Relative to plants, these higher mRNA and protein levels also resulted in a significantly increased capacity for ammonium uptake under both N-sufficient and N-deficient conditions (Loqu et al

Relative to plants, these higher mRNA and protein levels also resulted in a significantly increased capacity for ammonium uptake under both N-sufficient and N-deficient conditions (Loqu et al., 2006). Moreover, steady-state transcript levels were decreased after addition of ammonium or nitrate in N-deficient roots, suggesting a role for N availability in regulating transcript abundance. Nitrogen deficiency-dependent accumulation of IWP-4 mRNA was also observed in alone in N-dependent transcript accumulation was not found. However, transcript levels of did not accumulate in a N-dependent manner, even though the same T-DNA insertion line was used for expression. These results show that the accumulation of transcripts is usually regulated in a N- and organ-dependent manner and suggest mRNA turnover as an additional mechanism for the regulation of in response to the N nutritional status of plants. Membrane proteins of the AMT1 and AMT2 subfamilies are believed to represent the major pathways for high-affinity ammonium transport in plants (Loqu and von Wirn, 2004). When expressed in yeast (of Arabidopsis (mRNA levels in roots IWP-4 (Rawat et al., 1999). In addition to increased ammonium efflux (Kronzucker et al., 2001), such a rapid decrease of ammonium uptake capacities in roots might be required to avoid cellular ammonium toxicity (Britto and Kronzucker, 2002). Whether this rapid decrease in ammonium uptake capacity is brought about by posttranscriptional or posttranslational control remains an open question. So far, only a few studies investigated posttranscriptional control of herb nutrient transporters by the substrate or a downstream metabolite. For example, posttranscriptional regulation at the protein level was indicated by analysis of transgenic plants that constitutively expressed mRNA but accumulated IRT1 protein only in iron-deficient roots Efna1 (Connolly et al., 2002). In this case, it could even be shown that not only iron itself but also other substrates that are recognized by the transporter trigger a decrease of the IRT1 protein. More recently, it was shown that constitutively expressed green fluorescent protein (GFP)-tagged BOR1 transporter proteins in Arabidopsis roots were degraded upon resupply of boron to plants by a mechanism that involved endocytosis from the plasma membrane and subsequent degradation of the transporter protein (Takano et al., 2005). To analyze whether the AtAMT1;1 ammonium transporter is subject to posttranscriptional control, we uncoupled transcriptional control by ectopically expressing the gene under control of a cauliflower mosaic virus (CaMV) promoter in transgenic tobacco (transcripts accumulated in a N-dependent manner, indicating posttranscriptional regulation. The same type of mRNA regulation was also observed in shoots of transgenic Arabidopsis lines but not in roots, irrespective of whether a full-length cDNA or its 3-untranslated region (UTR)-deleted version was expressed. We then investigated mRNA accumulation of in mRNA levels is regulated in dependence of the herb organ, the homolog, and the N nutritional status of the plants. RESULTS Generation of Transgenic Tobacco Ectopically Expressing AtAMT1;1 Axenically grown tobacco seedlings were used for cDNA (Ninnemann et al., 1994) cloned in 5- to 3-orientation between a CaMV promoter sequence and an terminator sequence (H?fgen and Willmitzer, 1990). The cDNA contained 20 bp upstream and 174 bp downstream untranslated sequences with a 44-bp poly(A) tail and conferred ammonium uptake in yeast (Ninnemann et al., 1994; Gazzarrini et al., 1999). Transgenic plants were selected on medium containing kanamycin, allowed to self-pollinate, and impartial, homozygous T2 lines were selected for further analysis. To select for lines with high levels of gene expression, transgenic tobacco seeds were sterilized, germinated on agar, precultured on 2 mm ammonium nitrate, and harvested after culture on N-deficient nutrient solution for 3 d. A DNA fragment of open reading frame (ORF; Loqu et al., 2006) not cross-hybridizing with the endogenous tobacco AMTs was used as a probe. Three impartial lines (lines 18, 19, and 21) were selected that express mRNA in both roots and shoots (Fig. 1A). Interestingly, two different mRNAs with an approximate length of 1,700 and 1,900 nucleotides (nt) were IWP-4 detected. Probes specific for the 3-UTR of and the OCS terminator (and the 1,900-nt transcript as a read-through mRNA species made up of terminator sequences (Supplemental Fig. S1). The high transcript levels in the two transgenic lines 18 and 19 led to accumulation of large amounts of AtAMT1;1 protein, as determined by protein gel-blot analysis using a rabbit antibody targeted against.