Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata1

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a Km value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.

The Two Major Types of Plant Plasma Membrane H+-ATPases Show Different Enzymatic Properties and Confer Differential pH Sensitivity of Yeast Growth1

The proton-pumping ATPase (H+-ATPase) of the plant plasma membrane is encoded by two major gene subfamilies. To characterize individual H+-ATPases, PMA2, an H+-ATPase isoform of tobacco (Nicotiana plumbaginifolia), was expressed in Saccharomyces cerevisiae and found to functionally replace the yeast H+-ATPase if the external pH was kept above 5.0 (A. de Kerchove d'Exaerde, P. Supply, J.P. Dufour, P. Bogaerts, D. Thinès, A. Goffeau, M. Boutry [1995] J Biol Chem 270: 23828–23837). In the present study we replaced the yeast H+-ATPase with PMA4, an H+-ATPase isoform from the second subfamily. Yeast expressing PMA4 grew at a pH as low as 4.0. This was correlated with a higher acidification of the external medium and an approximately 50% increase of ATPase activity compared with PMA2. Although both PMA2 and PMA4 had a similar pH optimum (6.6–6.8), the profile was different on the alkaline side. At pH 7.2 PMA2 kept more than 80% of the maximal activity, whereas that of PMA4 decreased to less than 40%. Both enzymes were stimulated up to 3-fold by 100 μg/mL lysophosphatidylcholine, but this stimulation vanished at a higher concentration in PMA4. These data demonstrate functional differences between two plant H+-ATPases expressed in the same heterologous host. Characterization of two PMA4 mutants selected to allow yeast growth at pH 3.0 revealed that mutations within the carboxy-terminal region of PMA4 could still improve the enzyme, resulting in better growth of yeast cells.

Molecular Cloning and Expression Analysis of the Mitochondrial Pyruvate Dehydrogenase from Maize1

Four cDNAs, one encoding an α-subunit and three encoding β-subunits of the mitochondrial pyruvate dehydrogenase, were isolated from maize (Zea mays L.) libraries. The deduced amino acid sequences of both α- and β-subunits are approximately 80% identical with Arabidopsis and pea (Pisum sativum L.) homologs. The mature N terminus was determined for the β-subunit by microsequencing the protein purified from etiolated maize shoot mitochondria and was resolved by two-dimensional gel electrophoresis. This single isoelectric species comprised multiple isoforms. Both α- and β-subunits are encoded by multigene families in maize, as determined by Southern-blot analyses. RNA transcripts for both α- and β-subunits were more abundant in roots than in young leaves or etiolated shoots. Pyruvate dehydrogenase activity was also higher in roots (5-fold) compared with etiolated shoots and leaves. Both subunits were present at similar levels in all tissues examined, indicating coordinated gene regulation. The protein levels were highest in heterotrophic organs and in pollen, which contained about 2-fold more protein than any other organ examined. The relative abundance of these proteins in nonphotosynthetic tissues may reflect a high cellular content of mitochondria, a high level of respiratory activity, or an extra plastidial requirement for acetate.

Curvature Induced by Amyloplast Magnetophoresis in Protonemata of the Moss Ceratodon purpureus1

After gravistimulation of Ceratodon purpureus (Hedw.) Brid. protonemata in the dark, amyloplast sedimentation was followed by upward curvature in the wild-type (WT) and downward curvature in the wwr mutant (wrong way response). We used ponderomotive forces induced by high-gradient magnetic fields (HGMF) to simulate the effect of gravity and displace the presumptive statoliths. The field was applied by placing protonemata either between two permanent magnets at the edge of the gap, close to the edge of a magnetized ferromagnetic wedge, or close to a small (<1 mm) permanent magnet. Continuous application of an HGMF in all three configurations resulted in plastid displacement and induced curvature in tip cells of WT and wwr protonemata. WT cells curved toward the HGMF, and wwr cells curved away from the HGMF, comparable to gravitropism. Plastids isolated from protonemal cultures had densities ranging from 1.24 to 1.38 g cm−3. Plastid density was similar for both genotypes, but the mutant contained larger plastids than the WT. The size difference might explain the stronger response of the wwr protonemata to the HGMF. Our data support the plastid-based theory of gravitropic sensing and suggest that HGMF-induced ponderomotive forces can substitute for gravity.

