SUMMARYApproximately 20% of plant genes possess upstream open-reading frames (uORFs). The effect of uORFs on gene expression has mainly been studied at the translational level. Very little is known about the impact of plant uORFs on transcript content through the nonsense-mediated mRNA decay (NMD) pathway, which degrades transcripts bearing premature termination codons (PTCs). Here we examine the impact of the uORF of the Arabidopsis AtMHX gene on transcript accumulation. The suggestion that this uORF exposes transcripts containing it to NMD is supported by (i) the increase in transcript levels upon eliminating the uORF from constructs containing it, (ii) experiments with a modified uORF-peptide, which excluded peptide-specific degradation mechanisms, (iii) the increase in levels of the native AtMHX transcript upon treatment with cycloheximide, which inhibits translation and blocks NMD, and (iv) the sensitivity of transcripts containing the uORF of AtMHX to the presence of introns. We also showed that introns can increase NMD efficiency not only in transcripts having relatively short 3¢ untranslated regions (UTRs), but also in uORF-containing transcripts. AtMHX transcript levels were almost unaltered in mutants of the NMD factors UPF3 and UPF1. Possible reasons, including the existence of a NMD-compensatory mechanism, are discussed. Interestingly, the levels of UPF3 transcript were higher in upf1 mutants, suggesting a compensatory mechanism that links weak function of the NMD machinery to increased expression of UPF3. Our findings highlight that uORFs, which are abundant in plants, can not only inhibit translation but also strongly affect transcript accumulation.

AtMHX is an Arabidopsis tonoplast transporter that can exchange protons with Mg2+ and Zn2+ ions. This transporter, which may play a role in ion homeostasis of plants, is encoded by a single gene in Arabidopsis. The molecular mechanisms that regulate the expression of this transporter are practically unknown. While AtMHX transcript can be easily visualized, expression of the corresponding protein is apparently low. To understand whether AtMHX expression is repressed at the translational level, the 5' untranslated region (5' UTR) of this gene was fused to reporter genes. In vitro analyses showed that the 5' UTR of AtMHX can repress the translation of downstream coding sequences. The major cause of the repression was efficient initiation at an upstream open-reading-frame (uORF) included in the 5' UTR. Although the sequence context of the upstream AUG (uAUG) codon was highly unfavourable, it was recognized by over 90% of the scanning ribosomes both in vitro and in vivo. The inhibitory effect of the uORF was mediated by imposing the need for reinitiation and not by ribosome stalling, as the inhibition was not dependent on the amino-acid sequence of the uORF peptide. The efficiency of reinitiation was low. The in vivo studies, carried out with transiently transformed tobacco plants, indicated that alternations in the Mg2+ or Zn2+ levels did not affect the rate of translation. These data suggest that AtMHX expression is repressed by the 5' UTR of its gene.

AtMHX is a vacuolar transporter encoded by a single gene in Arabidopsis thaliana (L.) Heynh. It exchanges protons with Mg2+, Zn2+, and Fe2+ ions. Proper homeostasis of these metals is essential for photosynthesis and numerous enzymatic reactions. In particular, very little is known about mechanisms involved in Mg2+ homeostasis in plants. Expression analysis using reporter-gene constructs suggested that AtMHX functions in metal homeostasis mainly in tissues with photosynthetic potential. This balancing is conducted by expression in the vascular region, the cortex of stems, trichomes, and hydathodes. Expression in stems is developmentally regulated, suggesting that minerals are accumulated in the upper regions of young stems, and are released during silique development. Mineral content in different stem parts was consistent with this possibility. Expression was induced by auxin and ABA, but not by the metal content of the growth medium, suggesting that expression is mainly regulated by endogenous developmental programs. AtMHX exhibits two distinguished regulatory properties. Its leader intron is absolutely essential for expression, and mediates an 86-fold enhancement of expression. This enhancement is the highest reported thus far for any dicot intron. Another remarkable feature is that a repetitive genomic element of 530 bp (or part of it) functions as an enhancer.

Zn hyperaccumulator plants sequester Zn into their shoot vacuoles. To date, the only transporters implicated in Zn sequestration into the vacuoles of hyperaccumulator plants are cation diffusion facilitators (CDFs). We investigated the expression in Arabidopsis halleri of a homolog of AtMHX , an A. thaliana tonoplast transporter that exchanges protons with Mg, Zn and Fe ions. A. halleri has a single copy of a homologous gene, encoding a protein that shares 98% sequence identity with AtMHX . Western blot analysis with vacuolar-enriched membrane fractions suggests localization of AhMHX in the tonoplast. The levels of MHX proteins are much higher in leaves of A. halleri than in leaves of the non-accumulator plant A. thaliana . At the same time, the levels of MHX transcripts are similar in leaves of the two species. This suggests that the difference in MHX levels is regulated at the post-transcriptional level. In vitro translation studies indicated that the difference between AhMHX and AtMHX expression is not likely to result from the variations in the sequence of their 5 ¢ untranslated regions (5 ¢ UTRs). The high expression of AhMHX in A. halleri leaves is constitutive and not significantly affected by the metal status of the plants. In both species, MHX transcript levels are higher in leaves than in roots, but the difference is higher in A. halleri . Metal sequestration into root vacuoles was suggested to inhibit hyperaccumulation in the shoot. Our data implicate AhMHX as a candidate gene in metal accumulation or tolerance in A. halleri . Key-words :AtMHX ; hyperaccumulation; magnesium; metal transporter; vacuole; zinc; 5 ′ UTR. INTRODUCTIONA few plant species, generally referred to as metal hyperaccumulators, can accumulate and tolerate very high levels of certain metals (Baker & Brooks 1989). While Zn is an essential mineral, its excess leads to toxicity. Arabidopsis halleri and Thlaspi caerulescens ( Brassicaceae ) are among the 16 known plant species that can accumulate and tolerate high levels of Zn (Baker & Brooks 1989), and are the two main model species in which Zn hyperaccumulation has been studied to date (reviewed by Baker & Whiting 2002;Assuncao, Schat & Aarts 2003). Excessive accumulation of Zn in the shoot is a constitutive property of the A. halleri species . When different populations of A. halleri plants were grown hydroponically in the presence of high Zn levels, there were no differences in the accumulating abilities of populations from sites with different degrees of contaminations (Macnair 2002).The molecular basis of metal hyperaccumulation in A. halleri and T. caerulescens was addressed in a number of studies. Transcription of genes encoding Zn uptake transporters (that are presumably localized in the plasma membrane) is higher in both roots and shoots of T. caerulescens compared with the related non-accumulator species Thlaspi arvense Pence et al . 2000;Assuncao et al . 2001). Expression of these uptake transporters is down-regulated in the presence of 1 and 50 µ M Zn in T. arvense...

No abstract