Base excision repair (BER) is a critical genome defense pathway that deals with a broad range of non-voluminous DNA lesions induced by endogenous or exogenous genotoxic agents. BER is a complex process initiated by the excision of the damaged base, proceeds through a sequence of reactions that generate various DNA intermediates, and culminates with restoration of the original DNA structure. BER has been extensively studied in microbial and animal systems, but knowledge in plants has lagged behind until recently. Results obtained so far indicate that plants share many BER factors with other organisms, but also possess some unique features and combinations. Plant BER plays an important role in preserving genome integrity through removal of damaged bases. However, it performs additional important functions, such as the replacement of the naturally modified base 5-methylcytosine with cytosine in a plant-specific pathway for active DNA demethylation.

Six laccase genes, lcc1, lcc2, lcc3, lcc4, lcc5, and lcc9, of the vascular wilt fungus Fusarium oxysporum were isolated and characterized. All genes have the characteristic conserved domains for copper binding of phenol oxidase enzymes. Targeted inactivation of lcc1, lcc3, and lcc5 resulted in a significant decrease of extracellular laccase activity. Reverse transcription-polymerase chain reaction showed that lcc1, lcc2, and lcc9 were constitutively expressed in culture, whereas lcc3 and lcc5 appeared down and up-regulated, respectively, by PacC. Oxidative stress conditions and phenolic compounds altered the growth rate of the Δlcc3 mutant compared with the wild-type. lcc1, lcc3, and lcc9 were expressed in roots and stems during the infection process. However, inactivation of lcc1, lcc3, and lcc5 had no detectable effects on virulence on tomato plants.

Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l^+/−zdp^−/− mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis. DNA cytosine methylation (5-methylcytosine, 5-meC) is an important epigenetic mark, and methylation patterns are coordinately controlled by methylation and demethylation reactions during development and reproduction. In plants, REPRESSOR OF SILENCING (ROS1) is one of the well characterized 5-meC DNA glycosylases that initiate active DNA demethylation by 5-meC excision. Our previous work showed that a 3′-DNA phosphatase, ZDP, functions downstream of ROS1 during active DNA demethylation in Arabidopsis. Here we found that the apurinic/apyrimidinic endonuclease APE1L functions downstream of ROS1 in a ZDP-independent branch of the active DNA demethylation pathway in Arabidopsis. In plants, gene imprinting requires the 5-meC DNA glycosylase Demeter (DME) that has been proposed to initiate a base excision repair pathway for active DNA demethylation in the central cell in female gametophyte. However, besides DME, no other base excision repair enzymes have been found to be important for gene imprinting. Our results show that APE1L and ZDP act jointly downstream of DME to regulate gene imprinting in plants, and suggest that DME-initiated active DNA demethylation in the central cell and endosperm uses both APE- and ZDP-dependent mechanisms.

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.

Summary: Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell‐free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro . We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′‐ or 3′‐blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short‐patch BER) or several nucleotides (long‐patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.