ABSTRACT
Though it is an essential process, transcription can be a source of genomic instability. For instance, it may generate RNA:DNA hybrids as the nascent transcript hybridizes with the complementary DNA template. These hybrids, called R-loops, act as a major cause of replication fork stalling and DNA breaks. In this study, we show that lowering transcription and R-loop levels in plastids of Arabidopsis thaliana reduces DNA rearrangements and mitigates plastid genome instability phenotypes. This effect can be observed on a genome-wide scale, as the loss of the plastid sigma transcription factor SIG6 prevents DNA rearrangements by favoring conservative repair in the presence of ciprofloxacin-induced DNA damage or in the absence of plastid genome maintenance actors such as WHY1/WHY3, RECA1 and POLIB. Additionally, resolving R-loops by the expression of a plastid-targeted exogenous RNAse H1 produces similar results. We also show that highly-transcribed genes are more susceptible to DNA rearrangements, as increased transcription of the psbD operon by SIG5 correlates with more locus-specific rearrangements. The effect of transcription is not specific to Sigma factors, as decreased global transcription levels by mutation of heat-stress-induced factor HSP21, mutation of nuclear-encoded polymerase RPOTp, or treatment with transcription-inhibitor rifampicin all prevent the formation of plastid genome rearrangements, especially under induced DNA damage conditions.
Subject(s)
Arabidopsis/genetics , DNA Damage , DNA Repair , Gene Expression Regulation, Plant , Genome, Chloroplast , Genomic Instability , Arabidopsis Proteins/genetics , Cell Nucleus/metabolism , Chlorophyll/chemistry , Chloroplasts/genetics , DNA Primers/genetics , DNA Replication , DNA, Plant/genetics , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/genetics , Genome, Plant , Mutation , Phenotype , Plants, Genetically Modified/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombinases/genetics , Rifampin/pharmacology , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
In order to provide more insight into the function of aquaporins during pollination, we characterized NIP4;1 and NIP4;2, 2 pollen-specific aquaporins of Arabidopsis thaliana. NIP4;1 and NIP4;2 displayed high amino acid identity. RT-PCR and GUS promoter analysis showed that they have different expression patterns. NIP4;1 is expressed at low levels in mature pollen, while NIP4;2 is highly expressed only during pollen tube growth. Single T-DNA nip4;1 and nip4;2 mutants and double amiRNA nip4;1 nip4;2 knockdowns showed reduced male fertility due to deficient pollen germination and pollen tube length. Functional assays in oocytes showed that NIP4;1 and NIP4;2 transport water and nonionic solutes. Here, the participation of the different pollen aquaporins in pollen hydration and pollen tube growth is discussed.
