A tiling microarray for global analysis of chloroplast genome expression in cucumber and other plants.

Agnieszka Zmieńko,Wojciech Pląder,Piotr Formanowicz,Marek Figlerowicz,Magdalena Guzowska-Nowowiejska,Radosław Urbaniak

doi:10.1186/1746-4811-7-29

Abstract

Plastids are small organelles equipped with their own genomes (plastomes). Although these organelles are involved in numerous plant metabolic pathways, current knowledge about the transcriptional activity of plastomes is limited. To solve this problem, we constructed a plastid tiling microarray (PlasTi-microarray) consisting of 1629 oligonucleotide probes. The oligonucleotides were designed based on the cucumber chloroplast genomic sequence and targeted both strands of the plastome in a non-contiguous arrangement. Up to 4 specific probes were designed for each gene/exon, and the intergenic regions were covered regularly, with 70-nt intervals. We also developed a protocol for direct chemical labeling and hybridization of as little as 2 micrograms of chloroplast RNA. We used this protocol for profiling the expression of the cucumber chloroplast plastome on the PlasTi-microarray. Owing to the high sequence similarity of plant plastomes, the newly constructed microarray can be used to study plants other than cucumber. Comparative hybridization of chloroplast transcriptomes from cucumber, Arabidopsis, tomato and spinach showed that the PlasTi-microarray is highly versatile.

Highlights

Plastids form a large family of cellular organelles that occur in plants and algae
It has recently been shown that genes transcribed by plastid-encoded RNA polymerase (PEP) are down-regulated and genes transcribed by nucleus-encoded phage-type RNA polymerases (NEPs) are upregulated in tobacco ΔpsaA and ΔpsbA deletion mutants, which lack genes that code for core components of photosystem I and photosystem II, respectively
Our aim was to obtain a microarray probe set allowing for both expression studies of known plastid genes or conserved ORFs, as well as discovery of new RNAs transcribed from non-protein coding regions of the cucumber chloroplast genome

Summary

Introduction

Plastids form a large family of cellular organelles that occur in plants and algae. The most prominent members of the plastid family are chloroplasts. Sigma factors are proteins of nuclear origin that confer promoter specificity of plastid-encoded RNA polymerase (PEP) core subunits This specificity is one of the regulation mechanisms that modulates gene expression under changing environmental conditions [7,9,10]. It has recently been shown that genes transcribed by PEP are down-regulated and genes transcribed by NEP are upregulated in tobacco ΔpsaA and ΔpsbA deletion mutants, which lack genes that code for core components of photosystem I and photosystem II, respectively These mutations, located in the chloroplast genome, affect the expression of nuclear genes. The probes on the PlasTi-microarray do not overlap nor they are contiguous, this array has the highest resolution of the plastid arrays reported so far and covers both coding and non-coding regions on both strands of the plastome This array is an excellent versatile tool for global functional studies of plastid genomes. We demonstrate that the PlasTi-microarray can be used for analyzing the plastome transcriptome in cucumber and other flowering plants

Objectives

Methods

Results

Discussion

Conclusion