A tiling microarray for global analysis of chloroplast genome expression in cucumber and other plants
© Żmieńko et al; licensee BioMed Central Ltd. 2011
Received: 27 May 2011
Accepted: 28 September 2011
Published: 28 September 2011
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.
Plastids form a large family of cellular organelles that occur in plants and algae. The most prominent members of the plastid family are chloroplasts. Chloroplasts use light energy to convert carbon dioxide into organic compounds in a process called photosynthesis. Depending on tissue localization and environmental conditions, other types of plastids may develop. Plastids are also involved in various aspects of plant cell metabolism, e.g., they can store starch, lipids or proteins. Certain factors can induce mature plastids to transform from one type to another, as well as to revert back . The process of plastid biogenesis and interconversion is coupled with large structural and biochemical changes. This huge transformation potential of plastids is partly a result of the presence of their own genetic material (plastome) and inherent transcriptional and translation machinery. The first complete sequences of plastid genomes (from Nicotiana tabacum and Marchantia polymorpha) were determined in 1986. Currently, more than 200 plastome sequences are available in GenBank. Most of them (more than 170) are derived from flowering plants. The majority of plastomes were sequenced after 2006, when high throughput sequencing methods became more widely available and less expensive [2, 3]. The sequences of plastid genomes and their organization are highly conserved. Plastomes range in length from 120 to 200 Mbp. They usually contain two large inverted repeats (IR), namely IRA and IRB, separated by single copy regions. However, in some plants, such as Medicago truncatula, the plastomes lack one IR region. Genes encoded in the plastome can be divided into two categories: protein coding (about 70-100 genes, mostly coding for proteins related to the light-phase of photosynthesis or coding for ribosomal proteins), and RNA coding (about 30-50 rRNA and tRNA genes). There are also some conserved open reading frames (conserved ORFs), which have undefined or poorly defined functions. Some plastid genes overlap one another, and many genes are organized into operons, indicative of their prokaryotic origin. The latter are transcribed into polycistronic preRNAs, which are further processed into individual RNA species. The transcripts undergo extensive post-transcriptional modifications, including trans-splicing and RNA editing [4–7].
Plastids do not operate independently of nuclear genetic information. A large number of photosynthesis-related chloroplast proteins are encoded in the nucleus. Similarly, many proteins that are essential for post-transcriptional processing and stabilization of plastid transcripts are encoded in the nucleus and transported to plastids after their synthesis in the cytoplasm . For example, 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]. Apart from PEP, nucleus-encoded phage-type RNA polymerases (NEPs) are also engaged in transcription in plastids [11, 12]. It has recently been shown that genes transcribed by PEP are down-regulated and genes transcribed by NEP are up-regulated 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, also affect the expression of nuclear genes. Genes related to photosynthesis were down-regulated, and stress-responsive genes were up-regulated . This and many other works demonstrate that plastid genes act in concert with nuclear genome products, allowing plants to adapt quickly and flexibly to changing environmental and developmental conditions. However, although the overall structure and function of plastids are quite well known already, and individual plastid genes have often been subjected to intensive studies, few plastome-scale expression studies have been published so far [6, 9, 10, 13–22]. Moreover, most reported experiments focus on the gene-coding regions, but there is growing evidence that the so-called non-coding parts of genomes may play important regulatory roles in prokaryotes and in eukaryotic organelles [15, 23–27]. Therefore, based on cucumber plastid genome sequence, we constructed an oligonucleotide tiling microarray (PlasTi-microarray). Although 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. In this paper, we present the microarray design, as well as detailed protocols for chloroplast RNA (cpRNA) sample preparation and hybridization. We also propose general procedures that can be used for PlasTi-microarray data normalization and analysis. We demonstrate that the PlasTi-microarray can be used for analyzing the plastome transcriptome in cucumber and other flowering plants.
