R. R. Mendel

New GATEWAY vectors for high throughput analyses of protein-protein interactions by bimolecular fluorescence complementation.

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N- or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.

The role of abscisic acid and auxin in the response of poplar to abiotic stress

The plant hormones auxin and abscisic acid may at first sight appear to be a conflicting pair of plant regulators. Abscisic acid content increases during stress and protects plant water status. The content of free auxin in the developing xylem of poplar declines during stress, while auxin conjugates increase. This indicates that specific down-regulation of a signal transduction chain is important in plant adaptation to stress. Diminished auxin content may be a factor that adapts growth and wood development of poplar during adverse environmental conditions. To allow integration of environmental signals, abscisic acid and auxin must interact. Data are accumulating that abscisic acid-auxin cross-talk exists in plants. However, knowledge of the role of plant hormones in the response of trees to stress is scarce. Our data show that differences in the localisation of ABA synthesis exist between the annual, herbaceous plant Arabidopsis and the perennial woody species, poplar.

Identification of an Arabidopsis solute carrier critical for intracellular transport and inter-organ allocation of molybdate.

Plants represent an important source of molybdenum in the human diet. Recently, MOT1 has been identified as a transport protein responsible for molybdate import in Arabidopsis thaliana L.; however, the function of the homologous protein MOT2 has not been resolved. Interestingly, MOT2-GFP analysis indicated a vacuolar location of this carrier protein. By site directed mutagenesis at the N-terminal end of MOT2, we identified a di-leucine motif that is essential for driving the protein into the vacuolar membrane. Molybdate quantification in isolated vacuoles showed that this organelle serves as an important molybdate store in Arabidopsis cells. When grown on soil, leaves from mot2 T-DNA mutants contained more molybdate, whereas mot2 seeds contained significantly less molybdate than corresponding wild-type (Wt) tissues. Remarkably, MOT2 mRNA accumulates in senescing leaves and mot2 leaves from plants that had finished their life cycle had 15-fold higher molybdate levels than Wt leaves. Reintroduction of the endogenous MOT2 gene led to a Wt molybdate phenotype. Thus, mot2 mutants exhibit impaired inter-organ molybdate allocation. As total concentrations of the molybdenum cofactor (Moco) and its precursor MPT correlates with leaf molybdate levels, we present novel evidence for an adjustment of Moco biosynthesis in response to cellular MoO₄²⁻ levels. We conclude that MOT2 is important for vacuolar molybdate export, an N-terminal di-leucine motif is critical for correct subcellular localisation of MOT2 and activity of this carrier is required for accumulation of molybdate in Arabidopsis seeds. MOT2 is a novel element in inter-organ translocation of an essential metal ion.

The role of nitrate reduction in the anoxic metabolism of roots. I. Characterization of root morphology and normoxic metabolism of wild type tobacco and a transformant lacking root nitrate reductase

Two tobacco lines with (Nicotiana tabacum cv. Gatersleben, WT) or without (transformant LNR-H) nitrate reductase in roots were chosen as model systems to re-evaluate the role of root nitrate reduction for survival of anoxia. In this first paper, the two hydroponically grown lines were compared with respect to their root morphology, root respiration and the root content of inorganic cations, anions, and metabolites. Leaf transpiration in relation to root morphology was also determined. In comparison to WT roots containing NR, the NR-free LNR-H transformants had slightly shorter and thicker roots with a lower root surface area per g leaf FW. Consistent with that, LNR-H leaves had lower transpiration rates than WT. LNR-H-roots also showed consistently higher respiration and higher contents of ATP, starch and hexose monophosphates than WT roots. Concentrations of free sugars were only slightly higher in LNR-H roots. Total soluble protein content was identical in both lines, whereas amino acids were higher in LNR-H. Contents of major inorganic cations and anions were also almost identical in both lines. We conclude that WT versus LNR-H plants are a suitable tool to re-evaluate the role of nitrate reduction in flooding tolerance.

