DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families.

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted "S"-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.

Structural basis of high-affinity nuclear localization signal interactions with importin-α.

Classical nuclear localization signals (cNLSs), comprising one (monopartite cNLSs) or two clusters of basic residues connected by a 10-12 residue linker (bipartite cNLSs), are recognized by the nuclear import factor importin-α. The cNLSs bind along a concave groove on importin-α; however, specificity determinants of cNLSs remain poorly understood. We present a structural and interaction analysis study of importin-α binding to both designed and naturally occurring high-affinity cNLS-like sequences; the peptide inhibitors Bimax1 and Bimax2, and cNLS peptides of cap-binding protein 80. Our data suggest that cNLSs and cNLS-like sequences can achieve high affinity through maximizing interactions at the importin-α minor site, and by taking advantage of multiple linker region interactions. Our study defines an extended set of binding cavities on the importin-α surface, and also expands on recent observations that longer linker sequences are allowed, and that long-range electrostatic complementarity can contribute to cNLS-binding affinity. Altogether, our study explains the molecular and structural basis of the results of a number of recent studies, including systematic mutagenesis and peptide library approaches, and provides an improved level of understanding on the specificity determinants of a cNLS. Our results have implications for identifying cNLSs in novel proteins.

Chemical Synthesis, Herbicidal Activity, Crop Safety, and Molecular Basis of ortho-Fluoroalkoxy Substituted Sulfonylureas as Novel Acetohydroxyacid Synthase Inhibitors.

In the face of increasing resistance to the currently used commercial herbicides and the lack of success in identifying new herbicide targets, alternative herbicides need to be developed to control unwanted monocotyledon grasses in food crops. Here, a panel of 29 novel sulfonylurea-based compounds with ortho-fluoroalkoxy substitutions at the phenyl ring were designed and synthesized. Pot assays demonstrated that two of these compounds, 6d and 6u, have strong herbicidal activities against Echinochloa crus-galli, Eleusine indica, Alopecurus aequalis, and Alopecurus japonicus Steudel at a dosage of 15 g ha-1. Furthermore, these two compounds exhibited <5% inhibition against wheat at a dosage of 30 g ha-1 under post-emergence conditions. 6u also exhibited <5% inhibition against rice at a dosage of 30 g ha-1 under both post-emergence and pre-emergence conditions. A kinetics study demonstrated that 6d and 6u are potent inhibitors of Arabidopsis thaliana acetohydroxyacid synthase (AHAS; EC 2.2.1.6) with potent Ki values of 18 ± 1.1 and 11.9 ± 4.0 nM, respectively. The crystal structure of 6u in complex with A. thaliana (At)AHAS has also been determined at 2.7 Å resolution. These new compounds represent new alternative herbicide choices to protect wheat or rice from invading grasses.

Endocytosis-like protein uptake in the bacterium Gemmata obscuriglobus.

Endocytosis is a process by which extracellular material such as macromolecules can be incorporated into cells via a membrane-trafficking system. Although universal among eukaryotes, endocytosis has not been identified in Bacteria or Archaea. However, intracellular membranes are known to compartmentalize cells of bacteria in the phylum Planctomycetes, suggesting the potential for endocytosis and membrane trafficking in members of this phylum. Here we show that cells of the planctomycete Gemmata obscuriglobus have the ability to uptake proteins present in the external milieu in an energy-dependent process analogous to eukaryotic endocytosis, and that internalized proteins are associated with vesicle membranes. Occurrence of such ability in a bacterium is consistent with autogenous evolution of endocytosis and the endomembrane system in an ancestral noneukaryote cell.

DNA is taken up by root hairs and pollen, and stimulates root and pollen tube growth.

Phosphorus (P) enters roots as inorganic phosphate (P(i)) derived from organic and inorganic P compounds in the soil. Nucleic acids can support plant growth as the sole source of P in axenic culture but are thought to be converted into P(i) by plant-derived nucleases and phosphatases prior to uptake. Here, we show that a nuclease-resistant analog of DNA is taken up by plant cells. Fluorescently labeled S-DNA of 25 bp, which is protected against enzymatic breakdown by its phosphorothioate backbone, was taken up and detected in root cells including root hairs and pollen tubes. These results indicate that current views of plant P acquisition may have to be revised to include uptake of DNA into cells. We further show that addition of DNA to P(i)-containing growth medium enhanced the growth of lateral roots and root hairs even though plants were P replete and had similar biomass as plants supplied with P(i) only. Exogenously supplied DNA increased length growth of pollen tubes, which were studied because they have similar elongated and polarized growth as root hairs. Our results indicate that DNA is not only taken up and used as a P source by plants, but ironically and independent of P(i) supply, DNA also induces morphological changes in roots similar to those observed with P limitation. This study provides, to our knowledge, first evidence that exogenous DNA could act nonspecifically as signaling molecules for root development.