Construction of the PlasTi-microarray
Criteria for oligonucleotide probe selection
Oligo selection criterion
probes satisfying the rule [%]
Melting temperature = 70°C (+/- 8°C)
Nucleotide composition: oligonucleotide cannot have a contiguous single nucleotide base repeat longer than 8 bases AND probe GC content fits the 20-to-60% range AND each base cannot constitute more than 40% of the oligonucleotide sequence
Stem of potential hairpin structure cannot be longer than 8 bases
Minimum Hamming distance to non-target parts of genome > 25
Substring: not more than 20 contiguous bases common to other (non-target) parts of genome
Discrepancies from the probe genome perfect complementarities in inverted repeat regions
Number of probes affected
70/70 > Identity ≥ 65/65, 0 Mismatches, 0 Gaps
Identity = 69/70, 1 Mismatch, 0 Gaps
Identity = 69/70 or 70/71, 0 Mismatches, 1 Gap
Identity = 68/70 or 70/72, 0 Mismatches, 2 Gaps
Identity = 70/76, 0 Mismatches, 6 Gaps
Evaluation of experimental procedure
All microarray experiments described in this paper were performed using a two-color hybridization approach. Cucumber PlasTi-microarrays were produced with a SpotArray 24 instrument (PerkinElmer). All probes were printed in duplicate on the epoxide-coated glass slides (Corning) in sixteen 17 × 36 print-tip groups, together with Stratagene's SpotReport™ Alien™ cDNA Array Validation System oligonucleotide set and control buffer spots. The probes are complementary to the target sequences (they are also complementary to transcripts, not to cDNA). As a result, a method of direct chemical RNA labeling was chosen. For all experiments described in this paper, the Micromax ASAP RNA labeling kit (PerkinElmer) was used. In the original manufacturer's procedure, the total RNA is chemically modified with either Cy3 or Cy5 during a short incubation, and the labeled mRNA is further purified with Oligotex™ RNA kit (QIAGEN). Here, the total cpRNA was subjected to analysis, so the purification procedure was limited to the removal of non-incorporated dye, without fractionating the labeled RNA. Therefore, the miRNeasy Mini Kit (QIAGEN) was used for purification. This kit preserves shorter RNA molecules (tRNA transcripts, highly abundant in chloroplasts and presumptive regulatory RNAs) from washing out. Also, the amount of input RNA was lowered [see Methods for detailed labeling protocol]. Alternatively, the Micromax ASAP RNA labeling kit can be replaced by the Arcturus® Turbo Labeling™ Kit with Cy3/Cy5 (Applied Biosystems) (data not shown).
Data normalization and gene expression analysis
Evaluation of hybridization signal intensities on PlasTi-microarrays
Overall spot intensity characteristics
Norm. min (A min /A mean )
Norm. max (A max /A mean )
"Coding" probes with A probe > A mean
"Non-coding" probes with A probe > A mean
Non-specific signal characteristics
BUFFER (Abuff mean /Aneg mean )
1.021 ± 0.160
0.999 ± 0.020
1.002 ± 0.027
1.002 ± 0.026
1.001 ± 0.034
EMPTY (Aempty mean /Aneg mean )
0.916 ± 0.043
0.993 ± 0.018
0.989 ± 0.013
0.990 ± 0.014
0.987 ± 0.010
ALIEN (Aalien mean /Aneg mean )
1.031 ± 0.155
1.000 ± 0.026
1.001 ± 0.027
1.001 ± 0.022
1.002 ± 0.029
Probes with A probe > Athr
To permit better visualization of the expression patterns of functionally related genes, a graphic representation of the expression data was created with the MapMan software (Figure 3B). MapMan is a user-driven tool that superimposes large data sets, for example microarray data, on diagrams of metabolic pathways . For our dataset, a ChloroPlast_CustomArray pathway diagram  was modified to display all genes represented on a PlasTi-microarray, and specific mapping files were prepared. Those files were added to MapMan Store. They can be downloaded from the project website and used to visualize any gene expression data produced with PlasTi-microarrays .
