Establishing experimental conditions for microarray experiment 107

To determine optimal conditions for microarray analysis, the expression of seven genes was studied to identify the conditions which caused minimal stress, whilst still causing altered regulation of some genes. Plants were grown as outlined in Section 4.2.1 above for 14 days, after which, the plants were treated with gold for six hours and the tissues were harvested (Section 4.2.1). RNA was extracted and cDNA was synthesised (Sections 2.4.3 and 2.4.4). The expression of the seven genes was subsequently analysed via qPCR.

Previous microarray research in Cupriavidus metallidurans (Reith et al. 2009) investigated the expression of genes in the bacterium in response to gold. Of the 332 genes upregulated when C. metallidurans was treated with 100 µM gold for ten minutes, the top 25 were analysed (Table 4.3). These 25 genes were used to identify homologues in the Arabidopsis genome. The protein Basic Local Alignment Search Tool (BLASTP) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to probe the Arabidopsis genome using the C. metallidurans protein sequences (Altschul et al. 1990). Of the 25 upregulated genes, one had no homology to any proteins in Arabidopsis and eight were hypothetical proteins to which no function could be attached. These nine genes were therefore not suitable for use in establishing experimental conditions. Twelve of the genes returned hits when Arabidopsis was probed, but only small sections of the genes were conserved and therefore these genes were not suitable. Of the remaining genes, Rmet_3524, Rmet_4888 and Rmet_2922 had homologues in Arabidopsis which had previously been shown to be involved in metal homeostasis (highlighted in Table 4.3). Rmet_3524 is homologous to HMA5 (At1g63440), a heavy metal ATPase shown to be a copper transporter involved in copper detoxification (Andres-Colas et al. 2006). Rmet_4888 is a dihydrolipoamide dehydrogenase and the Arabidopsis homologue (mtLPD1, At1g48030) has been shown to respond to metals other than gold, including cadmium (Sarry et al.

2006) and more recently, zinc, copper and cobalt (Tan et al. 2010). Rmet_2922 is a heat shock chaperone protein and the Arabidopsis homologue (mtHSC70-2, At5g09590) has been shown to respond to cadmium (Sarry et al. 2006).

Chapter 4. Microarray study of the Arabidopsis response to gold

The Arabidopsis homologues of these genes were therefore chosen for analysis in response to gold in Arabidopsis in order to establish suitable experimental conditions for the microarray. In Arabidopsis, there are two members of the mtLPD family (Lutziger and Oliver 2001) and two mtHSC70 genes (Sung et al.

2001). Thus, both members of the mtLPD family and both members of the mtHSC70 family were tested for changes in expression. The HMA family comprises eight members (Axelsen and Palmgren 2001; Abdel-Ghany et al.

2005) (see Chapter 1). Of the eight HMA genes, HMA5 and HMA7 were tested because they are the two genes most closely related to Rmet_3524.

Chapter 4. Microarray study of the Arabidopsis response to gold

Table 4.3 Upregulated genes in C. metallidurans in response to gold

The 25 most upregulated genes in C. metallidurans in response to gold(III) (Reith et al. 2009). Cells highlighted green are the three genes whose homologues were chosen to study in this work.

Mage ID Regulation change

Annotation Arabidopsis Homologues?

Rmet_3525 87.08 copper chaperone, heavy metal ion binding (modular protein) Homologues but not conserved

Rmet_4187 83.13 hypothetical protein Hypothetical protein

Rmet_3524 67.73 copper-transporting P-type ATPase CopA Homologue is copper transporter

Rmet_4888 58.4 dihydrolipoamide dehydrogenase Conserved protein

Rmet_4685 49.94 hypothetical protein Hypothetical protein

Rmet_4684 47.39 hypothetical protein Hypothetical protein

Rmet_4889 36.59 glutaredoxin-like region Conserved protein

Rmet_4908 34.14 conserved hypothetical protein Hypothetical protein

Rmet_0332 33.6 putative lactoylglutathione lyase Homologues but not conserved

Rmet_3620 33.2 multifunctional enzyme (peptidase / oxidoreductase) (degP / mucDlike) Conserved protein

Rmet_0333 32.02 regulatory protein, ArsR Homologues but not conserved

Rmet_3523 30.14 DNA-binding transcriptional activator of copper-responsive regulon genes omologues but not conserved

