Carbohydrate Metabolism in Chromoplasts: Carotenoid and Lipid Biosynthesis Pathways

Chromoplasts sustain their active synthesis of lipids and carotenoids through carbohydrate metabolism rather than photosynthesis, drawing on energy sources and reducing equivalents such as NADH₂ and NADPH₂. Because the continuous synthesis and accumulation of lipid compounds and carotenoids constantly consumes precursors, these organelles must perpetually replenish their pool of precursor molecules, energy carriers, and reducing equivalents.

The chloroplasts of photosynthesizing tissues obtain these components directly from photosynthetic reactions. In contrast to chloroplasts, chromoplasts are metabolically heterotrophic organelles, meaning they cannot generate their own building blocks from light and carbon dioxide.

Isolated chromoplasts that are active in synthesizing lipids and carotenoids carry out almost no light-dependent incorporation of carbon dioxide. For this reason, the renewal of the pool of organic compounds — the precursors for carotenoid and lipid synthesis, along with the energy sources and reducing equivalents of these organelles — cannot be supplied by photosynthesis. The supply must instead come from imported metabolites and from the organelle's own carbohydrate-processing pathways.

How does carbohydrate metabolism change as chloroplasts become chromoplasts?

As chloroplasts transform into chromoplasts, they lose their light-dependent capacity to fix carbon dioxide and undergo substantial shifts in the activity of various carbohydrate-metabolism enzymes. H. Ziegler and colleagues documented these changes during the conversion of chloroplasts into chromoplasts in the fruit of the annual pepper plant.

The earliest steps of carbon dioxide fixation — those leading to the formation of glycerol-3-phosphate and malic acid — are blocked less during plastid transformation than the reactions that follow. Plastids with an intermediate structural organization (chlorochromoplasts) retained fairly active enzymes that convert the primary products of carbon dioxide fixation into glucose-6-phosphate, the substrate for the oxidative pentose phosphate cycle of carbohydrate conversion. H. Ziegler and co-workers went on to compare the activity of the key enzymes of the reductive pentose phosphate cycle and two enzymes of the subsequent conversions of photosynthetic products in isolated chloroplasts and chromoplasts.

The characteristic enzymes of the Calvin cycle lose roughly 80% of their activity during transformation, while fructose-bisphosphate aldolase activity stays unchanged. The enzymes that decline sharply are:

• ribulose-bisphosphate carboxylase
• phosphoribulokinase
• NADP⁺-dependent glyceraldehyde-phosphate dehydrogenase

The high aldolase activity in chromoplasts reflects the efficient use of hexose phosphates as substrates for the oxidative pentose phosphate cycle, according to the authors. Supporting evidence came from experiments showing that glucose-6-phosphate dehydrogenase — the key enzyme of this chain of carbohydrate conversions — had similarly high activities in both chloroplasts and chromoplasts, and was 1.4–1.5 times higher in organelles of intermediate organization.

Why is glucose-6-phosphate dehydrogenase no longer light-regulated in chromoplasts?

In photosynthetically active chloroplasts the activity of glucose-6-phosphate dehydrogenase is photoregulated, but in mature chromoplasts this light regulation does not occur. The loss of photoregulation is consistent with the chromoplast's shift toward a heterotrophic, dark-running carbohydrate metabolism that no longer depends on the light state of the plastid.

Where do chromoplasts obtain acetyl-CoA for lipid and carotenoid synthesis?

Chromoplasts use acetyl-CoA as the principal substrate for the synthesis of lipids and carotenoids. This acetyl-CoA can be generated from acetic acid by the enzyme acetyl-CoA synthetase, which was detected in functionally active plastids of the yellow daffodil.

The transformation of chloroplasts into chromoplasts alters the permeability of the plastid envelope membranes to certain compounds, including acetic acid, as shown by in vitro experiments with plastids (see The chromoplast envelope). Acetyl-CoA — along with ATP and NADH₂ — can also arise from glycolytic conversions of triose phosphates.

