10.3.2 Genome interactions

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Table 10.3

Plants are unique in having genomes in three cellular com-partments: the nucleus, plastids and mitochondria. Most DNA and hence most of the genes, an estimated 30 000 in higher plants, reside in the nucleus. However, several vital genes for photosynthesis and respiration are found in chloroplast and mitochondria, respectively (Table 10.3). DNA in organelles was first noted in the 1960s, and was subsequently shown to self-replicate, but not in the manner of nuclear DNA and not synchronised with the S phase. The nature of organelle DNA has also, among other characteristics of these organelles, lent much support to the notion that plastids and mitochondria had evolutionary origins as endosymbiotic prokaryotes. We now know that there are approximately 120 genes in plastids but only around 20 in mitochondria (Table 10.3).

(a)  Nuclear genome

The total amount of DNA per plant nuclear genome is highly variable, and ranges from 0.1 to 127 picograms per complete haploid chromosome set (a picogram is 10–12 g). As mentioned already, Arabidopsis has a very small genome: 0.1 pg represents approximately 105 kilobase pairs (kb) of DNA. Maize has over 10 times more — 106 kb — with most of the extra quantity being non-coding. The proportion of non-coding DNA affects molecular genetic studies such as cloning of genes based on molecular maps. In species with large sections interspersed between coding sequences, the probability of finding genes is much diminished.

(b)  Plastid genome

Plastid (chloroplast) genomes of higher plants range from 120 to 160 kb and complete chloroplast DNA (cpDNA) sequences have been determined for several species. In a leaf cell there is much more total DNA than that represented by a single chloroplast genome. For example, 20% of the DNA in the basal half of a 2 cm spinach leaf is cpDNA. At this stage of development, each of the approximately 16 chloroplasts per cell has about 200 cpDNA genome copies, but this number varies with devel-opment. Not surprisingly, most of the proteins coded by cpDNA relate to photosynthetic function, but some key enzymes, for example the small subunit of Rubisco (see below), are encoded by the nuclear genome.

(c)  Mitochondrial genome

In flowering plants, mitochondrial DNA (mtDNA) size varies from 200 to 2400 kb, a much greater range between species than for plastid DNA. Number of mtDNA copies per mito-chondrion varies widely, and total mtDNA per cell is also dependent on the number of mitochondria. Organisation of mtDNA is unusual, mostly existing as a series of subgenomic circles. The functions of some mitochondrial genes are listed in Table 10.3.

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Figure 10.27 Two genomes interact to generate a whole range of chlorplast proteinse. Nuclear-encoded genes are translated on 80S cytoplasmic ribosomes, whereas chlorplast genes are expressed using 70S plastid ribosomes. Nuclear-encoded proteins are targeted to specific locations in the chlorplast by special 'transit' peptides at the N-terminal end of the protein. In some instances, such as the major photosynthetic enzyme Rubisco (ribulose-1,5-biphosphate carboxylase/ oxygenase), one polypeptide is encoded by a nuclear gene (the small submit) and the other by a chloroplast gene (the large submit). Final assembly of the functional protein occurs within the chloroplast.

(Based on Keegstra 1989, reproduced with permission of Cell Press)

(d)  Genome cooperation

To enable normal cell functioning, all three genomes need to work together. Most of the interactions are nucDNA ⇔ cpDNA and nucDNA ⇔ mtDNA. As mentioned above, major protein complexes of photosynthetic membranes include some proteins encoded by nucDNA and some by cpDNA. Each genome uses its own gene expression machinery: nuc-leus and cytoplasmic ribosomes for nucDNA genes, but cpDNA expression is entirely contained within the plastid (Figure 10.27). Proteins synthesised in the cytoplasm are targeted to the chloroplast by virtue of specific amino acid sequences at the N terminus called transit peptides. Different transit peptides determine final locations within the chloroplast, that is, stroma or thylakoid, and even orientation within the membrane, that is, outward or inward facing. We can deduce that there is signalling between the two compartments to ensure appropriate quantities of each protein. The regulation of nuclear gene expression includes transcriptional and post-transcriptional mechanisms as described earlier, but we have little information on how chloroplasts signal to the nucleus. The major carboxylating enzyme, Rubisco, has two subunits, one coded by a nuclear gene rbcS for the small subunit, and one by the chloroplast gene rbcL for the large subunit. Large quantities of this enzyme are needed not just because it is the key carboxylating enzyme in most species, but also because it is actually a relatively inefficient catalyst. With Rubisco sometimes representing more than 50% of the total chloroplast protein, and indeed being the most abundant protein on earth, we can expect that this colossal scale of genome cooperation is quite tightly regulated.

