Evaluation of reference genes for RT-qPCR gene expression analysis in Arabidopsis thaliana exposed to elevated temperatures.

Increases in environmental temperature are directly linked to the issue of climate change and are known to significantly disrupt plant growth and development. Studies of gene expression in plants commonly include RT-qPCR but the reliability of the method depends on the use of suitable reference genes for data normalization. Despite this, no reference genes have been validated specifically for experiments in Arabidopsis thaliana employing treatments with elevated temperature. Here, ten genes were selected for expression stability analysis based on the screening of available literature and microarray data from temperature-treated A. thaliana. Expression levels of candidate reference genes were measured in 12-day-old seedlings, rosette leaves and flower buds of 5-week-old A. thaliana plants exposed to five different temperatures (22°C, 27°C, 32°C, 37°C and 42°C) and their expression stabilities were assessed using four statistical algorithms (BestKeeper, geNorm, NormFinder and comparative ΔCq method). This study provides reliable reference genes for use in A. thaliana RT-qPCR expression analyses employing elevated temperature treatments, namely OGIO and PUX7 in seedlings, UBC21 and PUX7 in leaves, TIP41 and UBC21 in buds, and TIP41 and UBC21 in all three tissues combined. Orthologues of these genes can be of potential use in less studied plants, especially agricultural species heavily affected by climate change.

Biochemical and epigenetic changes in phytoplasma-recovered periwinkle after indole-3-butyric acid treatment.

AIM: To elucidate the possible mechanism of phytoplasma elimination from periwinkle shoots caused by indole-3-butyric acid (IBA) treatment. METHODS AND RESULTS: It has been shown that a transfer of in vitro-grown phytoplasma-infected Catharanthus roseus (periwinkle) plantlets from medium supplemented with 6-benzylaminopurine (BA) to one supplemented with IBA can induce remission of symptoms and even permanent elimination of 'Candidatus Phytoplasma asteris' reference strain HYDB. Endogenous auxin levels and general methylation levels in noninfected periwinkles, periwinkles infected with two 'Candidatus Phytoplasma' species and phytoplasma-recovered periwinkles were measured and compared. After the transfer from cytokinin- to auxin-containing media, healthy shoots maintained their phenotype, methylation levels and hormone concentrations. Phytoplasma infection caused a change in the endogenous indole-3-acetic acid to IBA ratio in periwinkle shoots infected with two 'Candidatus Phytoplasma' species, but general methylation was significantly changed only in shoots infected with 'Ca. P. asteris', which resulted in the only phytoplasma species eliminated from shoots after transfer to IBA-containing medium. Both phytoplasma infection and treatment with plant growth regulators influenced callose deposition in phloem tissue, concentrations of photosynthetic pigments and soluble proteins, H(2) O(2) levels and activities of catalase (CAT) and ascorbate peroxidase (APX). CONCLUSION: Lower level of host genome methylation in 'Ca. P. asteris'-infected periwinkles on medium supplemented with BA was significantly elevated after IBA treatment, while IBA treatment had no effect on cytosine methylation in periwinkles infected with 'Candidatus Phytoplasma ulmi' strain EY-C. SIGNIFICANCE AND IMPACT OF THE STUDY: Hormone-dependent recovery is a distinct phenomenon from natural recovery. As opposed to spontaneously recovered plants in which elevated peroxide levels and differential expression of peroxide-related enzymes were observed, in hormone-dependent recovery changes in global host genome, methylation coincide with the presence/absence of phytoplasma.