Marek Dynowski

A Mycorrhizal-Specific Ammonium Transporter from Lotus japonicus Acquires Nitrogen Released by Arbuscular Mycorrhizal Fungi

In mycorrhizal associations, the fungal partner assists its plant host by providing nitrogen (N) in addition to phosphate. Arbuscular mycorrhizal (AM) fungi have access to inorganic or organic forms of N and translocate them via arginine from the extra- to the intraradical mycelium, where the N is transferred to the plant without any carbon skeleton. However, the molecular form in which N is transferred, as well as the involved mechanisms, is still under debate. NH4+ seems to be the preferential transferred molecule, but no plant ammonium transporter (AMT) has been identified so far. Here, we offer evidence of a plant AMT that is involved in N uptake during mycorrhiza symbiosis. The gene LjAMT2;2, which has been shown to be the highest up-regulated gene in a transcriptomic analysis of Lotus japonicus roots upon colonization with Gigaspora margarita, has been characterized as a high-affinity AMT belonging to the AMT2 subfamily. It is exclusively expressed in the mycorrhizal roots, but not in the nodules, and transcripts have preferentially been located in the arbusculated cells. Yeast (Saccharomyces cerevisiae) mutant complementation has confirmed its functionality and revealed its dependency on acidic pH. The transport experiments using Xenopus laevis oocytes indicated that, unlike other plant AMTs, LjAMT2;2 transports NH3 instead of NH4+. Our results suggest that the transporter binds charged ammonium in the apoplastic interfacial compartment and releases the uncharged NH3 into the plant cytoplasm. The implications of such a finding are discussed in the context of AM functioning and plant phosphorus uptake.

Plant plasma membrane water channels conduct the signalling molecule H2O2.

H(2)O(2) is a relatively long-lived reactive oxygen species that signals between cells and organisms. H(2)O(2) signalling in plants is essential for response to stress, defence against pathogens and the regulation of programmed cell death. Although H(2)O(2) diffusion across membranes is often considered as a passive property of lipid bilayers, native membranes represent significant barriers for H(2)O(2). In the present study we addressed the question of whether channels might facilitate H(2)O(2) conduction across plasma membranes. The expression of several plant plasma membrane aquaporins in yeast, including PIP2;1 from Arabidopsis (where PIP is plasma membrane intrinsic protein), enhanced the toxicity of H(2)O(2) and increased the fluorescence of dye-loaded yeast when exposed to H(2)O(2). The sensitivity of aquaporin-expressing yeast to H(2)O(2) was altered by mutations that alter gating and the selectivity of the aquaporins. The conduction of water, H(2)O(2) and urea was compared, using molecular dynamics simulations based on the crystal structure of SoPIP2;1 from spinach. The calculations identify differences in the conduction between the substrates and reveal channel residues critically involved in H(2)O(2) conduction. The results of the calculations on tetramers and monomers are in agreement with the biochemical data. Taken together, the results strongly suggest that plasma membrane aquaporin pores determine the efficiency of H(2)O(2) signalling between cells. Aquaporins are present in most species and their capacity to facilitate the diffusion of H(2)O(2) may be of physiological significance in many organisms and particularly in communication between different species.

Molecular mechanisms of ammonium transport and accumulation in plants

The integral membrane proteins of the ammonium transporter (AMT/Rh) family provide the major route for shuttling ammonium ( NH 4 + / NH 3 ) across bacterial, archaeal, fungal and plant membranes. These proteins are distantly related to the Rh (rhesus) glycoproteins, which are absent in higher plants, but are present in many species, including bacteria and mammals. It appears that the large nitrogen requirement of plants resulted in unique strategies to acquire, capture and/or release ammonium. The biological function of plant ammonium transporters will be discussed and compared to other AMT/Rh proteins.

Regulation of NH4+ Transport by Essential Cross-talk between AMT Monomers through the Carboxyl-tails

Ammonium transport across plant plasma membranes is facilitated by AMT/Rh-type ammonium transporters (AMTs), which also have homologs in most organisms. In the roots of the plant Arabidopsis (Arabidopsis thaliana), AMTs have been identified that function directly in the high-affinity NH4+ acquisition from soil. Here, we show that AtAMT1;2 has a distinct role, as it is located in the plasma membrane of the root endodermis. AtAMT1;2 functions as a comparatively low-affinity NH4+ transporter. Mutations at the highly conserved carboxyl terminus (C terminus) of AMTs, including one that mimics phosphorylation at a putative phosphorylation site, impair NH4+ transport activity. Coexpressing these mutants along with wild-type AtAMT1;2 substantially reduced the activity of the wild-type transporter. A molecular model of AtAMT1;2 provides a plausible explanation for the dominant inhibition, as the C terminus of one monomer directly contacts the neighboring subunit. It is suggested that part of the cytoplasmic C terminus of a single monomer can gate the AMT trimer. This regulatory mechanism for rapid and efficient inactivation of NH4+ transporters may apply to several AMT members to prevent excess influx of cytotoxic ammonium.

