BCH/PPA 503 -- Plant Biochemistry
Lecture Twelve
Carbohydrate Metabolism -- Polysaccharides, Starch Synthesis and Turnover

The purpose of today’s lecture is to discuss the biosynthesis and metabolism of oligo- and polysaccharides, particularly sucrose and starch, and the interaction of key points of carbohydrate metabolism with other major metabolic pathways in plants.

 

 

Reading Assignments for Lecture 12 discussion:

a)      REQUIRED:

1-     Vijn, I. and Smeekens, S. 1999. Fructan: More Than a Reserve Carbohydrate? Plant Physiology 120: 351-359.

2-     Chapter 13, pp 640-675, of the Biochemistry & Molecular Biology of Plants class text.

 

b)      OPTIONAL:

1-     Veramendi, J., Roessner, U. and Trethewey, R.N. 1999. Antisense Repression of Hexokinase 1 Leads to an Overaccumulation of Starch in Leaves of Transgenic Potato Plants But Not to Significant Changes in Tuber Carbohydrate Metabolism. Plant Physiology 121: 123-133.

2-     Smith, A. 1999. Making Starch. Curr. Opinion in Plant Biology 2:223-229.

3-     Bush, D. 1999. Sugar transporters in plant biology. Curr. Opinion in Plant Biology 2:187-191.

4-     Bustos, R., B. Fahy, C. M. Hylton, R. Seale, N. M. Nebane, A. Edwards, C. Martin and A. M. Smith. 2004. Starch granule initiation is controlled by a heteromultimeric isoamylase in potato tubers. PNAS 101: 2215-2220.

 

 

The structures and additional background reading can be found in general biochemistry texts such as the Garrett and Grisham 1995 text (available in the library), Chapt. 13 of the class text and General Organic and Biochemistry, Chapter 17 -- Carbohydrates.

 

Utilization of triose phosphate generated by photosynthesis in starch, sucrose and/or fructan synthesis is tightly controlled (see Chapt. 13 of class text). Since 5/6 of triose phosphate synthesized in photosynthesis is needed for regeneration of RuBP, a maximum of 1/6 of triose phosphate formed is available for synthesis of fructose and other molecules in the cytoplasm. In C3 plants only ~ 1/8 of triose phosphate is available for export from chloroplasts due to photorespiration. If more than 1/6 to 1/8 of the triose phosphate were removed from the Calvin-Benson cycle, RuBP could no longer be regenerated and the cycle would collapse.

 

 

 

However, CO2 fixation reactions of photosynthesis can only proceed if the triose phosphate formed is utilized in synthesis of molecules such as sucrose, starch or fructans. Thus formation of storage and transport carbohydrates can only but must proceed when sufficient photosynthesis occurs. Starch buildup in chloroplasts can only proceed so far after which photosynthesis shuts down. Thus for efficient plant growth photosynthesis must be coordinated with delivery of photosynthate to sinks. Some of the principal steps of carbohydrate metabolism were shown in lecture 11:

 

 

 

 

As mentioned in the last lecture reduced carbon (photosynthate) is transported from source to sink tissues in the form of sucrose. Also as mentioned in the last lecture, sucrose is not made in plastids. The principal avenue for transport of reduced carbon out of chloroplasts has been thought to be in the form of the triose phosphates, DHAP and G3P. These triose phosphates can be utilized in respiration, in the synthesis of other molecules in plants such as lipids or for synthesis of sucrose. G3P and DHAP can be condensed by an aldolase to form Fru-1,6-P2 in the cytoplasm as in the chloroplasts. Fru-1,6-P2 can be converted into Fru-6-P by PPi: fructose 6-P phosphotransferase. Fru-6-P can be isomerized to Glu-6-P which in turn can be converted into Glu-1-P by a mutase. Glu-6-P and Glu-1-P are the main forms in which photosynthate is delivered to plastids of sink tissues for accumulation of starch.

 

 

As mentioned in Lecture 11, sucrose can be synthesized from hexose monophosphates by sucrose synthase or sucrose phosphate synthase. Both enzymes are freely reversible in vitro, but in vivo sucrose phosphate synthase is associated with sucrose phosphatase which catalyzes an irreversible reaction rendering the coupled reaction only toward sucrose synthesis.

In the case of sucrose synthase the in vivo sucrose concentrations are nearly always much higher than fructose or UDP-glucose resulting in this reaction essentially always going in the direction of sucrose cleavage. Invertases on the other hand only catalyze the cleavage of sucrose into glucose and fructose.