Plants can use protein as a nitrogen source without assistance from other organisms.

Nitrogen is quantitatively the most important nutrient that plants acquire from the soil. It is well established that plant roots take up nitrogen compounds of low molecular mass, including ammonium, nitrate, and amino acids. However, in the soil of natural ecosystems, nitrogen occurs predominantly as proteins. This complex organic form of nitrogen is considered to be not directly available to plants. We examined the long-held view that plants depend on specialized symbioses with fungi (mycorrhizas) to access soil protein and studied the woody heathland plant Hakea actites and the herbaceous model plant Arabidopsis thaliana, which do not form mycorrhizas. We show that both species can use protein as a nitrogen source for growth without assistance from other organisms. We identified two mechanisms by which roots access protein. Roots exude proteolytic enzymes that digest protein at the root surface and possibly in the apoplast of the root cortex. Intact protein also was taken up into root cells most likely via endocytosis. These findings change our view of the spectrum of nitrogen sources that plants can access and challenge the current paradigm that plants rely on microbes and soil fauna for the breakdown of organic matter.

Importin-beta is a GDP-to-GTP exchange factor of Ran: implications for the mechanism of nuclear import.

Ran-GTP interacts strongly with importin-beta, and this interaction promotes the release of the importin-alpha-nuclear localization signal cargo from importin-beta. Ran-GDP also interacts with importin-beta, but this interaction is 4 orders of magnitude weaker than the Ran-GTP.importin-beta interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-beta promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-beta complex stabilizes the complex, which cannot be dissociated by importin-alpha. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-beta has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-beta. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-beta. Importin-alpha inhibits the GDP exchange reaction by sequestering importin-beta, whereas RanBP1 restores the GDP nucleotide exchange by importin-beta by forming a tetrameric complex with importin-beta, Ran, and importin-alpha. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.

Nitrogen affects cluster root formation and expression of putative peptide transporters.

Non-mycorrhizal Hakea actites (Proteaceae) grows in heathland where organic nitrogen (ON) dominates the soil nitrogen (N) pool. Hakea actites uses ON for growth, but the role of cluster roots in ON acquisition is unknown. The aim of the present study was to ascertain how N form and concentration affect cluster root formation and expression of peptide transporters. Hydroponically grown plants produced most biomass with low molecular weight ON>inorganic N>high molecular weight ON, while cluster roots were formed in the order no-N>ON>inorganic N. Intact dipeptide was transported into roots and metabolized, suggesting a role for the peptide transporter (PTR) for uptake and transport of peptides. HaPTR4, a member of subgroup II of the NRT1/PTR transporter family, which contains most characterized di- and tripeptide transporters in plants, facilitated transport of di- and tripeptides when expressed in yeast. No transport activity was demonstrated for HaPTR5 and HaPTR12, most similar to less well characterized transporters in subgroup III. The results provide further evidence that subgroup II of the NRT1/PTR family contains functional di- and tripeptide transporters. Green fluorescent protein fusion proteins of HaPTR4 and HaPTR12 localized to tonoplast, and plasma- and endomembranes, respectively, while HaPTR5 localized to vesicles of unknown identity. Grown in heathland or hydroponic culture with limiting N supply or starved of nutrients, HaPTR genes had the highest expression in cluster roots and non-cluster roots, and leaf expression increased upon re-supply of ON. It is concluded that formation of cluster roots and expression of PTR are regulated in response to N supply.

Crosstalk between sugarcane and a plant-growth promoting Burkholderia species.

Bacterial species in the plant-beneficial-environmental clade of Burkholderia represent a substantial component of rhizosphere microbes in many plant species. To better understand the molecular mechanisms of the interaction, we combined functional studies with high-resolution dual transcriptome analysis of sugarcane and root-associated diazotrophic Burkholderia strain Q208. We show that Burkholderia Q208 forms a biofilm at the root surface and suppresses the virulence factors that typically trigger immune response in plants. Up-regulation of bd-type cytochromes in Burkholderia Q208 suggests an increased energy production and creates the microaerobic conditions suitable for BNF. In this environment, a series of metabolic pathways are activated in Burkholderia Q208 implicated in oxalotrophy, microaerobic respiration, and formation of PHB granules, enabling energy production under microaerobic conditions. In the plant, genes involved in hypoxia survival are up-regulated and through increased ethylene production, larger aerenchyma is produced in roots which in turn facilitates diffusion of oxygen within the cortex. The detected changes in gene expression, physiology and morphology in the partnership are evidence of a sophisticated interplay between sugarcane and a plant-growth promoting Burkholderia species that advance our understanding of the mutually beneficial processes occurring in the rhizosphere.

