An alternatively spliced domain of the NDC1 NAD(P)H dehydrogenase gene strongly influences the expression of the ACTIN2 reference gene in Arabidopsis thaliana

In plant respiratory chains, alternative pathways for NAD(P)H oxidation are mediated by type II NAD(P)H dehydrogenases belonging to the NDA, NDB, and NDC families. For the latter type, Arabidopsis thaliana contains a single gene, NDC1, whose functional role has not previously been analyzed in the plant. We found that A. thaliana NDC1 is alternatively spliced. Four base pairs at the 3′ end of intron 5 are spliced out in NDC1–1, but retained in the NDC1–2 mRNA, which therefore contains a truncated reading frame. Both variants are conserved in dicotyledonous and monocotyledonous plants and their relative abundance varies between organs and in response to light. Three analyzed NDC1 T-DNA insertion lines all displayed an early bolting phenotype. A dramatic upregulation of ACTIN2 was characteristic of two lines containing T-DNA inserts upstream of intron 5, whereas a line with an insertion downstream of the NDC1–2 reading frame had an ACTIN2 expression level identical to the wildtype. Thus, the alternatively spliced 5′ domain of NDC1 strongly influences the expression of the functionally unrelated ACTIN2, which is a common reference gene for quantitative RT-PCR. Also for other reference genes, strong expressional effects were observed when comparing various mutants and wildtypes in microarray databases.

► NDC1 is spliced into two alternative mRNA variants that are widely conserved. ► T-DNA insertions up- and downstream of the alternative splice site cause different phenotypes. ► The expression of the ACTIN2 reference gene for quantitative RT-PCR is strongly influenced by NDC1. ► Genotypic changes exceed other cues in affecting expression of reference genes.