Wanda M. Waterworth

Repairing breaks in the plant genome: the importance of keeping it together

DNA damage threatens the integrity of the genome and has potentially lethal consequences for the organism. Plant DNA is under continuous assault from endogenous and environmental factors and effective detection and repair of DNA damage are essential to ensure the stability of the genome. One of the most cytotoxic forms of DNA damage are DNA double-strand breaks (DSBs) which fragment chromosomes. Failure to repair DSBs results in loss of large amounts of genetic information which, following cell division, severely compromises daughter cells that receive fragmented chromosomes. This review will survey recent advances in our understanding of plant responses to chromosomal breaks, including the sources of DNA damage, the detection and signalling of DSBs, mechanisms of DSB repair, the role of chromatin structure in repair, DNA damage signalling and the link between plant recombination pathways and transgene integration. These mechanisms are of critical importance for maintenance of plant genome stability and integrity under stress conditions and provide potential targets for the improvement of crop plants both for stress resistance and for increased precision in the generation of genetically improved varieties.

A plant DNA ligase is an important determinant of seed longevity

DNA repair is important for maintaining genome integrity. In plants, DNA damage accumulated in the embryo of seeds is repaired early in imbibition, and is important for germination performance and seed longevity. An essential step in most repair pathways is the DNA ligase-mediated rejoining of single- and double-strand breaks. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Here, we report the characterization of DNA LIGASE VI, which is only found in plant species. The primary structure of this ligase shows a unique N-terminal region that contains a β-CASP motif, which is found in a number of repair proteins, including the DNA double-strand break (DSB) repair factor Artemis. Phenotypic analysis revealed a delay in the germination of atlig6 mutants compared with wild-type lines, and this delay becomes markedly exacerbated in the presence of the genotoxin menadione. Arabidopsis atlig6 and atlig6 atlig4 mutants display significant hypersensitivity to controlled seed ageing, resulting in delayed germination and reduced seed viability relative to wild-type lines. In addition, atlig6 and atlig6 atlig4 mutants display increased sensitivity to low-temperature stress, resulting in delayed germination and reduced seedling vigour upon transfer to standard growth conditions. Seeds display a rapid transcriptional DNA DSB response, which is activated in the earliest stages of water imbibition, providing evidence for the accumulation of cytotoxic DSBs in the quiescent seed. These results implicate AtLIG6 and AtLIG4 as major determinants of Arabidopsis seed quality and longevity.

Arabidopsis DNA double-strand break repair pathways

DSBs (double-strand breaks) are one of the most serious forms of DNA damage that can occur in a cells genome. DNA replication in cells containing DSBs, or following incorrect repair, may result in the loss of large amounts of genetic material, aneuploid daughter cells and cell death. There are two major pathways for DSB repair: HR (homologous recombination) uses an intact copy of the damaged region as a template for repair, whereas NHEJ (non-homologous end-joining) rejoins DNA ends independently of DNA sequence. In most plants, NHEJ is the predominant DSB repair pathway. Previously, the Arabidopsis NHEJ mutant atku80 was isolated and found to display hypersensitivity to bleomycin, a drug that causes DSBs in DNA. In the present study, the transcript profiles of wild-type and atku80 mutant plants grown in the presence and absence of bleomycin are determined by microarray analysis. Several genes displayed very strong transcriptional induction specifically in response to DNA damage, including the characterized DSB repair genes AtRAD51 and AtBRCA1. These results identify novel candidate genes that encode components of the DSB repair pathways active in NHEJ mutant plants.

DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks

BackgroundDNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability.ResultsKnockout mutants of atlig1 are lethal. Therefore, RNAi lines with reduced levels of AtLIG1 were generated to allow the roles and importance of Arabidopsis DNA ligase 1 in DNA metabolism to be elucidated. Viable plants were fertile but displayed a severely stunted and stressed growth phenotype. Cell size was reduced in the silenced lines, whilst flow cytometry analysis revealed an increase of cells in S-phase in atlig1-RNAi lines relative to wild type plants. Comet assay analysis of isolated nuclei showed atlig1-RNAi lines displayed slower repair of single strand breaks (SSBs) and also double strand breaks (DSBs), implicating AtLIG1 in repair of both these lesions.ConclusionReduced levels of Arabidopsis DNA ligase 1 in the silenced lines are sufficient to support plant development but result in retarded growth and reduced cell size, which may reflect roles for AtLIG1 in both replication and repair. The finding that DNA ligase 1 plays an important role in DSB repair in addition to its known function in SSB repair, demonstrates the existence of a previously uncharacterised novel pathway, independent of the conserved NHEJ. These results indicate that DNA ligase 1 functions in both DNA replication and in repair of both ss and dsDNA strand breaks in higher plants.

The importance of safeguarding genome integrity in germination and seed longevity

Seeds are important to agriculture and conservation of plant biodiversity. In agriculture, seed germination performance is an important determinant of crop yield, in particular under adverse climatic conditions. Deterioration in seed quality is associated with the accumulation of cellular damage to macromolecules including lipids, protein, and DNA. Mechanisms that mitigate the deleterious cellular damage incurred in the quiescent state and in cycles of desiccation-hydration are crucial for the maintenance of seed viability and germination vigour. In early-imbibing seeds, damage to the embryo genome must be repaired prior to initiation of cell division to minimize growth inhibition and mutation of genetic information. Here we review recent advances that have established molecular links between genome integrity and seed quality. These studies identified that maintenance of genome integrity is particularly important to the seed stage of the plant lifecycle, revealing new insight into the physiological roles of plant DNA repair and recombination mechanisms. The high conservation of DNA repair and recombination factors across plant species underlines their potential as promising targets for the improvement of crop performance and development of molecular markers for prediction of seed vigour.