Transcriptional activity of non-coding regions
PlasTi-microarray in cross-species hybridization experiments
Sequence similarity of cucumber PlasTi-microarray probes to plastid genomes of other plants
PlasTi- microarray probes matching target plastid genome with E < 1 × 10-3
PlasTi- microarray probes matching target plastid genome with E ≤ 1 × 10-10
Summary of CSH microarray experiments
Green Channel (Cy3)
Red Channel (Cy5)
No. of probes with both spot replicates flagged as "good"
Cy5/Cy3 total intensity ratio of probes
Published plant plastome macro- and microarrays
leaves of wild-type vs transplastomic tobacco lacking PEP
cDNA microarray; 220 PCR probes (71 - 2373 bp), each corresponding to a single known gene or an intergenic region
light- or dark-grown seedlings, RIP-chip analysis of MatK-bound RNAs
cDNA microarray; 108 DNA fragments to detect all annotated plastid genes
analysis of knockout transformant for the arginine tRNA gene, trnR-CCG
cDNA microarray; 79 PCR probes (88 - 1646 bp) representing protein-coding genes
effects of the sig2 lesion on the global plastid gene expression
Maize (used also for barley in CSH studies)
cDNA microarray; 248 overlapping PCR products (73 - 1653 bp) covering the whole plastid genome
identification of RNAs associated with PPR proteins in maize (CRP1, PPR4, PPR5) or whirly1 in barley, by RIP-chip
cDNA microarray; PCR products (150 - 1500 bp) for 47 chloroplast, 9 mitochondrial, and 15 nuclear genes
analysis of nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene
Cyanidioschyzon merolae (red algae)
cDNA microarray; 193 PCR probes for protein coding genes and orfs
role of nuclear-encoded sigma factors in plastid transcriptome changes during the shift from dark to light
wheat (used also for barley and rice in CSH studies)
nylon macroarray; 67 PCR products (200 bp - 1259 bp) representing 60 wheat plastid genes (excluding tRNAs) and 7 nuclear genes related to plastid metabolism
germinating seeds and seedlings at three different
stages of development
cDNA microarray; PCR probes for 887 nuclear, 62 chloroplast, and 27 mitochondrial transcripts
comparison of chloroplasts and etioplasts in stage 2 semi-emerged leaf blades of one month-old plant
tobacco, potato, tomato
oligonucleotide microarray; 128 probes (68-71 bases) representing tobacco genes, ycfs and orfs (+ 5 probes designed for potato and tomato due to insufficient homology of tobacco probes)
tomato fruit development and chloroplast-to-chromoplast conversion; potato tuber amyloplasts vs leaf chloroplasts comparison
nylon macroarray; 96 PCR probes (75 - 400 bp), representing all Euglena genes, pseudogenes and orfs
12 different developmental stages and stress treatments
nylon macroarray; 94 PCR probes for genes encoding plastid proteins, tRNAs and rRNAs; data were complemented with analysis of published data from Affymetrix 22 K ATH1 array experiments
numerous nuclear Arabidopsis mutants affected in diverse chloroplast functions and wild-type plants subjected to various stresses and conditions
The PlasTi-microarray tiles the whole plastid genome with a resolution that is much higher than any plastid array reported so far. Still, this resolution is not comparable to that of regular high-density tiling arrays, where probes often overlap each other. We show that this moderate resolution can be beneficial for the simplicity of the data analysis pipeline. The small size of the plastid genomes and the perfect separation of the probes that target coding and intergenic regions allowed us to successfully adopt normalization methods dedicated to expression arrays. The use of these methods is further justified by recent findings that standard (sequence-independent) normalization methods perform equally to sequence-dependent methods even for high-density tiling arrays with short oligonucleotide probes . Also, the analysis of the non-coding regions can simply be based on the probe intensity threshold and can be performed with Excel or similar software. The interactive map that shows the localization of the probes on the cucumber genome sequence is presented as supplementary material [Additional File 2]. This map provides the possibility of distinguishing between the signals located near or opposite to gene coding regions and the signals that apparently are not linked to those parts of the genome. This kind of information can provide clues to the significance of transcripts detected in the intergenic regions.
