulation of metabolism, and acclimation to environmental conditions

It has been widely assumed that the enzymes that catalyse thermodynamically irreversible reactions represent the key sites for the control of metabolism. This bias has been accentuated because such enzymes are frequently subject to ‘fine’ regulation, and it has seemed plausible by analogy that they play the major role in the ‘coarse’ regulation of metabolism. It has also been assumed that enzymes that catalyse readily reversible reactions are expressed in excess, and are therefore unsuitable sites for ‘fine’ or ‘coarse’ regulation. The results in the present article directly contradict this view, and demonstrate that effective ‘coarse’ control of the expression of aldolase is of paramount importance for the acclimation of photosynthesis to different environmental conditions. First, even though plastid aldolase catalyses a reversible reaction and is not susceptible to ‘fine’ regulation, it is subject to marked ‘coarse’ regulation. A similar response is also found for transketolase, another ‘non-regulated’ enzyme that catalyses a reversible reaction. Second, plastid aldolase is never expressed in large excess, and makes an increasingly large contribution to the control of ambient photosynthesis as the growth irradiance, the carbon dioxide concentration, and the rate of ambient photosynthesis increase. ‘Non-regulated’ enzymes may exert increasing control as pathway flux increases because their activity can only be increased by a shift in the substrate/product ratio or by ‘coarse’ regulation, whereas the activity of ‘regulated’ enzymes can also be increased by relaxing a range of ‘fine’ feedback loops that act on the enzyme.

Two important implications follow for the design of experiments to understand acclimation and evolution, and of strategies to manipulate metabolism. First, there is no connection between the susceptibility or non-susceptibility of an enzyme to ‘fine’ regulation, and the importance of ‘coarse’ regulation during acclimation to new environmental conditions, or as a relevant trait for selection. Second, investigations of changes in gene expression during development or in response to changes in the environment should not be restricted to one or a small number of putatively ‘regulatory’ enzymes, but should also address the changes of a wider spectrum of enzymes including those that are not involved in ‘fine’ regulation.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant material and growth conditions

Wild-type potato plants and selected transformant lines were multiplied in tissue culture ( Haake et al. 1998 ), explanted to soil in growth chambers, and grown at an irradiance of 70 or 390 μmol photons sec–1 m–2 and 350–400 p.p.m. CO2 or 800 p.p.m. CO2, temperature of 25°C, relative humidity of 60%, in a 12-h day/12-h night photo-regime, and watered daily with nutrient solution ( Geiger et al. 1998 ).

Plant harvest

The first fully expanded leaf from 28-day-old plants was harvested 10 h into the photoperiod, transferred to liquid nitrogen under growth irradiance and carbon dioxide concentration, powdered at –180°C, stored at –80°C, subaliquots removed, weighed at –180°C, and extracted to determine transcript and protein levels, enzyme activities and metabolites. The remainder of the plant was weighed, frozen in liquid nitrogen and stored at –80°C.

Determination, enzyme activities, protein content, gas exchange and metabolites

These were carried out as in Haake et al. (1998 ).

Western blots

These were performed as in Haake et al. (1998 ) using the Luminol system (Pierce). Antibodies against aldolase and plastid FBPase (Professor R. Scheibe) were used at 1/10 000 dilution.

Northern blots

These were performed as in Geiger et al. (1998 ) using probes for aldolase ( Haake et al. 1998 ), RbcS ( Eckes et al. 1985 ), pFbp ( Kossmann et al. 1994 ) and Tkt (U. Sonnewald, IPK, Gatersleben).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We are grateful for support from BASF AG and the DFG (Sti78/3–1, Schwerpunktprogramm Stoffwechsel und Wachstum der Pflanze bei erhöhrter CO2 Konzentration). We are grateful to Professor R. Scheibe for providing us with the FBPase-antibody, to J. Kossmann for the pFbp-clone, and to U. Sonnewald for the Tkt-clone.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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