Yair Shachar-Hill

From genome to function: the Arabidopsis aquaporins

BackgroundIn the post-genomic era newly sequenced genomes can be used to deduce organismal functions from our knowledge of other systems. Here we apply this approach to analyzing the aquaporin gene family in Arabidopsis thaliana. The aquaporins are intrinsic membrane proteins that have been characterized as facilitators of water flux. Originally termed major intrinsic proteins (MIPs), they are now also known as water channels, glycerol facilitators and aqua-glyceroporins, yet recent data suggest that they facilitate the movement of other low-molecular-weight metabolites as well.ResultsThe Arabidopsis genome contains 38 sequences with homology to aquaporin in four subfamilies, termed PIP, TIP, NIP and SIP. We have analyzed aquaporin family structure and expression using the A. thaliana genome sequence, and introduce a new NMR approach for the purpose of analyzing water movement in plant roots in vivo.ConclusionsOur preliminary data indicate a strongly transcellular component for the flux of water in roots.

Plant one-carbon metabolism and its engineering.

The metabolism of one-carbon (C1) units is vital to plants. It involves unique enzymes and takes place in four subcellular compartments. Plant C1 biochemistry has remained relatively unexplored, partly because of the low abundance or the lability of many of its enzymes and intermediates. Fortunately, DNA sequence databases now make it easier to characterize known C1 enzymes and to discover new ones, to identify pathways that might carry high C1 fluxes, and to use engineering to redirect C1 fluxes and to understand their control better.

Carbon Partitioning, Cost, and Metabolism of Arbuscular Mycorrhizas

Colonization of roots by arbuscular mycorrhizal [AM] fungi results in many changes in the carbon partitioning and metabolism of the host plant. The rate of carbon assimilation, the export of photosynthates from leaves, and the sink strength of roots may be increased relative to that in uncolonized plants. Hexose is taken up by the obligately symbiotic fungus for its growth, maintenance, and reproduction. This can represent a significant cost to the host, most notably under conditions in which the fungus offers little nutritive benefit. The components of the carbon partitioning and cost of arbuscular mycorrhizas, as well as current knowledge of the carbon metabolism of germinating spores and infra- and extraradical hyphae of AM fungi are reviewed in this chapter.

The glyoxylate cycle in an arbuscular mycorrhizal fungus. Carbon flux and gene expression.

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of (13)C labeling of germinating spores and extraradical mycelium with (13)C(2)-acetate and (13)C(2)-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle.

Tracking metabolism and imaging transport in arbuscular mycorrhizal fungi

In the last few years the application of modern techniques to the study of arbuscular mycorrhizas has greatly increased our understanding of the mechanisms underlying carbon metabolism in these mutualistic symbioses. Arbuscular mycorrhizal (AM) monoxenic cultures, nuclear magnetic resonance spectroscopy together with isotopic labeling, and analyses of expressed sequence tags (ESTs) have shed light on the metabolic processes taking place in these interactions, particularly in the case of the mycobiont. More recently, in vivo multiphoton microscopy has provided us with some new insights in the allocation and translocation processes which play crucial roles in the distribution of host plant-derived C throughout the fungal colony. In this mini-review we highlight recent advances in these fields, with special attention to the visualization of oleosomes (i.e., lipid bodies) as they move along the long, coenocytic AM fungal hyphae. Volumetric measurements of such oleosomes have allowed us to estimate the flux of triacylglycerides from the intraradical to the extraradical phase of the AM fungal colony. We raise questions and postulate regulatory mechanisms for C metabolism and translocation within the arbuscular mycorrhizal fungal colony.

Isolation, Characterization, and Functional Expression of cDNAs Encoding NADH-dependent Methylenetetrahydrofolate Reductase from Higher Plants

Methylenetetrahydrofolate reductase (MTHFR) is the least understood enzyme of folate-mediated one-carbon metabolism in plants. Genomics-based approaches were used to identify one maize and two Arabidopsis cDNAs specifying proteins homologous to MTHFRs from other organisms. These cDNAs encode functional MTHFRs, as evidenced by their ability to complement a yeast met12 met13 mutant, and by the presence of MTHFR activity in extracts of complemented yeast cells. Deduced sequence analysis shows that the plant MTHFR polypeptides are of similar size (66 kDa) and domain structure to other eukaryotic MTHFRs, and lack obvious targeting sequences. Southern analyses and genomic evidence indicate thatArabidopsis has two MTHFR genes and that maize has at least two. A carboxyl-terminal polyhistidine tag was added to oneArabidopsis MTHFR, and used to purify the enzyme 640-fold to apparent homogeneity. Size exclusion chromatography and denaturing gel electrophoresis of the recombinant enzyme indicate that it exists as a dimer of ≈66-kDa subunits. Unlike mammalian MTHFR, the plant enzymes strongly prefer NADH to NADPH, and are not inhibited byS-adenosylmethionine. An NADH-dependent MTHFR reaction could be reversible in plant cytosol, where the NADH/NAD ratio is 10−3. Consistent with this, leaf tissues metabolized [methyl-14C]methyltetrahydrofolate to serine, sugars, and starch. A reversible MTHFR reaction would obviate the need for inhibition by S-adenosylmethionine to prevent excessive conversion of methylene- to methyltetrahydrofolate.