Choice of a start codon in a single transcript determines DNA ligase 1 isoform production and intracellular targeting in Arabidopsis thaliana.

DNA ligase 1 (AtLIG1) is the only essential DNA ligase activity in Arabidopsis and is implicated in the important processes of DNA replication, repair and recombination and in transgene insertion during Agrobacterium-mediated plant transformations. The mitochondrial and nuclear forms of DNA ligase 1 in Arabidopsis are translated from a single mRNA species through the control of translation initiation from either the first (M1) or second (M2) in-frame AUG codons respectively. Translation from a third in-frame AUG codon (M3) occurs on transcripts in which M1 and M2 are mutagenized to stop codons. Wild-type AtLIG1-GFP constructs (where GFP stands for green fluorescent protein) can be targeted in planta to both the nucleus and mitochondria. AtLIG1-GFP translation from M1 specifically targets the fusion protein only to mitochondria in planta, whereas translation from M2 or M3 targets the fusion protein only to the nucleus. Interestingly, the AtLIG1-GFP fusion protein in which translation is initiated from M1 contains both an N-terminal mtPS (mitochondrial targeting presequence) and a nuclear localization signal; nonetheless, this protein is only targeted to the mitochondria. This result raises intriguing questions on the translational control mechanisms that regulate how the protein products of a single transcript are targeted to more than one cellular compartment.

DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds

Significance The DNA checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED play crucial roles in the maintenance of genome stability, safeguarding cellular survival and the faithful transmission of genetic information. Here we show that ATM is an important factor that influences seed quality by linking progression through germination with genome integrity. Our findings provide insight into the roles of DNA damage responses, identifying their importance in regulating germination, a process critical for plant survival in the natural environment and crop production. Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

Dynamics of plant histone modifications in response to DNA damage

DNA damage detection and repair take place in the context of chromatin, and histone proteins play important roles in these events. Post-translational modifications of histone proteins are involved in repair and DNA damage signalling processes in response to genotoxic stresses. In particular, acetylation of histones H3 and H4 plays an important role in the mammalian and yeast DNA damage response and survival under genotoxic stress. However, the role of post-translational modifications to histones during the plant DNA damage response is currently poorly understood. Several different acetylated H3 and H4 N-terminal peptides following X-ray treatment were identified using MS analysis of purified histones, revealing previously unseen patterns of histone acetylation in Arabidopsis. Immunoblot analysis revealed an increase in the relative abundance of the H3 acetylated N-terminus, and a global decrease in hyperacetylation of H4 in response to DNA damage induced by X-rays. Conversely, mutants in the key DNA damage signalling factor ATM (ATAXIA TELANGIECTASIA MUTATED) display increased histone acetylation upon irradiation, linking the DNA damage response with dynamic changes in histone modification in plants.

A novel ATM‐dependent X‐ray‐inducible gene is essential for both plant meiosis and gametogenesis

DNA damage in Arabidopsis thaliana seedlings results in upregulation of hundreds of genes. One of the earliest and highest levels of induction is displayed by a previously uncharacterized gene that we have termed X-ray induced 1 (XRI1). Analysis of plants carrying a null xri1 allele revealed two distinct requirements for this gene in plant fertility. XRI1 was important for the post-meiotic stages of pollen development, leading to inviability of xri(-) pollen and abnormal segregation of the mutant allele in heterozygous xri1(+/-) plants. In addition, XRI1 was essential for male and female meiosis, as indicated by the complete sterility of homozygous xri1 mutants due to extensive chromosome fragmentation visible in meiocytes. Abolition of programmed DNA double-strand breaks in a spo11-1 mutant background failed to rescue the DNA fragmentation of xri1 mutants, suggesting that XRI1 functions at an earlier stage than SPO11-1 does. Yeast two-hybrid studies identified an interaction between XRI1 and a novel component of the Arabidopsis MND1/AHP2 complex, indicating possible requirements for XRI1 in meiotic DNA repair.

The physiology and molecular biology of peptide transport in seeds

Peptide transport plays a major role in the nitrogen nutrition of the cereal embryo during germination. During germination, enzymatic hydrolysis of storage proteins in the cereal grain endosperm forms a reservoir of small peptides and amino acids which are translocated across the scutellum to supply organic nitrogen to the growing embryo. Uptake of these solutes by the scutellum, a modified cotyledon which functions in nutrient transport, is mediated by carriers localized to the plasma membrane of the scutellar epithelium. To date the peptide transporter HvPTR1 is by far the best characterized example of a nutrient transporter involved in reserve mobilization during germination. Peptide transport in the barley scutellum has been relatively well-characterized biochemically, and in recent years the barley scutellar peptide transporter HvPTR1 has been cloned and characterized at the molecular level. Here, we review the physiological role and importance of peptide transport in germination, focusing on recent characterization of the barley peptide transporter HvPTR1. In barley, the uptake of small peptides by the scutellum appears to be mediated by a proton-coupled peptide transport system capable of handling peptides 2–4 amino acid residues in length.