Rubisco synthesis

The amino acid composition of storage proteins differs from that of the complete sprout [12, 13]. At least in the case of oilseed rape, alfalfa (Medicago sativa L.) and Camelina sativa, amino acids in the sprout are used mainly, either directly or indirectly, for the synthesis of the Rubisco proteins. Computer analysis shows that the amino acid composition of cruciferin and napin is completely different to the amino acid composition of Rubisco. This indicates that amino acids released from the seed storage proteins must be converted into other amino acids prior to Rubisco synthesis. 

To give a real example, have a closer look on main functions and cycle of magnesium in green plants. Control on autocatalysis depends on the principal functions of Mg, that is, on photosynthesis when substantial parts of Mg taken up by roots are allocated to chlorophyll and rubisco synthesis, less will be available for other metabolic pathways, reducing the turnovers there unless there are lots of Mg around like in marine plants. In addition, the tricarboxylate cycle (citrate cycle) requires Mg (besides Fe and Mn) to produce the enzymes hence some Mg (as well as Fe, Mn) must be invested to produce the citrate (malate, oxaloacetate (aspartate)) ions delivered by the roots to render Mg (and other metals) in turn bioavailable by means of complexation and resorption of almost neutral complex entities. Furthermore, the tricarboxylate cycle is coupled to biosynthesis of amino acids by redox transamination hence there will be both competition at the metal center(s) and possible extraction of metal ions from enzymes once NHj and electrons are... 

Fig. 3.2 Rapeseeds were germinated from 12to 168 h in airlift tank. The total soluble proteins were extracted and separated by 15% SDS-PAGE. The gel was stained with Coomassie blue. Between 36 and 60 h, the degradation of storage proteins and the de novo synthesis of Rubisco is clearly visible.

Fig. 3.3 A. Northern blot showing the synthesis of Rubisco SSU mRNA after 24-36 h of sprouting in an airlift tank. Total RNA was isolated from sprouts germinated from 12 to 168 hours. B. On the same filter, unlabelled Rubisco RNA produced by in vitro transcription was loaded as a control. The amount of control RNA is indicated in pg.

Transcriptional inhibitors could be used simultaneously. Rifampicin blocks chloroplast and mitocondrian RNA synthesis [23, 24], while tagetitoxin is a very specific inhibitor of chloroplast RNA polymerase [25]. Treatment with these antibiotics does not inhibit Rubisco SSU synthesis since the promoter is part of the nuclear genome, while the cytosolic ribosomes are not affected by streptomycin. Therefore SSU promoters can be used to drive transgene expression and facilitate the accumulation of recombinant proteins. Expressed proteins are targeted to a suitable cellular compartment, such as the cytoplasm, apoplastic space or chloroplast, depending on the nature of the protein. 

Fig. 3.7 Transgenic rapeseeds were sprouted in an airlift tank with (lane 1) and without (lane 2) of streptomycin at 100 mg L-1. Total proteins were extracted, separated by SDS-PAGE and stained with Coomassie blue. The synthesis of Rubisco large and small subunits was inhibited as clearly shown in lane 2.

The rubisco reaction forms part of a cycle of reactions, called the Calvin cycle, that leads to the regeneration of ribulose 1,5-bisphosphate (ready to fix another C02) and the net production of glyceraldehyde 3-phosphate for the synthesis... 

In the brown alga Lamionaria saccharina, RubisCo is also inhibited by cadmium (Kremer and Markham, 1982). In-vivo, no direct interaction of cadmium with the enzyme was found, but de novo protein synthesis was inhibited. It was concluded that... 

Although the bundle sheath chloroplasts contain all the enzymes of the RPP cycle, there is now evidence that some of the 3-PGA formed by the activity of rubisco is exported to the mesophyll cells [9]. Bundle sheath chloroplasts of maize are deficient in photosystem II activity and apparently cannot produce sufficient NADPH to reduce all of the 3-PGA formed to triose phosphate. Responsibility for this step is thus shared with the mesophyll chloroplasts which recycle triose phosphate to the bundle sheath as DHAP. This transport of 3-PGA from bundle sheath to mesophyll permits the synthesis of sucrose in the mesophyll cell cytoplasm. The evidence suggests that the mesophyll cells are the major site of sucrose synthesis [10-13]. Sucrose phosphate synthetase, one of the regulatory enzymes of sucrose synthesis and fructose 6-phosphate, 2-kinase (Fru-6-P,2K), the enzyme synthesizing fructose 2,6-bisphosphate — a potent regulator of cytoplasmic sucrose synthesis (see Section 5.4.1) — are both almost completely confined to the mesophyll cells. 

A further aspect of molecular synthesis in a high-temperature environment is the recognition of "heat-shock proteins," proteins whose synthesis is promoted by heat stress. One particular group of heat-shock proteins is seen as particularly relevant to the development of photosynthesis. These are the chaperonins, which play an important role in binding and shaping the enzyme rubisco. 

The genes that code for ribulose- 1,5-bisphosphate carboxylase (rubisco) are found within the chloroplast (the L subunit) and the nucleus (the S subunit). The activation of these genes is mediated by an increase in light intensity (illumination). Phytochrome also appears to play a role in this activating process. Once the S subunit is transported from the cytoplasm into the chloroplast, both subunits assemble to form the L8S8 holoenzyme. A protein called the large subunit-binding protein appears to assist in the assembly of the holoenzyme. When illumination is low, the synthesis of both subunits is rapidly depressed. 

Among the proteins found in the chloroplast stroma are the enzymes of the Calvin cycle, which functions in fixing carbon dioxide into carbohydrates during photosynthesis (Chapter 8). The large (L) subunit of ribulose 1,5-bisphosphate carboxylase (rubisco) is encoded by chloroplast DNA and synthesized on chloroplast ribosomes in the stromal space. The small (S) subunit of rubisco and all the other Calvin cycle enzymes are encoded by nuclear genes and transported to chloroplasts after their synthesis in the cytosol. The precursor forms of these stromal proteins contain an N-terminal stromal-import sequence (see Table 16-1). 

Experiments with isolated chloroplasts, similar to those with mitochondria illustrated in Figure 16-25, have shown that they can import the S-subunit precursor after its synthesis. After the unfolded precursor enters the stromal space, it binds transiently to a stromal Hsc70 chaperone and the N-terminal sequence is cleaved. In reactions facilitated by Hsc60 chaperonins that reside within the stromal space, eight S subunits combine with the eight L subunits to yield the active rubisco enzyme. 

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