Tracking metabolism and imaging transport in arbuscular mycorrhizal fungi. Metabolism and transport in AM fungi

In the last few years the application of modern techniques to the study of arbuscular mycorrhizas has greatly increased our understanding of the mechanisms underlying carbon metabolism in these mutualistic symbioses. Arbuscular mycorrhizal (AM) monoxenic cultures, nuclear magnetic resonance spectroscopy together with isotopic labeling, and analyses of expressed sequence tags (ESTs) have shed light on the metabolic processes taking place in these interactions, particularly in the case of the mycobiont. More recently, in vivo multiphoton microscopy has provided us with some new insights in the allocation and translocation processes which play crucial roles in the distribution of host plant-derived C throughout the fungal colony. In this mini-review we highlight recent advances in these fields, with special attention to the visualization of oleosomes (i.e., lipid bodies) as they move along the long, coenocytic AM fungal hyphae. Volumetric measurements of such oleosomes have allowed us to estimate the flux of triacylglycerides from the intraradical to the extraradical phase of the AM fungal colony. We raise questions and postulate regulatory mechanisms for C metabolism and translocation within the arbuscular mycorrhizal fungal colony.

Application of in vitro methods to study carbon uptake and transport by AM fungi

Just as multi-compartmented root chambers have advantages over standard plastic pots for the study of nutrient uptake by arbuscular mycorrhizal [AM] fungi in soil, so the split-plate in vitro system has advantages over the standard dual culture system for the study of the physiology of AM fungi. We used the split-plate culture system of Ri T-DNA transformed Daucus carota L. roots and Glomus intraradices Schenck & Smith, in which only the fungus has access to the distal compartment, to study the ability of germ tubes and extraradical and intraradical hyphae to take up 13C-labeled substrates. Labeled substrates were added to one side of the plate divider and plates were incubated for 8 weeks while the fungus proliferated on the side from which the root was excluded. Tissues then were recovered from the plate and examined via NMR spectroscopy. Results showed that the morphological phases of the fungus differed in their ability to take up these substrates, most notably that intraradical hyphae take up hexose while extraradical hyphae cannot. In addition, NMR studies indicated that intraradical hyphae actively synthesized lipids while extraradical hyphae did not. These data show that eventual axenic culture of AM fungi is more than a matter of finding the proper substrate for growth. Genetic regulation must be overcome to make extraradical hyphae behave like intraradical hyphae in terms of C uptake and metabolism.

Expression in an arbuscular mycorrhizal fungus of genes putatively involved in metabolism, transport, the cytoskeleton and the cell cycle

Arbuscular mycorrhizal (AM) fungi are multinucleate, coenocytic, obligate symbionts with no known sexual stages and very wide host and habitat ranges. While contributing vitally to the growth of land plants they face unique challenges in metabolism, transport, growth and development. To provide clues to the strategies that AM fungi have adopted, random sequencing of cDNAs from Glomus intraradices was undertaken. Putative genes for enzymes, transporters, structural proteins and cell-cycle regulatory factors were discovered. Among the ESTs of particular interest are sequences with homology to known trehalase, arsenite transporter, cysteine synthase, tubulins, actin, dynein, cell cycle regulatory proteins, and three meiosis-related proteins. The significance of these sequences is discussed in the context of what is known about AM metabolism, transport, growth and phylogeny.

Magnetic susceptibility shift selected imaging (MESSI) and localized 1H2O spectroscopy in living plant tissues

Maize root segments permeated with aqueous solutions of the paramagnetic agents GdDTPA2− or DyDTPA‐BMA display two well‐resolved NMR peaks corresponding to the signals from intracellular and extracellular 1H2O, which arise from well‐understood bulk magnetic susceptibility effects. This allows each component to be studied separately. Images obtained at each frequency with MESSI editing, and single‐ and multiple‐voxel (‘spectroscopic imaging’) localized spectra, clearly indicate that the agents permeate into the interstitial spaces, and into the longitudinal (xylem/phloem) channels in the stele (core) of the root, confirming earlier assessments. We believe these are the first images of a multicellular tissue acquired in vivo exclusively from the intracellular water proton resonance. This method can be further exploited to study water transport in similar systems. Copyright