The upstream open reading frame of the Arabidopsis AtMHX gene has a strong impact on transcript accumulation through the nonsense-mediated mRNA decay pathway.

Approximately 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.

High expression in leaves of the zinc hyperaccumulator Arabidopsis halleri of AhMHX, a homolog of an Arabidopsis thaliana vacuolar metal/proton exchanger.

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.

AtMHX is an auxin and ABA-regulated transporter whose expression pattern suggests a role in metal homeostasis in tissues with photosynthetic potential.

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.