Nucleic Acids Research, 2008, Vol. 36, No. 22 6983 Figure 4. Molecular characterization of transgenic AtGRP8-RQ ox lines. (A) WT, AtGRP8-ox and AtGRP8-RQ-ox plants were harvested at zt3 and zt11. The RNA gel blot was hybridized with the AtGRP8 cDNA to determine the total transcript level (top) and with the gene-speciﬁc probe to monitor the endogenous AtGRP8 transcript (middle). The position of the pre-mRNA, as_AtGRP8 retaining the ﬁrst half of the intron and the mRNA are indicated. Boxes represent exons, lines represent the ﬁrst and the second half of the intron, respectively. The ethidium-bromide stained gel shows equal loading (bottom). (B) The immunoblot with total protein of WT plants, the AtGRP8-ox lines 5 and 21, and the AtGRP8-RQ-ox lines 1, 4 and 5, harvested at zt11, was probed with the AtGRP8 antibody (top) and an antibody against LHCP as loading control (bottom). (C) The RNA gel blot shown in (A) was stripped and rehybridized with the AtGRP7 probe. (D) The immunoblot with the same protein extracts as shown in (B) was probed with the AtGRP7 antibody (top) and the LHCP antibody (bottom). and thus do not act entirely redundant, as their expression pattern is not fully congruent. To determine whether the exposure to low temperatures may inﬂuence the alternative splicing, the pattern of the alternative splice forms and mature mRNAs in 2-week-old plants exposed to cold for 1, 2 or 5 days was compared to that in untreated plants (Figure S3). as_AtGRP7 accumu- lated in parallel with the RNA. While the AtGRP8 mRNA only weakly and transiently increased, a stronger upregu- lation was observed for as_AtGRP8. Thus, only for AtGRP8 a slight change in the ratio of the alternative splice forms is observed in the cold. Previous studies have demonstrated changes in the alternative splicing pat- tern of Arabidopsis SR genes upon cold treatment which may give rise to proteins with diﬀerent domains and, con- sequently, changes in splicing of downstream targets (31,32). as_AtGRP8 and as_AtGRP7 are direct targets of the NMD pathway Degradation of as_AtGRP8 and as_AtGRP7 is dependent on AtUPF1 and AtUPF3, key components of the NMD pathway of mRNA surveillance that ensures clearance of PTC-containing mRNAs from the cellular transcriptome (12). Recently, NMD has emerged as a widespread regulatory mechanism of physiological gene expression (33–35). In the Arabidopsis lba (low -amylase) mutant which has a point mutation in the UPF1 gene a suite of transcripts show higher steady-state abundance, as expected for NMD substrates (36). But additionally sev- eral transcripts are down-regulated with a high proportion of sugar-inducible mRNAs, implicating AtUPF1 in sugar signalling (36). By analogy, the elevated as_AtGRP8 and as_AtGRP7 levels in upf1 and upf3 mutants (12) may be an indirect consequence of the reduced AtUPF1 or AtUPF3 levels. To distinguish such an indirect eﬀect of AtUPF1 and AtUPF3 on transcription from a direct eﬀect on transcript abundance, we assayed the pre-mRNA levels by RT–PCR. AtGRP8 pre-mRNA steady-state abundance was not changed in the upf1 and upf3 mutants (Figure 6A) and also the AtGRP7 pre-mRNA level was indistinguishable from WT (Figure 6B) in contrast to the strongly elevated as_AtGRP7 and as_AtGRP8 levels found in upf1-5, upf3-1 and upf3-2 (12). This indicates that the AtUPF1- and AtUPF3-dependent reduction of as_AtGRP8 and as_AtGRP7 is due to post-transcriptional destabilization rather than an indirect consequence of transcriptional inhibition. In higher plants, an increasing number of PTC-containing transcripts have been found to be stabilized when UPF1 and/or UPF3 functions are impaired (36–39). It has not been investigated whether some of them may be inﬂuenced either through an NMD-independent function of AtUPF1 and AtUPF3 or as a consequence of a cognate transcription factor under- going NMD. In humans, in a survey of potential NMD targets more than 5% of the genes detected on Aﬀymetrix GeneChips were aﬀected by the presence or absence of UPF1 (40). For 15 out of 16 selected transcripts the pre- mRNA level was also changed, however, suggesting that the vast majority of those transcripts are aﬀected indir- ectly through altered transcription rather than being bona ﬁde NMD targets (40). If the unproductive AtGRP8 and AtGRP7 splicing elic- ited by elevated levels of AtGRP7 and AtGRP8 protein indeed is functionally relevant, this may be reﬂected at the level of sequence conservation. The gene structure of the small glycine-rich RNA-binding proteins with a single RRM is well conserved among diﬀerent plant species, har- boring a single intron of similar size between RNP2 and RNP1, and a predicted cryptic 50 splice site (8,19).