Irina Berezin

Overexpression of AtMHX in tobacco causes increased sensitivity to Mg2+, Zn2+, and Cd2+ ions, induction of V-ATPase expression, and a reduction in plant size.

AtMHX is a vacuolar transporter encoded by a single gene in Arabidopsis. Electrophysiological analysis showed that it exchanges protons with Mg2+, Zn2+, and Fe2+ ions. The physiological impact of AtMHX was examined so far only in tissue-culture grown seedlings of tobacco plants overexpressing this transporter. Here we investigated the impact of AtMHX on growth, response to different metals, and metal accumulation of mature tobacco plants, as well as Arabidopsis plants in which we overexpressed this transporter. The analyses were carried out in hydroponic growth-systems, in which the mineral composition could be effectively controlled, and the metal content of roots could be examined. Transformed tobacco plants showed necrotic lesions and apical burnings upon growth with increased levels of Mg2+, Zn2+, and Cd2+ ions. This suggested that AtMHX can carry in planta not only Mg2+ and Zn2+ ions, as previously deduced based on observations in tissue-culture, but also Cd2+ ions. Transformed plants of both tobacco and Arabidopsis showed a reduction in plant size. However, the overall response of Arabidopsis to AtMHX overexpression was minor. No change was detected in the mineral content of any organ of the transgenic tobacco or Arabidopsis plants. The necrotic lesions in tobacco resembled those seen in plants with perturbed proton balancing, raising the assumption that AtMHX can affect the proton homeostasis of cells. In agreement with this assumption, the transformed tobacco plants had increased expression and activity of the vacuolar H+-ATPase. The relative significance of AtMHX for metal and proton homeostasis still has to be elucidated.

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.

Phylogeny and a structural model of plant MHX transporters.

BackgroundThe Arabidopsis thaliana MHX gene (AtMHX) encodes a Mg2+/H+ exchanger. Among non-plant proteins, AtMHX showed the highest similarity to mammalian Na+/Ca2+ exchanger (NCX) transporters, which are part of the Ca2+/cation (CaCA) exchanger superfamily.ResultsSequences showing similarity to AtMHX were searched in the databases or sequenced from cDNA clones. Phylogenetic analysis showed that the MHX family is limited to plants, and constitutes a sixth family within the CaCA superfamily. Some plants include, besides a full MHX gene, partial MHX-related sequences. More than one full MHX gene was currently identified only in Oryza sativa and Mimulus guttatus, but an EST for more than one MHX was identified only in M. guttatus. MHX genes are not present in the currently available chlorophyte genomes. The prevalence of upstream ORFs in MHX genes is much higher than in most plant genes, and can limit their expression. A structural model of the MHXs, based on the resolved structure of NCX1, implies that the MHXs include nine transmembrane segments. The MHXs and NCXs share 32 conserved residues, including a GXG motif implicated in the formation of a tight-turn in a reentrant-loop. Three residues differ between all MHX and NCX proteins. Altered mobility under reducing and non-reducing conditions suggests the presence of an intramolecular disulfide-bond in AtMHX.ConclusionsThe absence of MHX genes in non-plant genomes and in the currently available chlorophyte genomes, and the presence of an NCX in Chlamydomonas, are consistent with the suggestion that the MHXs evolved from the NCXs after the split of the chlorophyte and streptophyte lineages of the plant kingdom. The MHXs underwent functional diploidization in most plant species. De novo duplication of MHX occurred in O. sativa before the split between the Indica and Japonica subspecies, and was apparently followed by translocation of one MHX paralog from chromosome 2 to chromosome 11 in Japonica. The structural analysis presented and the identification of elements that differ between the MHXs and the NCXs, or between the MHXs of specific plant groups, can contribute to clarification of the structural basis of the function and ion selectivity of MHX transporters.

Overexpression of the vacuolar metal/proton exchanger AtMHX in tomato causes decreased cell expansion and modifications in the mineral content

AtMHX is an Arabidopsis vacuolar transporter that exchanges protons with Mg2+, Zn2+ and Fe2+ ions. Tobacco (Nicotiana tabacum (L.)) plants that overexpressed AtMHX showed necrotic lesions, similar to those shown by plants having increased proton influx from the apoplast into the cytosol. This raised the assumption that AtMHX affects the proton homeostasis of cells. Here, we expressed AtMHX in tomato (Lycopersicon esculentum Mill.). The results clarified that the common response of all plant species in which AtMHX was overexpressed thus far was a reduction in plant mass. Transformed tomato plants, in which this reduction was greater compared with tobacco or Arabidopsis thaliana (L.), exhibited reduced cell expansion and a reduction in potassium content. Modifications were also seen in the content of other minerals, including not only metals that can be carried by AtMHX. These changes may thus reflect not only direct metal transport by AtMHX but also the consequences of reduction in cell size. Decreased cell expansion characterises plants with diminished expression of vacuolar proton pumps, presumably due to reduction in the proton-motive force (PMF) necessary to drive solute (mainly potassium) influx into vacuoles and consequently water uptake. This supported a model in which AtMHX-mediated proton efflux from vacuoles affects the PMF, potassium influx, and cell expansion.