Glycerol-3-phosphate and pyruvic acid can serve as precursors of fatty acids in the chromoplasts of the yellow daffodil, as the work of H. Kleinig and B. Liedvogel demonstrated. Evidently, chromoplasts run a chain of glycolytic conversions leading from dihydroxyacetone phosphate to acetyl-CoA.

How are triose phosphates and ATP supplied to the chromoplast?

Triose phosphates can enter the chromoplast from the cytoplasm in exchange for inorganic phosphate via the phosphate translocator in the chromoplast envelope. The same envelope also contains an adenylate translocator, which can supply these organelles with ATP synthesized in the cytoplasm.

NADPH₂ serves as a cofactor at individual stages of the biochemical conversions in the synthesis of fatty acids and carotenoids. NADPH₂ is most likely produced within chromoplasts through the reactions of the oxidative pentose phosphate cycle. The high aldolase and glucose-6-phosphate dehydrogenase activities found in chromoplasts are arguments in favour of this assumption. The carbohydrate metabolism is best presented as a diagram. Figure 1 — Scheme of some important metabolic processes carried out in chromoplasts:

1 — phosphate translocator; 2 — adenylate translocator; 3 — penetration of acetic and mevalonic acids through the envelope membranes of chlorochromoplasts and chromoplasts; 4 — acetyl-CoA synthetase; 5 — glycolysis; 6–10 — biosynthesis of carotenoids from isopentenyl pyrophosphate; 11 — aldolase; 12 — glucose-6-phosphate dehydrogenase; 13 — NAD⁺-dependent triose-phosphate dehydrogenase.

On the basis of these data, H. Ziegler and colleagues proposed a scheme of the most important metabolic processes in chromoplasts (Fig. 1). H. Kleinig and B. Liedvogel then investigated the effectiveness of different exogenous energy sources in the synthesis of fatty acids. Besides ATP, the chromoplasts of the daffodil corona used ADP relatively efficiently as an energy source, achieving 74% incorporation of [1-¹⁴C]-acetic acid into fatty acids relative to ATP.

Which energy sources drive fatty acid synthesis in chromoplasts?

The high efficiency of ADP is attributed to adenylate kinase functioning within chromoplasts, an enzyme previously shown in the photosynthesizing organelles of various organisms. Among the nucleotide triphosphates tested, the relative effectiveness ranked as follows:

• ATP — the reference energy source (100%)
• ADP — relatively effective (74% of ATP), via adenylate kinase
• UTP, CTP and GTP — weak stimulating effect (15–32% of ATP)
• AMP — practically no stimulation of label incorporation into fatty acids

The glycolytic reactions leading from dihydroxyacetone phosphate to acetyl-CoA pass through high-energy organic molecules as intermediates. H. Kleinig and B. Liedvogel tested these high-energy intermediates of the glycolytic chain as possible energy sources for fatty acid synthesis.

Dihydroxyacetone phosphate, glyceraldehyde-3-phosphate and glycerol-3-phosphate showed activity comparable to ATP. Glycerol-2-phosphate and phosphoenolpyruvic acid stimulated fatty acid synthesis more strongly still — 2.3–2.7 times more effectively than ATP.

How does phosphoenolpyruvate boost ATP levels inside chromoplasts?

Chromoplasts contain pyruvate kinase, which catalyses the formation of ATP from phosphoenolpyruvate. Isolated chromoplasts contained only 50 pmol ATP per mg protein, corresponding to 5 µM. After phosphoenolpyruvate was added to the chromoplast suspension, the amount of ATP increased 7–8 fold through phosphorylation of endogenous ADP or AMP, reaching 380 pmol ATP per mg protein, or 36 µM.

The intermediates of the oxidative pentose phosphate cycle can therefore act as effective energy sources for the biosynthesis of lipids and carotenoids. If this also occurs in chromoplasts in vivo, then the phosphate translocator in the chromoplast envelope may run in reverse — that is, it could deliver triose phosphates into the plastid stroma in exchange for inorganic phosphate.

The reverse function of the phosphate translocator has already been noted to operate in chloroplasts under non-photosynthesizing conditions, lending plausibility to the same mechanism in chromoplasts.