Journal of Experimental Botany, Vol. 54, No. 391, pp. 2343-2349,
October 1, 2003
© 2003 Oxford University Press
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
Received 16 January 2003; Accepted 15 June 2003
Zentrum für Molekularbiologie der Pflanzen, Allgemeine Genetik, University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany
* To whom correspondence should be sent. Fax: +49 7071 295042. E-mail: friedrich.schoeffl{at}zmbp.uni-tuebingen.de
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 °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 °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.
Key words: Actin, heat-shock, internal standard, mRNA quantification, real-time PCR, ribosomal protein.
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