WHIRLY1 is a major organizer of chloroplast nucleoids.

Karin Krupinska,Maria Mulisch,Svenja Oetke,Anke Schã¤Fer,Gã¶Tz Hensel,Jochen Kumlehn,Christine Desel,Julien Hollmann

doi:10.3389/fpls.2014.00432

Abstract

WHIRLY1 is an abundant protein of chloroplast nucleoids, which has also been named pTAC-1 with regard to its detection in the proteome of transcriptionally active chromosomes (TAC). In barley primary foliage leaves, expression of the WHIRLY1 gene is highest at the base whereas protein accumulation is highest in the middle of the leaf where young developing chloroplasts are found. In order to elucidate the function of WHIRLY1 in chloroplast nucleoids, transgenic barley plants with an RNAi-mediated knock-down of the HvWHIRLY1 gene (RNAi-W1) were generated. The homozygous RNAi-W1-7 plants, barely containing traces of the WHIRLY1 protein, were chosen for detailed analyses of nucleoids. Nucleic acid specific-staining with YO-PRO®-1 revealed that in comparison to wild type chloroplasts, which have multiple small nucleoids attached to thylakoids, chloroplasts of the transgenic plants contain large irregularly formed patches of DNA besides nucleoids that are similar in size and shape to those of wild type chloroplasts. In large electron lucent areas, filamentous structures were detected by conventional transmission electron microscopy. Analyses of ptDNA levels by both DNA dot-blot hybridization and quantitative PCR showed that leaves of the transgenic plants have a two- to three-fold higher level of ptDNA than the wild type. The higher ptDNA level in RNAi-W1 plants coincided with an enhanced expression of the gene encoding a putative organelle targeted DNA polymerase in the mid part of primary foliage leaves. Furthermore, overexpression of the barley WHIRLY1 gene in E. coli cells revealed a higher compaction of bacterial nucleoids. These results suggest that WHIRLY1 belongs to the group of plastid nucleoid associated proteins (ptNAP) having a function in compacting a subpopulation of chloroplast nucleoids thereby affecting DNA replication.

Highlights

WHIRLY1 belongs to a small family of single-stranded DNA binding proteins, which contains two members in most plants such as barley, whereas Arabidopsis thaliana has three WHIRLY proteins
These results suggest that WHIRLY1 belongs to the group of plastid nucleoid associated proteins having a function in compacting a subpopulation of chloroplast nucleoids thereby affecting DNA replication
The developmental changes in expression of the HvSVR4 gene closely follow the changes in HvWHIRLY1 expression, which is in accordance with a role of WHIRLY1 in DNA transaction processes required for early chloroplast development

Summary

Introduction

WHIRLY1 belongs to a small family of single-stranded DNA (ssDNA) binding proteins, which contains two members in most plants such as barley, whereas Arabidopsis thaliana has three WHIRLY proteins. Maize mutants with severely reduced levels of the WHIRLY1 protein are impaired in chloroplast development due to greatly diminished levels of ribosomal RNA (Prikryl et al, 2008). The Arabidopsis mutant why1why lacking both plastid located WHIRLY proteins was shown to have variegated green/white/yellow leaves in 5% of the progeny. In such leaves ptDNA molecules with aberrations resulting from illegitimate recombination were detected (Maréchal et al, 2009), indicating that WHIRLY proteins have a function in repair of organelle DNA (Maréchal and Brisson, 2010). Plants resulting from a cross between the Arabidopsis double mutant why1why and a mutant impaired in organelle DNA polymerase IB (polIB) had a more severe phenotype and increased DNA rearrangements than the why1why mutant suggesting that DNA polymerase IB and WHIRLY proteins act synergistically in maintenance of plastid genome stability (Parent et al, 2011; Lepage et al, 2013)

Methods

Results

Conclusion