Susan M. Howitt

Structure, function and regulation of ammonium transporters in plants.

Ammonium is an important source of nitrogen for plants. It is taken up by plant cells via ammonium transporters in the plasma membrane and distributed to intracellular compartments such as chloroplasts, mitochondria and vacuoles probably via different transporters in each case. Ammonium is generally not used for long-distance transport of nitrogen within the plant. Instead, most of the ammonium transported into plant cells is assimilated locally via glutamine synthetases in the cytoplasm and plastids. Ammonium is also produced by plant cells during normal metabolism, and ammonium transporters enable it to be moved from intracellular sites of production to sites of consumption. Ammonium can be generated de novo from molecular nitrogen (N(2)) by nitrogen-fixing bacteria in some plant cells, such as rhizobia in legume root nodule cells, and at least one ammonium transporter is implicated in the transfer of ammonium from the bacteria to the plant cytoplasm. Plant physiologists have described many of these ammonium transport processes over the last few decades. However, the genes and proteins that underlie these processes have been isolated and studied only recently. In this review, we consider in detail the molecular structure, function and regulation of plant ammonium transporters. We also attempt to reconcile recent discoveries at the molecular level with our knowledge of ammonium transport at the whole plant level.

Identification of a SulP-type bicarbonate transporter in marine cyanobacteria

Cyanobacteria possess a highly effective CO2-concentrating mechanism that elevates CO2 concentrations around the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). This CO2-concentrating mechanism incorporates light-dependent, active uptake systems for CO2 and HCO–3. Through mutant studies in a coastal marine cyanobacterium, Synechococcus sp. strain PCC7002, we identified bicA as a gene that encodes a class of HCO–3 transporter with relatively low transport affinity, but high flux rate. BicA is widely represented in genomes of oceanic cyanobacteria and belongs to a large family of eukaryotic and prokaryotic transporters presently annotated as sulfate transporters or permeases in many bacteria (SulP family). Further gain-of-function experiments in the freshwater cyanobacterium Synechococcus PCC7942 revealed that bicA expression alone is sufficient to confer a Na+-dependent, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} uptake activity. We identified and characterized three cyanobacterial BicA transporters in this manner, including one from the ecologically important oceanic strain, Synechococcus WH8102. This study presents functional data concerning prokaryotic members of the SulP transporter family and represents a previously uncharacterized transport function for the family. The discovery of BicA has significant implications for understanding the important contribution of oceanic strains of cyanobacteria to global CO2 sequestration processes.

Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants

We have cloned and characterized the first member of a novel family of ammonium transporters in plants: AtAMT2 from Arabidopsis thaliana. AtAMT2 is more closely related to bacterial ammonium transporters than to plant transporters of the AMT1 family. The protein was expressed and functionally characterized in yeast. AtAMT2 transported ammonium in an energy‐dependent manner. In contrast to transporters of the AMT1 family, however, AtAMT2 did not transport the ammonium analogue, methylammonium. AtAMT2 was expressed more highly in shoots than roots and was subject to nitrogen regulation.

The cyanobacterial CCM as a source of genes for improving photosynthetic CO2 fixation in crop species

Crop yields need to nearly double over the next 35 years to keep pace with projected population growth. Improving photosynthesis, via a range of genetic engineering strategies, has been identified as a promising target for crop improvement with regard to increased photosynthetic yield and better water-use efficiency (WUE). One approach is based on integrating components of the highly efficient CO(2)-concentrating mechanism (CCM) present in cyanobacteria (blue-green algae) into the chloroplasts of key C(3) crop plants, particularly wheat and rice. Four progressive phases towards engineering components of the cyanobacterial CCM into C(3) species can be envisaged. The first phase (1a), and simplest, is to consider the transplantation of cyanobacterial bicarbonate transporters to C(3) chloroplasts, by host genomic expression and chloroplast targeting, to raise CO(2) levels in the chloroplast and provide a significant improvement in photosynthetic performance. Mathematical modelling indicates that improvements in photosynthesis as high as 28% could be achieved by introducing both of the single-gene, cyanobacterial bicarbonate transporters, known as BicA and SbtA, into C(3) plant chloroplasts. Part of the first phase (1b) includes the more challenging integration of a functional cyanobacterial carboxysome into the chloroplast by chloroplast genome transformation. The later three phases would be progressively more elaborate, taking longer to engineer other functional components of the cyanobacterial CCM into the chloroplast, and targeting photosynthetic and WUE efficiencies typical of C(4) photosynthesis. These later stages would include the addition of NDH-1-type CO(2) pumps and suppression of carbonic anhydrase and C(3) Rubisco in the chloroplast stroma. We include a score card for assessing the success of physiological modifications gained in phase 1a.

