2. MARCO TEÓRICO Y ESTADO DEL ARTE
2.3 ESTADO DEL ARTE
The nuclear genomes of higher plants and eukaryotic algae contain
innumerable genes which are translated in the cell cytoplasm and the products transported
Over the course of evolutionary time these genes have been translocated from the chloroplast genome to the nucleus. The chloroplast genome encodes only 1-5% of the protein-coding genes of a cyanobacterial genome. The process by which genes are lost from the chloroplast is called endosymbiotic gene transfer, and the pre-requisite for this to occur was the development of apparatus in the chloroplast membrane which allowed the import of proteins produced in the cytoplasm into the organelle. In addition, the transferred genes gain an extra protein sequence (usually at the N- terminus of the protein) which targets the protein to the chloroplast. Machinery must therefore be present to remove this extra protein sequence before the protein will be functional in the organelle.
Although gene regulatory processes in the nucleus are much more complex than those in the chloroplast, this in itself does not explain why genes are transferred to the nucleus to such an extent. The chloroplast is able to tightly regulate transcription and translation, and the nucleus also has control at these levels. For example one of the plastid RNA polymerases (NEP) is nuclear-encoded in higher plants, and the other (PEP) is regulated by nuclear-encoded a factors. It could be that genes are transferred to the nucleus because plastids reproduce asexually, this means that harmful mutations cannot be recombined out (Muller's Ratchet). Chioroplasts can partially overcome the effects of Müller’s ratchet by recombination between genomes within the same chloroplast, but transfer of genes to the nucleus would also prevent some of the harmful effects (Race et al., 1999). Plastid genomes are also AT rich in both coding and non-coding regions. This could be due to the nature of the DNA damage to these genomes. Consequently the high AT content of these genes affects the amino acid composition of plastid proteins. For example plastid genes contain more codons for Phe, lie, Lys, Asn and Tyr and lack certain codons for Ala, Gly and Pro. These changes in amino acid content may be deleterious for some proteins and a transfer to the nucleus may be advantageous (Howe at a!., 2000).
_________________________________________________________________ Chapter 1 In answering the question “W hy are genes transferred to the nucleus?” we raise the question “W hy are genes retained by the chloroplast?” It was previously thought that some proteins were too hydrophobic to be transported across the chloroplast membrane, or that codon usage of some chloroplast genes is too different to allow nuclear expression. However the case of the
rbcL gene, encoding for the large subunit of Rubisco, disproves these theories. rbcL is always chloroplast-encoded, despite encoding for a soluble, hydrophilic protein. In addition, the gene can be transferred to the nucleus of tobacco and the transformant will grow phototrophically (reviewed by Race et al, 1999). The codons used in the rbcL gene can be recognised by cytosolic ribosomes. It is possible that some chloroplast-encoded genes are toxic to the cell in the cytoplasm and ultimately all genes will be lost, as we are not seeing the end point of an evolutionary process. This is certainly the case for hydrogenosomes. This organelle is also of endosymbiotic origin, but it totally lacks a genome (Palmer, 1997). However, in the case of the chloroplast it could be that genes are retained in order to rapidly respond to changes in redox balance. This explains why genes encoding for structural proteins involved in maintaining redox balance across the membranes, along with genes for transcription and translation are retained. Also, most chloroplast genes are lost independently in different lineages, so the remaining genes have been retained in different lineages independently, suggesting a selection pressure at work. It also explains the vast reduction in the plastid genome of parasites such as E. virginiana which no longer carry out photosynthesis. Hydrogenosomes have no electron transport across membranes. There is evidence for transcriptional regulation in response to redox state, for example the redox state of plastoquinone controls transcription of genes encoding for components of PSI and PSIl (Race at a i,
1999).
consequently the protein is produced by both genomes. Then, the organellar copy becomes degenerate and is ultimately lost. There are no known examples of genes which are functionally produced from both the nuclear and the organellar genome. However, in the case of mitochondria, which are thought to have evolved by the same mechanism, there are examples of newly transferred genes in the nucleus and degenerate genes in the organelle. For example the mitochondrial genome of Arabidopsis thaliana
contains a defective copy of the rps19 gene, and the nucleus contains a functional newly transferred copy (reviewed by Martin & Hermann, 1998). It is assumed that the targeting sequence is acquired almost simultaneously with integration of the gene into the nucleus, allowing rapid degeneration of the organellar gene copy. This is consistent with evidence that acquisition of the targeting sequence is not the rate limiting step in any gene transfer event. However, any period during which the gene is integrated into the nucleus and stably expressed, but not targeted to the organelle, would have a number of consequences for the newly transferred genes as they would be either freed from selective pressures, or subject to new ones. Both of these situations could result in the accumulation of mutations and account, in part, for the differences in organelle-encoded and nuclear-encoded genes.
To suggest that genes are lost from the chloroplast to the nucleus and then the protein products targeted back to the chloroplast would be over simplifying the process. There are cases which seem to result from loss of genes from the organelle to the nucleus and the protein products never acquire the chloroplast-targeting sequence to allow the protein to function in the organelle. The function is transferred to the cytoplasm and continues to occur at that location. For example, the glycolytic pathway in higher plants appears to be of eubacterial origin, the proteins are of organellar origin and have replaced the endogenous pathway of the original “host”. Also, there are chloroplast proteins which appear to result from the transfer of a gene from the mitochondrion to the nucleus, followed by a gene duplication event and the acquisition of a chloroplast targeting sequence onto the protein, which The transcriptional apparatus of Chlamvdomonas chioroplasts___________________________ ^
_________________________________________________________________ Chapter 1 redirects the protein to the chloroplast. This is then followed by loss of the gene from the chloroplast genome. Usually this occurs for proteins with functions common to both chioroplasts and mitochondria, but has also been suggested to be the origin of the chloroplast nuclear-encoded RNA polymerase (NEP), which is not present in cyanobacteria.
Genes with regulatory functions are transferred to the nucleus more readily than those with enzymatic or structural function. For example the CIp protease consists of two subunits. CIpP is the catalytic subunit and is chloroplast-encoded, and CIpC is the regulatory subunit and is nuclear- encoded (Martin & Herrmann, 1998).