Nitrogen fertilizer dose alters fungal communities in sugarcane soil and rhizosphere.

Fungi play important roles as decomposers, plant symbionts and pathogens in soils. The structure of fungal communities in the rhizosphere is the result of complex interactions among selection factors that may favour beneficial or detrimental relationships. Using culture-independent fungal community profiling, we have investigated the effects of nitrogen fertilizer dosage on fungal communities in soil and rhizosphere of field-grown sugarcane. The results show that the concentration of nitrogen fertilizer strongly modifies the composition but not the taxon richness of fungal communities in soil and rhizosphere. Increased nitrogen fertilizer dosage has a potential negative impact on carbon cycling in soil and promotes fungal genera with known pathogenic traits, uncovering a negative effect of intensive fertilization.

Thermodynamic non-ideality as an alternative source of the effect of sucrose on the thrombin-catalyzed hydrolysis of peptide p-nitroanilide substrates.

The inhibitory effect of sucrose on the kinetics of thrombin-catalyzed hydrolysis of the chromogenic substrate S-2238 (D-phenylalanyl-pipecolyl-arginoyl-p-nitroanilide) is re-examined as a possible consequence of thermodynamic non-ideality-an inhibition originally attributed to the increased viscosity of reaction mixtures. However, those published results may also be rationalized in terms of the suppression of a substrate-induced isomerization of thrombin to a slightly more expanded (or more asymmetric) transition state prior to the irreversible kinetic steps that lead to substrate hydrolysis. This reinterpretation of the kinetic results solely in terms of molecular crowding does not signify the lack of an effect of viscosity on any reaction step(s) subject to diffusion control. Instead, it highlights the need for development of analytical procedures that can accommodate the concomitant operation of thermodynamic non-ideality and viscosity effects.

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products.

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products.

The effect of protein supplied in the growth medium on plant pathogen resistance.

Externally supplied protein (bovine serum albumin, BSA) affects root development of Arabidopsis, increasing root biomass, root hair length, and root thickness. While these changes in root morphology may enhance access to soil microenvironments rich in organic matter, we show here that the presence of protein in the growth medium increases the plant's resilience to the root pathogen Cylindrocladium sp.

Effects of externally supplied protein on root morphology and biomass allocation in Arabidopsis.

Growth, morphogenesis and function of roots are influenced by the concentration and form of nutrients present in soils, including low molecular mass inorganic N (IN, ammonium, nitrate) and organic N (ON, e.g. amino acids). Proteins, ON of high molecular mass, are prevalent in soils but their possible effects on roots have received little attention. Here, we investigated how externally supplied protein of a size typical of soluble soil proteins influences root development of axenically grown Arabidopsis. Addition of low to intermediate concentrations of protein (bovine serum albumen, BSA) to IN-replete growth medium increased root dry weight, root length and thickness, and root hair length. Supply of higher BSA concentrations inhibited root development. These effects were independent of total N concentrations in the growth medium. The possible involvement of phytohormones was investigated using Arabidopsis with defective auxin (tir1-1 and axr2-1) and ethylene (ein2-1) responses. That no phenotype was observed suggests a signalling pathway is operating independent of auxin and ethylene responses. This study expands the knowledge on N form-explicit responses to demonstrate that ON of high molecular mass elicits specific responses.

A new species of Burkholderia isolated from sugarcane roots promotes plant growth.

Sugarcane is a globally important food, biofuel and biomaterials crop. High nitrogen (N) fertilizer rates aimed at increasing yield often result in environmental damage because of excess and inefficient application. Inoculation with diazotrophic bacteria is an attractive option for reducing N fertilizer needs. However, the efficacy of bacterial inoculants is variable, and their effective formulation remains a knowledge frontier. Here, we take a new approach to investigating diazotrophic bacteria associated with roots using culture-independent microbial community profiling of a commercial sugarcane variety (Q208(A) ) in a field setting. We first identified bacteria that were markedly enriched in the rhizosphere to guide isolation and then tested putative diazotrophs for the ability to colonize axenic sugarcane plantlets (Q208(A) ) and promote growth in suboptimal N supply. One isolate readily colonized roots, fixed N2 and stimulated growth of plantlets, and was classified as a new species, Burkholderia australis sp. nov. Draft genome sequencing of the isolate confirmed the presence of nitrogen fixation. We propose that culture-independent identification and isolation of bacteria that are enriched in rhizosphere and roots, followed by systematic testing and confirming their growth-promoting capacity, is a necessary step towards designing effective microbial inoculants.

Bacterial and plant ketol-acid reductoisomerases have different mechanisms of induced fit during the catalytic cycle.