Visualization and quantification of protein interactions in the biosynthetic pathway of molybdenum cofactor in Arabidopsis thaliana

The molybdenum cofactor (Moco) is the active compound at the catalytic site of molybdenum enzymes. Moco is synthesized by a conserved four-step pathway involving six proteins in Arabidopsis thaliana. Bimolecular fluorescence complementation was used to study the subcellular localization and interaction of those proteins catalysing Moco biosynthesis. In addition, the independent split-luciferase approach permitted quantification of the strength of these protein–protein interactions in vivo. Moco biosynthesis starts in mitochondria where two proteins undergo tight interaction. All subsequent steps were found to proceed in the cytosol. Here, the heterotetrameric enzyme molybdopterin synthase (catalysing step two of Moco biosynthesis) and the enzyme molybdenum insertase, which finalizes Moco formation, were found to undergo tight protein interaction as well. This cytosolic multimeric protein complex is dynamic as the small subunits of molybdopterin synthase are known to go on and off in order to become recharged with sulphur. These small subunits undergo a tighter protein contact within the enzyme molybdopterin synthase as compared with their interaction with the sulphurating enzyme. The forces of each of these protein contacts were quantified and provided interaction factors. To confirm the results, in vitro experiments using a technique combining cross-linking and label transfer were conducted. The data presented allowed the outline of the first draft of an interaction matrix for proteins within the pathway of Moco biosynthesis where product–substrate flow is facilitated through micro-compartmentalization in a cytosolic protein complex. The protected sequestering of fragile intermediates and formation of the final product are achieved through a series of direct protein interactions of variable strength.

Nonsynonymous SNPs in MARC1 and MARC2 in Healthy Caucasian and In Vitro Characterization of the Encoded Protein Variants

Human molybdenum-containing enzyme mitochondrial amidoxime reducing component (mARC), cytochrome b5 type B, and NADH cytochrome b5 reductase form an N-reductive enzyme system that is capable of reducing N-hydroxylated compounds. Genetic variations are known, but their functional relevance is unclear. Our study aimed to investigate the incidence of nonsynonymous single nucleotide polymorphisms (SNPs) in the mARC genes in healthy Caucasian volunteers, to determine saturation of the protein variants with molybdenum cofactor (Moco), and to characterize the kinetic behavior of the protein variants by in vitro biotransformation studies. Genotype frequencies of six SNPs in the mARC genes (c.493A>G, c.560T>A, c.736T>A, and c.739G>C in MARC1; c.730G>A and c.735T>G in MARC2) were determined by pyrosequencing in a cohort of 340 healthy Caucasians. Protein variants were expressed in Escherichia coli. Saturation with Moco was determined by measurement of molybdenum by inductively coupled mass spectrometry. Steady state assays were performed with benzamidoxime. The six variants were of low frequency in this Caucasian population. Only one homozygous variant (c.493A; MARC1) was detected. All protein variants were able to bind Moco. Steady state assays showed statistically significant decreases of catalytic efficiency values for the mARC-2 wild type compared with the mARC-1 wild type (P < 0.05) and for two mARC-2 variants compared with the mARC-2 wild type (G244S, P < 0.05; C245W, P < 0.05). After simultaneous substitution of more than two amino acids in the mARC-1 protein, N-reductive activity was decreased 5-fold. One homozygous variant of MARC1 was detected in our sample. The encoded protein variant (A165T) showed no different kinetic parameters in the N-reduction of benzamidoxime.

Sexual Transfer of Transgene (Bar) into Non-Transformed Wheat Genotypes and Cell-Level Selection of the Marker Gene in Microspore- and Anther Culture

Fertile transgenic wheat (Triticum aestivum L.) plants have been produced during the past few years (Vasil et. al 1993, Weeks et al. 1993, Becker et al., Nehra et. al 1994). At the end of 20th century the fundamental discoveries made in the field of molecular biology and cell and tissue culture have initiated a biological revolution in plant breeding and agricultural production. The first genetically engineered food crops have been introduced into the market in some countries (Nehra et al 1995), but several questions have been under discussion. The fate of transgene is an up-to-date question in plant genetic and breeding research.