Expression analysis of the Arabidopsis peroxidase multigenic family

Abstract

The expression of all Arabidopsis Class III peroxidase genes was analysed using specific macroarrays. Many genes were expressed differentially in roots, stems, leaves and flowers, including some genes with no reported ESTs. Our approach will allow an exhaustive study of this family in a wide range of conditions. Class III peroxidases form a numerous multigenic family in higher plants, whose expression is particularly sensitive to internal or external events. Arabidopsis thaliana genome harbours 73 genes encoding peroxidases. Since they exhibit homologies ranging from 28% to 93% at the nucleotide level, the risk of cross-hybridisation may be important when measuring the level of transcripts by blotting techniques, using whole cDNA sequences. We developed a procedure to assess the expression of all peroxidase genes on one membrane, with a high specificity. The method was based on the determination for each gene of a short specific sequence (amplicon) exhibiting at the most 70% homology with any other sequences of the Arabidopsis genome. Amplicons specific for each of the 73 peroxidase genes and two pseudogenes were blotted on a nylon membrane that was hybridised with radiolabelled cDNA libraries prepared from mRNAs of Arabidopsis roots, stems, leaves and flowers. Many genes were expressed at a low level, often in all organs, while sixteen genes were rather strongly expressed, in two to four organs. Some genes with no ESTs reported in databases were found to be expressed and this was confirmed by RT-PCR. Isoelectric focusing analysis revealed that the isoperoxidase pattern was similar in leaves, stems and flowers, but was quite different in roots. To our knowledge, only one similar study has been performed on the cytochrome P450 family, using microarrays, but this is the first work describing the expression profile of a whole large multigenic family using specific macroarrays.