Characterization of PitA and PitB from Escherichia coli.

Escherichia coli contains two major systems for transporting inorganic phosphate (P(i)). The low-affinity P(i) transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external P(i) concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P(i) transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P(i) transporter that may be repressed at low P(i) levels by the pho regulon. While a pitB(+) cosmid clone allowed growth on medium containing 500 microM P(i), E. coli with wild-type genomic pitB (pitA Delta pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB Delta pstC345 triple mutant. The mutation Delta pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB(+) pitA Delta pstC345 strain to utilize P(i), with P(i) uptake rates significantly higher than background levels. In addition, the apparent K(m) of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.

The proton pore in the Escherichia coli F0F1-ATPase: A requirement for arginine at position 210 of the a-subunit

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).

HvALMT1 from barley is involved in the transport of organic anions

Members of the ALMT gene family contribute to the Al3+ resistance of several plant species by facilitating malate efflux from root cells. The first member of this family to be cloned and characterized, TaALMT1, is responsible for most of the natural variation of Al3+ resistance in wheat. The current study describes the isolation and characterization of HvALMT1, the barley gene with the greatest sequence similarity to TaALMT1. HvALMT1 is located on chromosome 2H which has not been associated with Al3+ resistance in barley. The relatively low levels of HvALMT1 expression detected in root and shoot tissues were independent of external aluminium or phosphorus supply. Transgenic barley plants transformed with the HvALMT1 promoter fused to the green fluorescent protein (GFP) indicated that expression of HvALMT1 was relatively high in stomatal guard cells and in root tissues containing expanding cells. GFP fused to the C-terminus of the full HvALMT1 protein localized to the plasma membrane and motile vesicles within the cytoplasm. HvALMT1 conferred both inward and outward currents when expressed in Xenopus laevis oocytes that were bathed in a range of anions including malate. Both malate uptake and efflux were confirmed in oocyte assays using [14C]malate as a radiotracer. It is suggested that HvALMT1 functions as an anion channel to facilitate organic anion transport in stomatal function and expanding cells.

The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit.

The δ and b subunits are both involved in binding the F1 to the F0 part in theEscherichia coli ATP synthase (ECF1F0). The interaction of the purified δ subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified δ binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1,i.e. in the presence of ECF1-δ, δ, and bsol interact strongly, and a complex of ECF1·bsol can be isolated by native gel electrophoresis. Both δ subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the δ subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of δ as δ(1–134) cannot replace intact δ in the binding experiments. As purified, bsol is a stable dimer with 80% α helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem.271, 7038–7042). Monomeric bsol has less α helix,i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to δ in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124–131 likely forming a pair of parallel α helices.

Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (Km for ammonium and methylammonium of 8 μM and 31 μM, respectively) matched those of AtAMT1;1 expressed in yeast (Km for methylammonium of 32 μM and Ki for ammonium of 1–10 μM). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (Km for ammonium of 46 μM). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a Km of 36 μM, a low-affinity phase with a Km for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.

Membrane topology of the cyanobacterial bicarbonate transporter, BicA, a member of the SulP (SLC26A) family.

Abstract We have completed the first comprehensive transmembrane topology determination for a member of the ubiquitous and important SulP/SLC26 family of coupled anion transporters found in eukaryotes and prokaryotes. The prokaryotic member that we have mapped, namely BicA from Synechococcus PCC7002, is an important Na+-dependent bicarbonate transporter that is likely to play a major role in global primary productivity via the CO2 concentrating mechanism in cyanobacteria. We experimentally determined the topology based on phoA-lacZ topology mapping combined with reference to a range of predictive models based on hydropathy analysis and positive charge distribution. The 12-TMH structure for BicA is characterized by tight turns between several pairs of TMH and it features a prominent cytoplasmically-located STAS domain that is characteristic of the SulP family. A key difference from previous predicted models is that we identify a cytoplasmic loop between helices 8 and 9 where previous models suggested a TMH. This region includes a highly conserved motif that defines the SulP family. The identification of this region as cytoplasmic, rather than transmembrane, has implications for the function and perhaps regulation of SulP family members. This finding is used to reinterpret mutagenesis data relating to highly conserved residues in this region from both plant and human SulP transporters.