Rmet_1959 27.06 protein disaggregation chaperone Conserved protein

Rmet_4026 25.5 conserved hypothetical protein Hypothetical protein

Rmet_4102 24.04 putative transcriptional regulator, TetR familiy Homologues but not conserved Rmet_3619 23.86 organic hydroperoxide resistance transcriptional regulator, MarR family Homologues but not conserved

Rmet_4188 23.39 transcriptional regulator, TetR family Homologues but not conserved

Rmet_2280 22.63 conserved hypothetical protein; putative hemin uptake protein Homologues but not conserved

Rmet_1951 21.74 alkyl hydroperoxide reductase D Homologues but not conserved

Rmet_3522 18.4 lysophospholipase Conserved protein

Rmet_0720 16.83 hypothetical protein Hypothetical protein

Rmet_3456 15.95 transcriptional regulator, MerR-family Homologues but not conserved

Rmet_2922 13.78 chaperone Hsp70, co-chaperone with DnaJ Conserved protein

Rmet_0085 12.81 conserved hypothetical protein Hypothetical protein

Chapter 4. Microarray study of the Arabidopsis response to gold

4.3.1.2 Analysis of gene expression

The cDNA synthesised for the optimisation studies was used to test for the expression of the genes described above (Section 4.3.1). Relative gene expression was calculated for the 0.125 mM and 0.25 mM samples compared to the no gold treatment samples as described in Section 2.4.5 (Figure 4.2). All data were compared to the no gold control, and were normalised using ACTIN2 as a constitutively expressed control. For the 0.25 mM treatments, all six genes were upregulated. In the 0.125 mM treatments, all except for HSC1 were upregulated.

The data presented here demonstrate upregulation of Arabidopsis homologues of genes upregulated in C. metallidurans in response to gold. From these results 0.125 mM gold was selected for the microarray experiment. Additionally, at this concentration, changes in gene expression as a general stress response are likely to be lower than for 0.25 mM gold.

Figure 4.2 Results from qPCR to determine optimum microarray conditions

Changes in gene expression of in Arabidopsis root tissue treated with 0.125 or 0.25 mM gold. Error bars represent the standard error of the mean from four replicates. ACTIN2 was used as a constitutively expressed control to normalise the data.

Chapter 4. Microarray study of the Arabidopsis response to gold

4.3.2 Microarray experiment

Arabidopsis seedlings were grown and subsequently treated with gold as described in Section 4.2.3. After six hours of treatment, there was some purple colouration of the root tissues suggesting reduction of gold(III) to gold(0). RNA was extracted from plant tissues and analysed using the bioanalyser system. Six RNA samples were tested for integrity (Figure 4.4). Distinct bands show that the RNA integrity had been kept during the extraction procedure. This was further confirmed by studying the fluorescence of the samples (Figure 4.5). The six samples tested showed good integrity (Figure 4.5a is a representative graph).

Had the samples had lower integrity, the RNA would have been more fragmented (Figure 4.5b). The six samples described in Figure 4.4 were subsequently transcribed to cDNA and gene expression was measured using the ATH1 microarray. Statistical analysis of the microarray data was carried out as described in Section 4.2.3 and genes that were up or downregulated more than two-fold were selected.

Chapter 4. Microarray study of the Arabidopsis response to gold

Figure 4.3 Treatment of Arabidopsis plants for the microarray experiment

14-day-old Arabidopsis seedlings both prior to and 6 hours post treatment with either 0 or 0.125 mM KAuCl₄. Seedlings were germinated on ½MS(A) plugs and grown into Richard’s medium. This medium was changed after two weeks with either water, or KAuCl4at pH 5.7 for six hours.

Chapter 4. Microarray study of the Arabidopsis response to gold

Figure 4.4 Integrity of the RNA used for the microarray experiment Quality control of RNA extracted from Arabidopsis plants to check for RNA

integrity using an Aligent 2100 bioanalyser. No gold control samples are indicated by 0 and 0.125 mM KAuCl4samples are indicated by 1. Letters indicate separate biological replicates.

Chapter 4. Microarray study of the Arabidopsis response to gold

Figure 4.5 Integrity of the RNA used for the microarray experiment Quality control of RNA extracted from Arabidopsis plants to check for RNA integrity using an Aligent 2100 bioanalyser. A) Good quality RNA. B) RNA with high levels of fragmentation.