Using the PlasTi-microarray, we compared the patterns of gene expression in the cucumber plastids isolated from female flowers and from mature leaves. We observed that profiles of functionally related gene expression are congruent, e.g., in flowers; the expression of plastid genetic machinery-related genes is enhanced and the expression of photosynthesis-related genes is weakened. Also, the co-transcribed genes, for example genes of the rpl23-rpoA operon, often shared a similar expression pattern. These results are consistent with observations made by Cho and coworkers during their DNA macroarray-based studies of plastid gene expression in Arabidopsis flowers and leaves . They reported significant down-regulation of many photosynthesis-related genes and up-regulation of the plastid genetic system-related genes. The relative fold changes were higher in Cho's Arabidopsis studies than in our study. However, the PlasTi-microarrays are sensitive enough to detect even minor changes in gene expression. The expression profiles of functionally related genes generated with our microarray were more congruent than those obtained by Cho et al. (except for tRNA genes, see Figure 3). In Cho's experiments, the expression profiles of genes coding for particular components of protein complexes were often contradictory. For example, of the genes encoding the ATP-synthase protein complex, two (atpA and atpF) were up-regulated, and the other two (atpH and atpI) were down-regulated. Similarly, among the Photosystem I complex coding genes, psaA and psaB were up-regulated, whereas psaC, psaI and psaJ were down-regulated .
The high sequence similarity of plant plastomes allows the application of the PlasTi-microarray in cross-species hybridization studies. The probe length (70 nt) ensures high specificity and low sensitivity to single nucleotide mismatches, which frequently occur during inter-species hybridization. Accordingly, for Arabidopsis, spinach and tomato, we were able to obtain a high number of signals with SNR ≥ 3, the value widely used as a threshold for flagging spots of bad quality. Moreover, we showed that filtering the microarray probe set based on the homology of the probes to the analyzed genome can further increase the ratio of probes with high SNR values. Homology-based probe filtering is a common practice in the inter-species microarray studies [cited in ]. As the number of published plastid genome sequences is large and still growing, initial in silico evaluation of the PlasTi-microarray probe specificity can easily be performed before cross-species hybridization experiments.
We have presented in detail the PlasTi-microarray design and hybridization procedure. We show that this procedure can provide high-resolution data without the need for sophisticated analysis. The PlasTi-microarray allows the expression of both coding and non-coding plastome regions to be surveyed and generates data with at a level of resolution that has not been reported for previous plastid arrays. The microarrays are available from the authors on a cost-reimbursement basis, to academic and non-profit institutions.
Plant growing and material collection
All cucumber plants were in var. Borszczagowski background. Seeds were imbibed in water for several hours and sterilized in a regular bleaching reagent (Domestos, 25% v/v sol., 10 min.), followed by extensive washing with the sterile water. Seeds were grown for 4 days on plates with damp blotting-paper. After that seedlings were transferred to watered peat Jiffy pellets (Agrowit, Poland) and grown as follows: (i) experiments A, B and C - wt plants were grown in versatile chamber MLR-350H, Sanyo at with 16 h light at 25°C/8 h dark at 23°C cycles (120-160 μmol*m2*s-1 light), with Jiffy pellets 2/3 immersed in water. 1st and 2nd leaves of 3-4 week old plants were collected; (ii) experiment D - plants (wt, msc16 and tch03 lines) were grown in phytotron at 25°C/8 h dark at 23°C cycles (160-200 μmol*m2*s-1 light). Plants were transferred to 8-cm pots after 2 weeks. Five fully developed leaves were collected from each of 6-week old plants; (iii) experiment E - wt plants were grown in greenhouse (natural light, supported by sodium lamps (16 h light/8 h dark cycles, 100-300 μmol*m2*s-1). Plants were transferred to 30-cm pots after ten days. Material (young and mature leaves, female flowers, growing points, young fruits) was collected from 9-week old plants. For etiolated seedlings (experiment E), seeds were grown for 4 days at dark, at 28°C, before seedling collection.