Ketol-acid reductoisomerase (KARI) is the second enzyme in the branched-chain amino acid biosynthesis pathway, which is found in plants, fungi and bacteria but not in animals. This difference in metabolism between animals and microorganisms makes KARI an attractive target for the development of antimicrobial agents. Herein we report the crystal structure of Escherichia coli KARI in complex with Mg(2+) and NADPH at 2.3Å resolution. Ultracentrifugation studies confirm that the enzyme exists as a tetramer in solution, and isothermal titration calorimetry shows that the binding of Mg(2+) increases structural disorder while the binding of NADPH increases the structural rigidity of the enzyme. Comparison of the structure of the E. coli KARI-Mg(2+)-NADPH complex with that of enzyme in the absence of cofactors shows that the binding of Mg(2+) and NADPH opens the interface between the N- and C-domains, thereby allowing access for the substrates to bind: the existence of only a small opening between the domains in the crystal structure of the unliganded enzyme signifies restricted access to the active site. This observation contrasts with that in the plant enzyme, where the N-domain can rotate freely with respect to the C-domain until the binding of Mg(2+) and/or NADPH stabilizes the relative positions of these domains. Support is thereby provided for the idea that plant and bacterial KARIs have evolved different mechanisms of induced fit to prepare the active site for catalysis.

DNA uptake by Arabidopsis induces changes in the expression of CLE peptides which control root morphology.

The presence of externally supplied DNA in the growth medium enhances growth of lateral roots and root hairs in Arabidopsis. This phenomenon cannot be attributed to phosphorus (P) limitation because it is independent of the plants' P status. Rather, we hypothesized that DNA triggers a currently unknown signaling pathway. Analyzing the transcriptional changes of genes induced by externally supplied DNA, we show that 7 of the 17 studied CLAVATA3/ESR-related (CLEs) genes were differentially regulated. CLEs are known peptide hormones that affect plant development including root morphology. While previous research had shown that over-expression of these CLE genes alters root morphology, changes in gene expression had not been linked to environmental triggers. The differential expression of these CLEs genes and accompanied changes of the root phenotype are indicative of a DNA-elicited signal pathway which affects root development. We conclude that DNA acts as a signaling compound which induces root proliferation in a way that would enhance the plant's ability to acquire nutrients from soil organic matter. Our study further confirms the importance of CLEs for controlling root morphology in response to specific environmental conditions, and draws attention to a novel role of DNA as a signaling compound.

Uptake of non-pathogenic E. coli by Arabidopsis induces down-regulation of heat shock proteins.

We recently demonstrated that non-pathogenic and non-symbiotic microbes E. coli and yeast are taken up by roots and used as a source of nutrients by the plant. Although this process appears to be beneficial for the plant, the nutritional gain of microbe incorporation has to exceed the energy expense of microbe uptake and digestion, and the question remains whether the presence of microbes triggers pathogen- and other stress-induced responses. Here, we present evidence that digesting microbes is accompanied by strong down-regulation of genes linked to stress response in Arabidopsis. Genome-wide transcription analysis shows that uptake of E. coli by Arabidopsis roots is accompanied by a pronounced down-regulation of heat shock proteins. Plants up-regulate heat shock proteins in response to environmental stresses including temperature, salt, light and disease agents including microbial pathogens. The pronounced down-regulation of heat shock proteins in the presence of E. coli indicates that uptake and subsequent digestion of microbes does not induce stress. Additionally it suggests that resources devoted to stress resistance in control plants may be re-allocated to the process of microbe uptake and digestion. This observation adds evidences to the notion that uptake of microbes is an active, purposeful and intentional behavior of the plant.

Turning the table: plants consume microbes as a source of nutrients.

Interactions between plants and microbes in soil, the final frontier of ecology, determine the availability of nutrients to plants and thereby primary production of terrestrial ecosystems. Nutrient cycling in soils is considered a battle between autotrophs and heterotrophs in which the latter usually outcompete the former, although recent studies have questioned the unconditional reign of microbes on nutrient cycles and the plants' dependence on microbes for breakdown of organic matter. Here we present evidence indicative of a more active role of plants in nutrient cycling than currently considered. Using fluorescent-labeled non-pathogenic and non-symbiotic strains of a bacterium and a fungus (Escherichia coli and Saccharomyces cerevisiae, respectively), we demonstrate that microbes enter root cells and are subsequently digested to release nitrogen that is used in shoots. Extensive modifications of root cell walls, as substantiated by cell wall outgrowth and induction of genes encoding cell wall synthesizing, loosening and degrading enzymes, may facilitate the uptake of microbes into root cells. Our study provides further evidence that the autotrophy of plants has a heterotrophic constituent which could explain the presence of root-inhabiting microbes of unknown ecological function. Our discovery has implications for soil ecology and applications including future sustainable agriculture with efficient nutrient cycles.