Nucleotide Depletion and Chloroplast Division

Abstract

We have described the identification of crinkled leaves 8 (cls8) which contains a mutation within the gene encoding the large subunit of ribonucleotide reductase (RNR), the enzyme that catalyses the rate limiting step in the synthesis of deoxyribonucleotide triphosphates (dNTPs) for DNA synthesis and repair. The mutation resulted in plants with altered leaf and flower morphology, reduced root growth, bleached leaf sectors and reduced levels of dNTPs. An interesting consequence of the mutation was its effect on chloroplast division. Mutant plants had fewer, larger chloroplasts and a reduced number of chloroplast genomes compared to wild type plants. The morphological phenotype may be a consequence of altered chloroplast replication.

DNA replication is crucial to life: in the duplication of the genome, in its rearrangements, and in its repair when damaged.1 RNR, the enzyme catalysing the rate limiting step in the de novo synthesis of dNTPs, reducing the four ribonucleosides (NDPs) to their corresponding dNDPs,2 plays a crucial role in this process. The eukaryotic enzyme is a heterodimeric α₂β₂ type, comprising two large (R1) subunits and two small (R2) subunits.2 The R1 subunit binds allosteric effectors and contains the substrate binding site for the reduction reaction, whereas the R2 subunit contains a tyrosyl radical and a di-nuclear non-heme iron centre which provides energy for the reaction. The tobacco and Arabidopsis thaliana enzymes have been purified and their activity characterised.3–4

Recently the characterisation of the three members of the Arabidopsis R2 (small subunit) gene family (TSO2, RNR2A and RNR2B) has been published.5 Although there is a degree of functional redundancy between these genes TSO2 alone is sufficient to support normal plant development. We have described the isolation and characterisation of the Arabidopsis cls8 mutant which is mutated in the single gene encoding the R1 (large) subunit of RNR and results in a reduction in the level of dNTPs.6 cls8 cotyledons appear normal whilst the first true leaves and subsequent vegetative and floral organs show a degree of developmental aberration. Emerging leaves are bleached and crinkled. As the leaves expand they remain crinkled but become greener showing patches of white at the edges. Flowers can be asymmetric and petals crinkled. Vigour and seed set are not compromised. Additional cls8 alleles confirm that RNR2 activity is essential since these mutants are not viable beyond the 2–4 leaf stage.

Depleted dNTps Effect Chloroplast Division

Perhaps the most interesting effect of depleted levels of dNTPs in cls8 was on the chloroplasts. Relatively little is known about chloroplast DNA replication and the role of dNTP levels in its regulation. DNA replication is generally considered to be independent of plastid division as plastid and cell division can be uncoupled, however, recent evidence suggests that the two share regulatory elements.7 It is thought that as a leaf cell undergoes expansion plastid division is triggered to maintain the balance between cell size and plastid density and that DNA replication occurs at some point before division.8

The chloroplasts in green areas of cls8 plants were reduced in number but larger in size than in wild type cells whilst in pale areas chloroplast were reduced in number but also small in size. In both cases microscopic examination of the chloroplasts showed they contained apparently normal thylakoid and grana structures and cell size did not appear to be altered. The reduced dNTP pool appears to be a limiting factor in chloroplast genome replication since the number of genomes was reduced by a third in cls8.

The effect of compromised chloroplast development on plant leaf morphology has previously been established.9–11 Whilst the arc (accumulation and replication of chloroplasts) mutants demonstrate normal leaf development in plants with a reduced number of chloroplasts per cell.12 In the arc mutants, there is an inverse relationship between chloroplast number and their size such that the chloroplastic complement of the cell is maintained.13 Reduced DNA replication, mediated via RNAi silencing of the prereplication factor AtCDT1, has also been demonstrated to produce a reduced number of enlarged chloroplasts.7

Although the cls8 phenotype is to some degree a product of depleted dNTP levels on genomic DNA replication and hence cell division, its similarity to various chloroplast mutants9–11 suggests the phenotype may be, at least partially, mediated via the disruption of chloroplast division and development. We postulate that the enlarged chloroplasts in green areas have undergone sufficient DNA replication to allow expansion but not to trigger division whilst the small chloroplasts in the bleached areas have undergone insufficient DNA replication to allow expansion. Presumably, unlike the arc mutants, there is a reduction in the cellular chloroplast complement which effects leaf development. The greening of plants as leaves mature suggests that the effects of dNTP depletion are most severe during periods of rapid cell expansion and perhaps less critical outside these times.

The relationship between plastid DNA replication and plastid division is not clear but it seems likely that DNA replication is a prerequisite for division. As leaves develop there is an increase in the number of chloroplasts per cell requiring plastid genome replication and a pool of available dNTPs. Changes in the amount of chloroplast DNA can be significant. The chloroplast DNA content of cultured spinach leaf discs was shown to increase from 14% to 36% of the total cellular DNA following growth in the light.14 Chloroplast DNA replication might, therefore, require a substantial pool of the dNTPs, particularly during periods of rapid cell expansion, making plastid genome replication particularly sensitive to their depletion.

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