Whenever necessary (experiments B and C), plants were stressed before collection (see Additional file 3 for description, details will be described elsewhere). All material was roughly grounded in liquid nitrogen and stored at -80°C.
For CSH studies, Arabidopsis, tomato and spinach seedlings were grown as described above. Arabidopsis and spinach plants were further grown in peat Jiffy pellets, in a phytotron at 20°C with 8 h light/16 h dark cycles (160-200 μmol×m2×s-1 light). Mature leaves were collected from 6-week-old plants. Tomato plants were cultured in a greenhouse in mineral wool. Fully developed leaves were collected from 12-week-old fruiting plants. The material was roughly ground in liquid nitrogen and stored at -78°C.
cpRNA isolation and labeling
Chloroplasts were isolated according to  with the following modifications: the sorbitol concentration was increased to 0.4 M in buffers SGB (Sorbitol Grinding Buffer) and SDB (Sorbitol Dilution Buffer). Chloroplast isolation was conducted on ice, and samples were centrifuged at 4°C. Briefly, plant tissue was soaked and ground using a mortar and pestle in homogenization buffer (SGB; 0.4 M sorbitol, 50 mM HEPES, 2 mM EDTA, 1 mM MgCl2, 0.1% bovine serum albumin, 1% PVP; pH 7.4). The homogenate was filtered through Miracloth and centrifuged at 3,000 × g for 5 min. The pellet was resuspended and washed twice in sorbitol dilution buffer (SDB; 0.4 M sorbitol, 20 mM HEPES, 2 mM EDTA, 1 mM MgCl2, 0.1% bovine serum albumin, pH 7.4). The chloroplasts were loaded onto step gradients (30%:70% Percoll in SDB) and centrifuged for 30 min at 4°C at 1,500 × g. The chloroplasts were recovered from the Percoll interface and washed twice in SDB buffer. Only intact chloroplasts were used for cpRNA isolation.
The cpRNA was extracted from the isolated chloroplasts with the use of an miRNeasy Mini Kit (Qiagen) following the manufacturer's instructions. Potential DNA contamination was removed with TURBO DNA-free™ (Ambion) according to the manufacturer's instructions with the following modifications: i) 0.5 U of enzyme was used per 100 μl reaction, and ii) the time of incubation was shortened to 10 minutes. The cpRNA was precipitated with ethanol and redissolved in water to achieve a final concentration of ~ 500 ng/μl. Then, 2 μg of cpRNA was used per 20 μl labeling reaction with MICROMAX ASAP RNA Labeling Kit (PerkinElmer). The reaction mixture included 2 μl of Cy3 or Cy5 dye and 10 μl of labeling buffer. After a 15 min incubation at 85°C in a TC-3000 thermocycler (Techne), the mixture was rapidly cooled to 4°C. A 5 μl amount of Stop Solution was then added, and the samples were cleaned up, as described below.
For removing the unbound dye, Cy3 and Cy5 corresponding reactions were combined and cleaned up with an miRNeasy Mini Kit (Qiagen), following the isolation protocol (starting with the ethanol addition step). RNA was eluted twice with 35 μl water that had been preheated to 55°C. Labeling efficiency was controlled on Nanodrop 1000. Samples were then concentrated in a SpeedVac, to < 10 μl volume, denatured at 68°C for 5 min and mixed with 115 μl of SlideHyb #3 Buffer (Ambion) that had been preheated to 68°C. All samples were incubated at 43°C (for 5 - 30 min) prior to hybridization.
