Heat-stress-dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR

Abstract

The potential of different house-keeping genes for their use as internal standards of gene expression under changing environmental conditions and in different organs of plants was assessed. Using real-time PCR mRNA levels were precisely quantified for preselected actin and ribosomal protein genes in Arabidopsis thaliana (L.) Heinh. and Nicotiana tabacum L. grown at normal temperature and following heat stress. In tobacco leaves the mRNA levels of the constitutively expressed ribosomal protein gene Nt-L25 and the actin genes Nt-ACT9 and At-ACT66 were strongly reduced (to approximately 10%) during heat stress. Heat stress applied at the temperature optimum (37 degrees C) for elicitation of a heat stress response to Arabidopsis leaves resulted in a strong induction (several thousand-fold) of the mRNA heat shock protein genes, At-HSP17.6 and At-HSP18.2. Concomitantly, the mRNA levels of constitutively expressed actin 2 (At-ACT2) and ribosomal protein L23 (At-L23a) genes were reduced to approximately 50% of the levels in leaves incubated at room temperature. Conversely, under severe heat stress conditions (44 degrees C), the induction of At-HSP17.6 and At-HSP18.2 mRNAs was insignificant, the mRNA levels of At-ACT2 remained at approximately the same levels as in leaves incubated at room temperature, whereas the mRNA level of At-L23 declined. The mRNA levels of At-ACT2 and At-L23a examined in stem, flower and siliques of Arabidopsis plants grown under non-stress condition showed differential alterations; the mRNA level of ribosomal protein L23 correlates with the metabolic activity of tissues. The potential use of house-keeping gene expression as standards in expression profiling and the mechanisms modulating the mRNA levels are discussed.

Cytosol-Localized Heat Shock Factor-Binding Protein, AtHSBP, Functions as a Negative Regulator of Heat Shock Response by Translocation to the Nucleus and Is Required for Seed Development in Arabidopsis

“…3B) was AtPP2A (At1g13320; Czechowski et al, 2005), and AtACT2 (At3g18780) was used for heat treatments (Figs. 3A and 8A;Volkov et al, 2003;Charng et al, 2007).…”

Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.)

“…Relative AtBBM gene expression levels were determined using the comparative 2 ¡ CT method (Livak and Schmittgen2001). The expression level of the AtBBM gene in diVerent transgenic lines was normalized using the N. tabacum RIBOSOMAL L25 gene (GenBank accession L18908) as the reference (Volkov et al 2003) and the normalized AtBBM expression value from the transgenic line with the lowest AtBBM expression level as the calibrator.…”

Towards a Systematic Validation of References in Real-Time RT-PCR

“…It is generally assumed that housekeeping genes encoding proteins required for basal cell activities, such as central carbon metabolism, protein translation, cytoskeleton maintenance, and protein turnover, are expressed uniformly in different tissues and organs. However, it has been found that the expression of a number of such housekeeping genes, although constant in some experimental conditions, varies considerably in other cases (Volkov et al, 2003;Czechowski et al, 2005;Nicot et al, 2005;Remans et al, 2008). Therefore, their systematic use as reference genes may result in the misinterpretation of results.…”