The feedback control of UPF3 is crucial for RNA surveillance in plants

Nonsense-mediated-decay (NMD) is a eukaryotic RNA surveillance mechanism that controls the levels of both aberrant and normal transcripts. The regulation of this process is not well understood. The Arabidopsis NMD factor UPF3 is regulated by a negative feedback-loop that targets its own transcript for NMD. We investigated the functional significance of this control for the overall regulation of NMD in Arabidopsis. For this, we tested the ability of NMD-sensitive and -insensitive forms of UPF3, expressed under the control of UPF3 promoter, to complement NMD functionality in NMD-mutant plants and investigated their impact in wild-type (WT) plants. The sensitivity of UPF3 transcript to NMD was essential for efficient complementation of NMD in upf3 mutants. Upregulated UPF3 expression in WT plants resulted in over-degradation of certain transcripts and inhibited degradation of other transcripts. Our results demonstrate that, in contrast to mammalian cells, a delicate balance of UPF3 transcript levels by its feedback loop and by restriction of its transcription, are crucial for proper NMD regulation in Arabidopsis. Interestingly, the levels of many small-nucleolar-RNAs (snoRNAs) were decreased in upf1 and upf3 mutants and increased upon enhanced UPF3 expression. This suggests that proper snoRNA homeostasis in Arabidopsis depends on the integrity of the NMD pathway.

The Arabidopsis NMD Factor UPF3 Is Feedback-Regulated at Multiple Levels and Plays a Role in Plant Response to Salt Stress.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA surveillance mechanism that degrades aberrant transcripts and controls the levels of many normal mRNAs. It was shown that balanced expression of the NMD factor UPF3 is essential for the maintenance of proper NMD homeostasis in Arabidopsis. UPF3 expression is controlled by a negative feedback loop that exposes UPF3 transcript to NMD. It was shown that the long 3′ untranslated region (3′ UTR) of UPF3 exposes its transcript to NMD. Long 3′ UTRs that subject their transcripts to NMD were identified in several eukaryotic NMD factors. Interestingly, we show here that a construct that contains all the regulatory regions of the UPF3 gene except this long 3′ UTR is also feedback-regulated by NMD. This indicates that UPF3 expression is feedback-regulated at multiple levels. UPF3 is constitutively expressed in different plant tissues, and its expression is equal in leaves of plants of different ages. This finding is in agreement with the possibility that UPF3 is ubiquitously operative in the Arabidopsis NMD pathway. Expression mediated by the regulatory regions of UPF3 is significantly induced by salt stress. We found that both a deficiency and a strong excess of UPF3 expression are detrimental to plant resistance to salt stress. This indicates that UPF3 plays a role in plant response to salt stress, and that balanced expression of the UPF3 gene is essential for coping with this stress.

The Use of Hydroponic Growth Systems to Study the Root and Shoot Ionome of Arabidopsis thaliana

Plants provide an important source of minerals for human and animal consumption. There is a general worldwide deficiency of human intake of several essential minerals, including iron, zinc, copper, calcium, magnesium, selenium, and iodine (White & Broadley, 2009). This deficiency exists not only in developing countries, but also in the developed world. On the other hand, excessive amounts of heavy metals in crop plants can endanger the health of humans and livestock. It is, therefore, important to develop crop plants with balanced levels of minerals. Understanding of the genes and processes that control the mineral content of plants can provide the basis for engineering crops with the optimal concentrations of minerals in various organs and tissues. In recent years, there has been increasing interest in determining the elemental composition (ionome) of various plant species and identifying the genes that dominate this composition [reviewed by Baxter (2009), Salt et al. (2008), and Williams & Salt (2009)]. The agriculturally related plants whose ionomes were investigated include the crops rice (Norton et al., 2010) and Brassica napus (Liu et al., 2009), as well as the plant Lotus japonicus, which serves as a model species for legume crops (Chen et al., 2009).