Oligonucleotide probe design and selection
The oligonucleotide probe design and selection algorithm was written in C++ Builder. The cucumber plastid genome sequence, deposited in GenBank under accession number AJ970307, was used as a template. At the beginning of the design procedure, all possible 70-mer nucleotides (with a 1-nucleotide shift) were generated for each strand. We analyzed their nucleotide composition, melting temperature (Tm, calculated from the equation 64.9+(41 × (GC-16.4))/N, where N is probe length and GC is the sum of G and C occurrences in the probe) and secondary structure (determined by the hairpin detection algorithm, followed by mFold analysis of best candidates). To evaluate oligonucleotide specificity, the Hamming distance and substring length in comparison to all other possible 70-mers within the genome were calculated for each candidate sequence. Oligonucleotides not satisfying the threshold criteria, described in Table 1 of the Results section, were penalized: S - 20 points, H - 8 points, T - 5 points and C - 2 points. The best set of probes was chosen within the desired locations (contiguous coding/non coding regions), with an assumed distance between neighboring probes of 140 +/- 10 bases (and up to 4 probes per coding region) by selection of probes with the highest D parameters and 0 penalty points. If there were no oligonucleotides satisfying those criteria, probes with i) the lowest number of penalty points and ii) the highest D parameter were chosen.
Microarray preparation and hybridization
Probes (20 μM) (Amino-C6 70-mers, Operon) in Epoxide Spotting Buffer (IDT) were spotted in duplicate onto epoxide-coated slides (Corning) using SpotArray24 arrayer (PerkinElmer), with 16 pins. As controls, SpotReport Alien Oligo Array Validation System oligonucleotides (Stratagene) were spotted, each in 24 or 32 replicates. Also, 274 buffer spots were printed. The array design has been deposited in the Array Express database under accession ID A-MEXP-2057. Prior to use, microarrays were cross-linked in a UVIlink crosslinker (UVITEC Cambridge) with 150 mJ energy. Pre-hybridization and hybridization were performed in an automatic HybArray12 station (PerkinElmer). Prehybridization (in 5 × SSC, 0.1% SSC, 0.1 mg/ml BSA) was performed at 42°C for 45 min, followed by washing at 25°C with 0.1 × SSC (30 s flow, 1 min hold, 3 cycles) and water (15 s flow, 15 s hold, 2 cycles). Slides were drained by centrifugation in a High-Speed Centrifuge (ArrayIt) and loaded into clean hybridization chambers. Hybridization was conducted at 43°C for 17 hours, followed by washing with: LS Buffer (2 × SSC, 0.1% SDS) at 43°C, MS buffer (0.5 × SSC) at 30°C and HS buffer (0.005 × SSC) at 25°C, each for 5 cycles (20 s flow, 40 s hold). Slides were then removed, immersed in 0.05 × SSC, drained and scanned on a ScanArray Express scanner (PerkinElmer).
Microarray data analysis
For quantitative analysis filtering, GenePix Pro v. 6.1 (Axon Instruments) was used. Spots with more than 10% pixels with signal saturation in both channels, as well as visually bad or contaminated spots, were flagged out. Further data analysis was performed with R/Bioconductor packages. Hybridization quality was assessed with ArrayQuality  and ArrayQualityMetrix . Limma was used for normalization and assessing gene differential expression . Data filtering and other calculations were made in Microsoft Office Excel 2003 and 2007. Microarray data have been deposited to ArrayExpress Database and assigned accessions E-MEXP-3227 (analysis of organ-specific gene expression in cucumber) and E-MEXP-3220 (CSH validation experiment).
The PlasTi-microarray probes in fasta format were used as queries for homology searches via the NCBI web page , using the blastn algorithm, with the following parameters: word size = 11; Expect threshold = 0.001; Match/Mismatch scores = 1,-1; Gap costs: Existence 2; Extension 1. Search results were further analyzed in Excel 2003.
The study was supported by the Ministry of Science and Higher Education, Poland (grant PBZ-MNiSW-2/3/2006/32) and The National Centre for Research and Development (grant NCBiR N R12 002704). We are grateful to dr Luiza Handschuch and mgr Michał Góralski for their help with the arrayer setup, microarray scanning and stimulating discussions and to Marc Lohse for help with uploading data to MapMan Store.
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