Arabidopsis

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.

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional polle...

Choline (Cho) is an essential nutrient for humans as well as the precursor of glycine betaine (GlyBet), an important compatible solute in eukaryotes that protects cells from osmotic stress caused by dehydrating conditions. The key enzyme for plant Cho synthesis is phosphoethanolamine N-methyltransferase (PEAMT), which catalyzes all three methylation steps, including the rate-limiting N-methylation of phosphoethanolamine. Herein, we report that the beneficial soil bacterium Bacillus subtilis (strain GB03) enhances Arabidopsis Cho and GlyBet synthesis associated with enhanced plant tolerance to osmotic stress. When stressed with 100 mM exogenous mannitol, GB03-exposed plants exhibit increased transcript level of PEAMT compared with stressed plants without bacterial exposure. Endogenous Cho and GlyBet metabolite pools were elevated by more than two- and fivefold, respectively, by GB03 treatment, consistent with increased stress tolerance. Moreover, in the xipotl mutant line with reduced Cho production, a loss of GB03-induced drought tolerance is observed. Osmotic-stressed plants with or without GB03 exposure show similar levels of abscsisic acid (ABA) accumulation in both shoots and roots, suggesting that GB03-induced osmoprotection is ABA independent. GB03 treatment also improves drought tolerance in soil-grown plants as characterized by phenotypic comparisons, supported by an elevated accumulation of osmoprotectants. These results provide a biological strategy to enhance Cho biosynthesis in plants and, in turn, increase plant tolerance to osmotic stress by elevating osmoprotectant accumulation.