Requirement for Abasic Endonuclease Gene Homologues in Arabidopsis Seed Development

Arabidopsis thaliana has three genes, Ape1L, Ape2, and Arp, that show homology to abasic (apurinic/apyrimidinic) endonuclease genes of bacterial, yeast, or animal cells. In bacteria, yeast, and animals, abasic endonucleases function in base excision repair of oxidized and other modified DNA bases. Here we report that plants with knock-out mutations in any one of Ape1L, Ape2, or Arp show no apparent differences from wild type in growth rate, growth habit, and fertility. However, coincident knock-out mutations in Ape1L and Ape2 are lethal and lead to abortion of developing embryos. Mutations of Arp are not deleterious, even in combination with one of the other two mutations. The results are consistent with the interpretation that the process of base excision repair, involving at least one intact copy of Ape1L or Ape2, is required in the process of embryogenesis

Requirement for Abasic Endonuclease Gene Homologues in Arabidopsis Seed Development

Arabidopsis thaliana has three genes, Ape1L, Ape2, and Arp, that show homology to abasic (apurinic/apyrimidinic) endonuclease genes of bacterial, yeast, or animal cells. In bacteria, yeast, and animals, abasic endonucleases function in base excision repair of oxidized and other modified DNA bases. Here we report that plants with knock-out mutations in any one of Ape1L, Ape2, or Arp show no apparent differences from wild type in growth rate, growth habit, and fertility. However, coincident knock-out mutations in Ape1L and Ape2 are lethal and lead to abortion of developing embryos. Mutations of Arp are not deleterious, even in combination with one of the other two mutations. The results are consistent with the interpretation that the process of base excision repair, involving at least one intact copy of Ape1L or Ape2, is required in the process of embryogenesis

Expression of <i>Ape1L</i> (top), <i>Ape2</i> (middle), and <i>Arp</i> (bottom) genes.

<p>RT-PCR used template RNAs extracted from a selection of strains. Genotype of each strain, indicated by PCR analysis of extracted DNA, is shown at the top. In each strain with a T-DNA insertion, <i>ape1L-1</i>, <i>ape2-1</i>, and <i>arp-2</i>, accumulation of transcript of the corresponding gene was undetectable. RNA accumulation differed among the <i>arp-1</i> base-substitution strains.</p

Figure 1

<p>A. Relative sizes and structures of the genes for abasic endonucleases <i>Ape1L</i>, <i>Ape2</i>, and <i>Arp</i>. Diagrams show intron/exon structures, the positions of primers used to assay the identities of alleles, the locations of T-DNA inserts and, for <i>Arp</i>, the mis-sense mutation (*). For <i>Ape2</i>, only the predicted coding regions of gene model At4g36050.1 are shown. B. Relative sizes and predicted domains of the APE1L, APE2, and ARP proteins. xth, Xth exodeoxyribonuclease III; sap, DNA binding; zfp, GRF-type zinc-finger protein.</p

Development of <i>ape1L-1</i>, <i>ape2-1</i> embryos in siliques at different stages of development¹.

1<p>Siliques were taken from self-fertilized <i>ape1L-l^+/−</i>, <i>ape2-1^−/−</i> plants.</p>2<p>Silique stage was scored based on the stage of development of the largest class of embryos in the seeds of the silique.</p>3<p>Numbers indicate the proportion (%) of embryos of each stage found in mutant siliques; numbers in parentheses indicate the proportion (%) of seeds found in wild-type siliques at the same stage of development.</p

Development of <i>ape1L-1</i>, <i>ape-2-1</i> mutant seeds.

<p>A. Immature silique produced by self-cross of a plant heterozygous for <i>ape1L-1</i> and homozygous for <i>ape2-1</i>. B. Cleared pre-globular stage seeds showing one normally developing seed (left) and one aberrant (right) embryo proper and suspensor phenotype from the same silique; scale bar = 80 µm. A number of seeds continue to develop to the heart stage (C) while some seeds remain at the pre-globular stage of development (arrow); scale bar = 85 µm. D. Close-up of (C) showing a seed arrested at the pre-globular stage of development; scale bar = 15 µm. By the bent cotyledon stage (E) some seeds within the same silique show no visible embryo within the central vacuole (*); scale bar = 120 µm. E, embryo proper; S, suspensor.</p

Genotypes of progeny from the self-cross of a plant triply heterozygous for <i>ape1L-1</i>, <i>ape2-1</i>, <i>and arp-1</i> and their wild-type alleles.¹

1<p>Note the lack of progeny that are doubly homozygous for <i>ape1L-1</i> and <i>ape2-1</i> (last three rows).</p