Similar interactions between mitochondrial and nuclear genomes generate many of the respiratory complexes for the mitochondrial membranes. In addition, mitochondria depend on import of some nuclear-encoded tRNAs for mitochondrial protein synthesis. Cytoplasmic male sterility is a developmental phenomenon, best studied in maize, which illustrates the intricacies of nuclear and mitochondrial genome interaction. One particular mitochondrial gene cmsT causes male sterility due to inhibition or prevention of normal stamen development. This gene codes for a 13 kDa polypeptide which becomes an integral membrane protein within the inner mitochondrial membrane. We do not yet understand the exact mechanism, but failure of stamen development may relate to the high energy demand of this developing tissue.

The nucleus clearly has a dominant role in coordinating overall cellular activity, including the contributions of the plastid and mitochondrial genomes. But is there any inter-action between chloroplasts and mitochondria? There is no direct evidence for this, but there are some intriguing DNA sequence homologies in the two genomes. Chloroplast DNA sequences occur in both nucleus and mitochondria, for example partial sequences of the rbcL gene. However, the mitochondrial sequences are non-coding and may simply reflect ancient evolutionary history.

(e)  Endosymbiont theory: how did chloroplasts and mitochondria originate in eukaryotic cells?

There is very strong evidence that chloroplasts and mito-chondria were both originally prokaryotic endosymbionts within primitive eukaryotic cells. The progenitor of chloroplasts of higher plants was most likely an ancient member of the Cyanobacteria which contained chlorophyll and had photosystem I and II activity. Mitochondria probably came from aerobically respiring bacteria, and bear most resemblance to purple sulphur types in the Eubacteria. Recent comparative DNA sequence analysis of ribosomal and protein coding genes of these prokaryotes and the organelles has confirmed these views. These organelles are semiautonomous with their own genome replication and division cycles, and their protein synthesising machinery uses prokaryote-type 70S ribosomes. Although isolated chloroplasts can exist and continue to photosynthesise in a test tube for a few hours, eventually they require new proteins which are encoded in the nucleus.

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Figure 10.28 During evolution of bacteria and plants, some common genes such as those for the photosynthetic enzyme Rubisco large (rbcL) and small (rbcS) subunits have been transferred between genomes and between organisms. The thick arrows denote the endosymbiotic origin of chlorplasts (1) and mitochondria (2). Based on DNA sequence similarities, we deduce that gene transfer subsequently occurred from chlroplast to nucleus (4, representing the transfer of the rbcS gene in ancestral green algae). It has also been possible to genetically engineer tobacco with an rbcL gene inserted into its nuclear genome (5). Similar paths of transfer are known for mitochondrial genes, but originating from proteobacterium groups. A second form of rbcL occurs in anaerobic proteobacteria and has been transferred into nuclear genomes of dinoflagellates (3), a group of algae involved in coral reefs and responsible for toxic 'red tides'. Perhaps unexpected is the finding that modern plant mitochondria contain remnant non-functional rbcL genes, suggesting at least five instances of organelle transfer (6-10).

If the endosymbiont theory is correct, why does so little DNA remain in present-day chloroplasts and mitochondria? The answer relates to gene transfer into the nucleus, and this is consitent with the presence of cpDNA and mtDNA sequences in the nuclear genome. This can explain the genetic organisation of multi-submit proteins such as Rubisco. Through evolutionary time, there has probably been lateral gene transfer between bacteria and eukaryotes, and between comparments within eukaryotic cells (Figure 10.28).

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