CLC-b-Mediated NO−3/H+ Exchange Across the Tonoplast of Arabidopsis Vacuoles

Nitrate is frequently the major nitrogen source for plants and is generally assimilated during the day. In the absence of light, nitrate can transiently accumulate in the vacuolar lumen via tonoplast transporters. CLC-a, a member of the CLC family of anion transporters, is critically involved in this nitrate storage in the vacuole, while other CLC family members apparently have different roles in diverse cell organelles. Here, CLC-b, a close relative of CLC-a, was functionally expressed in oocytes and analyzed. CLC-b conducted strongly outwardly rectifying anionic currents that were largest in the presence of nitrate. Fluorescence ratio changes of oocytes loaded with a pH-dependent fluorescent dye suggested that NO(-)(3) transport is associated with H(+) exchange. CLC-b was localized at the tonoplast, as was CLC-c, when tagged with the green fluorescent protein. CLC-b expression was strongest in young roots, hypocotyl and cotyledons. The physiological role of CLC-b was analyzed using two independent knock-out alleles. Both lines grew as the wild type in various conditions. The total chloride and nitrate content was identical in clcb lines and the wild type, potentially suggesting that mutants were able to compensate the loss of CLC-b.

Molecular determinants of ammonia and urea conductance in plant aquaporin homologs

Aquaporins and/or aquaglyceroporins regulate the permeability of plant membranes to water and small, uncharged molecules. Using molecular simulations with a plant plasma membrane aquaporin tetramer, the residues in the channel constriction region were identified as the crucial determinants of ammonia and urea conductance. The impact of these residues was experimentally verified using AtPIP2;1 pore mutants. Several, but not all, mutants with a NIP‐like selectivity filter promoted yeast growth on urea or ammonia as sole sources of nitrogen. TIP‐like mutants conducted urea but not NH3, and a residue without direct contact to the pore lumen was critical for conduction in the mutants.

VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP 8 and Jasmonate-Dependent Defenses in Arabidopsis

The inositol pyrophosphate InsP8 plays an important role in plant defenses against herbivorous insects and necrotrophic fungi and is a key cofactor of the jasmonate receptor complex. Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.

Plant aquaporin selectivity: where transport assays, computer simulations and physiology meet.

Plants contain a large number of aquaporins with different selectivity. These channels generally conduct water, but some additionally conduct NH3, CO2 and/or H2O2. The experimental evidence and molecular basis for the transport of a given solute, the validation with molecular dynamics simulations and the physiological impact of the selectivity are reviewed here. The aromatic/arginine (ar/R) constriction is most important for solute selection, but the exact pore requirements for efficient conduction of small solutes remain difficult to predict. Yeast growth assays are valuable for screening substrate selectivity and are explicitly shown for hydrogen peroxide and methylamine, a transport analog of ammonia. Independent assays need to address the relevance of different substrates for each channel in its physiological context. This is emphasized by the fact that several plant NIP channels, which conduct several solutes, are specifically involved in the transport of metalloids, such as silicic acid, arsenite, or boric acid in planta.

Alanine Zipper-Like Coiled-Coil Domains Are Necessary for Homotypic Dimerization of Plant GAGA-Factors in the Nucleus and Nucleolus

GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.

Channel-like NH3 flux by ammonium transporter AtAMT2

Prokaryotes, plants and animals control ammonium fluxes by the regulated expression of ammonium transporters (AMTs) and/or the related Rhesus (Rh) proteins. Plant AMTs were previously reported to mediate electrogenic transport. Functional analysis of AtAMT2 from Arabidopsis in yeast and oocytes suggests that NH 4 + is the recruited substrate, but the uncharged form NH3 is conducted. AtAMT2 partially co‐localized with electrogenic AMTs and conducted methylamine with low affinity. This transport mechanism may apply to other plant ammonium transporters and explains the different capacities of AMTs to accumulate ammonium in the plant cell.