Orit Shaul

Magnesium transport and function in plants: the tip of the iceberg

The maintenance of Mg2+ homeostasis in the plant is essential for viability. This review describes Mg2+ functions and balancing in plants, with special focus on the existing knowledge of the involved transport mechanisms. Mg2+ is essential for the function of many cellular enzymes and for the aggregation of ribosomes. Mg2+ concentrations also modulate ionic currents across the chloroplast and the vacuolar membranes, and might thus regulate ion balance in the cell and stomatal opening. The significance of Mg2+ homeostasis has been particularly established with regard to Mg2+s role in photosynthesis. Mg2+ is the central atom of the chlorophyll molecule, and fluctuations in its levels in the chloroplast regulate the activity of key photosynthetic enzymes. Relatively little is known of the proteins mediating Mg2+ uptake and transport in plants. The plant vacuole seem to play a key role in Mg2+ homeostasis in plant cells. Physiological and molecular evidence indicate that Mg2+ entry to the vacuole is mediated by Mg2+/H+ exchangers. The Arabidopsis vacuolar Mg2+/H+ exchanger, AtMHX, is highly transcribed at the vascular tissue, apparently most abundantly at the xylem parenchyma. Inclusion of Mg2+ ions into the vacuoles of this tissue may determine their partitioning between the various plant organs. Impacts of Mg2+ imbalance are described with respect for both plant physiology and for its nutritional value to animal and human.

Cloning and characterization of a novel Mg2+/H+ exchanger

Cellular functions require adequate homeostasis of several divalent metal cations, including Mg2+ and Zn2+. Mg2+, the most abundant free divalent cytoplasmic cation, is essential for many enzymatic reactions, while Zn2+ is a structural constituent of various enzymes. Multicellular organisms have to balance not only the intake of Mg2+ and Zn2+, but also the distribution of these ions to various organs. To date, genes encoding Mg2+ transport proteins have not been cloned from any multicellular organism. We report here the cloning and characterization of an Arabidopsis thaliana transporter, designated AtMHX, which is localized in the vacuolar membrane and functions as an electrogenic exchanger of protons with Mg2+ and Zn2+ ions. Functional homologs of AtMHX have not been cloned from any organism. Ectopic overexpression of AtMHX in transgenic tobacco plants render them sensitive to growth on media containing elevated levels of Mg2+ or Zn2+, but does not affect the total amounts of these minerals in shoots of the transgenic plants. AtMHX mRNA is mainly found at the vascular cylinder, and a large proportion of the mRNA is localized in close association with the xylem tracheary elements. This localization suggests that AtMHX may control the partitioning of Mg2+ and Zn2+ between the various plant organs.

Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells

Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY‐2 cell suspension cultures. Sixteen different cytokinins, indole‐3‐acetic acid (IAA) and abscisic acid (ABA) were extracted using solid‐phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry. No significant correlation could be identified for IAA and ABA. In contrast, there were sharp peaks in the levels of specific cytokinins (zeatin‐ and dihydrozeatin‐type) at the end of the S phase and during mitosis. The levels of other cytokinins analyzed, including zeatins N‐ and O‐glucosides, remained low, suggesting that the increased amounts of their corresponding non‐glucosylated from resulted from de novo synthesis. These findings suggest that zeatin‐ and dihydrozeatin‐type cytokinins might play a specific regulatory role in the progression of the plant cell cycle. One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine.

Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase

The essential amino acids lysine and threonine are synthesized in higher plants by two separate branches of a common pathway. This pathway is primarily regulated by three key enzymes, namely aspartate kinase (AK), dihydrodipicolinate synthase (DHPS) and homoserine dehydrogenase (HSD), but how these enzymes operate in concert is as yet unknown. Addressing this issue, we have expressed in transgenic tobacco plants high levels of bacterial AK and DHPS, which are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Such expression of the bacterial DHPS by itself resulted in a substantial overproduction of lysine, whereas plants expressing only the bacterial AK overproduced threonine. When both bacterial enzymes were expressed in the same plant, the level of free lysine exceeded by far the level obtained by the bacterial DHPS alone. This increase, however, was accompanied by a significant reduction in threonine accumulation compared to plants expressing the bacterial AK alone. Our results suggested that in tobacco plants the synthesis of both lysine and threonine is under a concerted regulation exerted by AK, DHPS, and possibly also by HSD. We propose that the balance between lysine and threonine synthesis is determined by competition between DHPS and HSD on limiting amounts of their common substrate 3-aspartic semialdehyde, whose level, in turn, is determined primarily by the activity of AK. The potential of this molecular approach to increase the nutritional quality of plants is discussed.

Developmental expression of the Arabidopsis thaliana CycA2;1 gene.

Abstract. The associations of cyclins with highly conserved cyclin-dependent kinases are key events in the regulation of cell cycle progression. The spatio-temporal expression of an Arabidopsis thaliana (L.) Heynh. mitotic cyclin, Arath;CycA2;1, was studied by histochemical β-glucuronidase (GUS) analysis and in-situ hybridizations. The CycA2;1 promoter was active in the egg apparatus before fertilization. During embryogenesis, CycA2;1:gus expression was found in the embryo and the developing endosperm. Throughout plant development, CycA2;1 transcripts were found in both dividing and non-dividing cells, indicating that the expression of this cyclin is not a limiting factor for cell division. In the pericycle and stelar parenchyma, CycA2;1 transcripts were located at the xylem poles, a position that can be correlated with competence for lateral root formation. In addition, CycA2;1:gus expression was upregulated in roots by auxins and in the shoot apex by cytokinins. Transcription of CycA2;1 was shown by reverse transcription–polymerase chain reaction to be strongly induced by sucrose in A. thaliana cell suspensions.

Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.

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.

The Arabidopsis thaliana MHX gene includes an intronic element that boosts translation when localized in a 5′ UTR intron

The mechanisms that underlie the ability of some introns to increase gene expression, a phenomenon called intron-mediated enhancement (IME), are not fully understood. It is also not known why introns localized in the 5′-untranslated region (5′ UTR) are considerably longer than downstream eukaryotic introns. It was hypothesized that this extra length results from the presence of some functional intronic elements. However, deletion analyses studies carried out thus far were unable to identify specific intronic regions necessary for IME. Using deletion analysis and a gain-of-function approach, an internal element that considerably increases translational efficiency, without affecting splicing, was identified in the 5′ UTR intron of the Arabidopsis thaliana MHX gene. Moreover, the ability of this element to enhance translation was diminished by a minor downstream shift in the position of introns containing it from the 5′ UTR into the coding sequence. These data suggest that some of the extra length of 5′ UTR introns results from the presence of elements that enhance translation, and, moreover, from the ability of 5′ UTR introns to provide preferable platforms for such elements over downstream introns. The impact of the identified intronic element on translational efficiency was augmented upon removal of neighbouring intronic elements. Interference between different intronic elements had not been reported thus far. This interference may support the bioinformatics-based idea that some of the extra sequence of 5′ UTR introns is also necessary for separating different functional intronic elements.

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.