 

 

Sucrose transport to different plant parts is not a passive process and is instead modulated by a variety of sucrose and hexose transporters that fulfill a variety of essential roles in partitioning of assimilate between source and sink tissues. In the case of sucrose loading into phloem tissue, symport is driven by a proton motive force that is generated by a proton-ATPase as in chloroplasts and mitochondria but consuming rather than generating ATP. There is evidence for at least six sucrose symporter genes in Arabidopsis (Fig. 1 Bush 1999).

In some plant sink tissues sucrose is the principal form of reduced carbon storage and sucrose can build up to high levels in vacuoles of such storage tissues. Extracellular invertases are a major route for metabolism of sucrose and this is influenced by a number of signals and biotic or abiotic stresses (Fig. 2 Roitsch, 1999). Isozymes of invertase can be found in the cytosol and vacuoles in addition to the apoplast (Sturm, 1999). Further metabolism of hexoses generated by invertase or sucrose synthase requires their conversion back into hexose phosphates by hexokinases. Hexose phosphates are key intermediates in metabolism of carbohydrates:

 

The formation of hexose phosphates by hexokinase is therefore a very important reaction in the metabolism of carbohydrates. Transgenic studies with a cytoplasmic hexokinase in potatoes by Veramendi et al. (1999) provides useful insight on in vivo sugar metabolism. They were able to achieve large increases and decreases in hexokinase activity in leaves and tubers using sense and antisense constructs. This did not affect tuber growth, starch accumulation or other metabolism of tubers. However antisense suppression of hexokinase in leaves resulted in a 3- fold increase in starch, a 2-fold increase in glucose and a decrease in sucrose after a dark period. It has long been thought that export of reduced carbohydrate from chloroplasts was only in the form of triose phosphates. While this may be true in the light, the export of assimilate from transient starch turnover in chloroplasts in the dark appears to be predominately in the form of glucose as indicated in these studies.

 

 

As mentioned previously the energy is stored in plants (and animals) as polysaccharides or triglycerides.  The principal storage carbohydrate in plants is the polysaccharide, starch.  Starch is composed of 2 polymers, amylose, amylopectin and in some cases phytoglycogen.  Natural starch is usually ~10-30% amylose and 70-90% amylopectin.  US cornstarch is normally ~25% amylose and 75% amylopectin.  Amylose molecules are linear chains of D-glucose in alpha-1,4 linkages with molecular weights of several thousand to 500,000.  Amylopectin is a highly branched chain of glucose monomers (amylose often has a few branches, 1-5, usually near the reducing end of the molecule).  Most of the glucose linkages in amylopectin are alpha-1,4 as for amylose, whereas the branches are alpha-1,6.  Branches occur ~every 12- 30 residues.  Amylopectin molecular weights range up to 100 x 106 with a typical molecule being ~200-400 nm long.  Some starch (e.g., from potato) is phosphorylated with up to 1 Pi per 300 glucose residues.  Structures of amylose and amylopectin are given in Figs. 1 and 2 of Smith 1999:

 

1    2  

 

Starch is synthesized and stored in plastids; chloroplasts in leaves and specialized organelles in storage tissues termed amyloplasts.  The starch is densly packed in structures known as starch granules, often as starch crystals.  Starch granules, which contain 0.1-1% lipid and 0.05-0.5% protein, vary in size from < 1 µm to > 100 µm.  Starch granule structure and synthesis is shown in Fig. 1 of Smith (1999):

 

 

 

 

 

Both amylose and amylopectin synthesis begins with synthesis of ADP-glucose from glucose-1-phosphate and ATP by ADP-glucose pyrophosphorylase with liberation of pyrophosphate (see Fig. 2 Smith 1999).  Next starch synthase catalyzes the formation of an alpha-1,4 linkage between the non-reducing end of a preexisting glucose chain and the glucosyl moiety of ADP-glucose with release of ADP. 

 

 

Finally, the alpha-1,6 linkages of the branches are synthesized by starch branching enzyme that hydrolyzes an alpha-1,4 linkage within the chain and then forms an alpha-1,6 linkage between the reducing end of the spliced glucan chain and another glucose residue. 

 

 

These branches have an average length of ~20 glucan units.

 

Starch is catabolized by 5 enzymes:  a- amylase, ß-amylase, a-glucosidase, starch phosphorylase, and a-dextrin 6-glucanohydrolase (debranching enzyme) (see Chapt. 13 of class text):