Ectopic Expression of the Maize Homeobox Gene Liguleless3 Alters Cell Fates in the Leaf1

The semidominant mutation Liguleless3-O (Lg3-O) causes a blade-to-sheath transformation at the midrib region of the maize (Zea mays L.) leaf. We isolated a full-length lg3 cDNA containing a knotted1-like family homeobox. Six Lg3-O partial revertant alleles caused by insertion of a Mutator (Mu) transposon and two deletion derivatives were isolated and used to verify that our knotted1-like cDNA corresponds to the LG3 message. In wild-type plants the LG3 mRNA is expressed in apical regions but is not expressed in leaves. In mutant plants harboring any of three dominant lg3 alleles (Lg3-O, -Mlg, and -347), LG3 mRNA is expressed in leaf sheath tissue, indicating that the Lg3 phenotype is due to ectopic expression of the gene. The Lg3-O revertant alleles represent two classes of Lg3 phenotypes that correlate well with the level of ectopic Lg3 expression. High levels of ectopic LG3 mRNA expression results in a severe Lg3 phenotype, whereas weak ectopic Lg3 expression results in a mild Lg3 phenotype. We propose that ectopic Lg3 expression early in leaf development causes the blade-to-sheath transformation, but the level of expression determines the extent of the transformation.

ADP-Dependent Phosphorylation Regulates Association of a DNA-Binding Complex with the Barley Chloroplast psbD Blue-Light-Responsive Promoter1

The chloroplast gene psbD encodes D2, a chlorophyll-binding protein located in the photosystem II reaction center. Transcription of psbD in higher plants involves at least three promoters, one of which is regulated by blue light. The psbD blue-light-regulated promoter (BLRP) consists of a −10 promoter element and an activating complex, AGF, that binds immediately upstream of −35. A second sequence-specific DNA-binding complex, PGTF, binds upstream of AGF between −71 and −100 in the barley (Hordeum vulgare) psbD BLRP. In this study we report that ADP-dependent phosphorylation selectively inhibits the binding of PGTF to the barley psbD BLRP. ATP at high concentrations (1–5 mm) inhibits PGTF binding, but in the presence of phosphocreatine and phosphocreatine kinase, this capacity is lost, presumably due to scavenging of ADP. ADP inhibits PGTF binding at relatively low concentrations (0.1 mm), whereas other nucleotides are unable to mediate this response. ADP-mediated inhibition of PGTF binding is reduced in the presence of the protein kinase inhibitor K252a. This and other results suggest that ADP-dependent phosphorylation of PGTF (or some associated protein) inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles.

The Interaction between Cold and Light Controls the Expression of the Cold-Regulated Barley Gene cor14b and the Accumulation of the Corresponding Protein1

We report the expression of the barley (Hordeum vulgare L.) COR (cold-regulated) gene cor14b (formerly pt59) and the accumulation of its chloroplast-localized protein product. A polyclonal antibody raised against the cor14b-encoded protein detected two chloroplast COR proteins: COR14a and COR14b. N-terminal sequencing of COR14a and expression of cor14b in Arabidopsis plants showed that COR14a is not encoded by the cor14b sequence, but it shared homology with the wheat (Triticum aestivum L.) WCS19 COR protein. The expression of cor14b was strongly impaired in the barley albino mutant an, suggesting the involvement of a plastidial factor in the control of gene expression. Low-level accumulation of COR14b was induced by cold treatment in etiolated plants, although cor14b expression and protein accumulation were enhanced after a short light pulse. Light quality was a determining factor in regulating gene expression: red or blue but not far-red or green light pulses were able to promote COR14b accumulation in etiolated plants, suggesting that phytochrome and blue light photoreceptors may be involved in the control of cor14b gene expression. Maximum accumulation of COR14b was reached only when plants were grown and/or hardened under the standard photoperiod. The effect of light on the COR14b stability was demonstrated by using transgenic Arabidopsis. These plants constitutively expressed cor14b mRNAs regardless of temperature and light conditions; nevertheless, green plants accumulated about twice as much COR14b protein as etiolated plants.

Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots. II. Role of Cortical Microtubules and Cellulose Microfibrils1

We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101–111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized.

The Wheat Peptidyl Prolyl cis-trans-Isomerase FKBP77 Is Heat Induced and Developmentally Regulated1

We isolated a cDNA encoding a 568-amino acid, heat-stress-induced peptidyl prolyl isomerase belonging to the FK506-binding-protein (FKBP) family. The open reading frame encodes for a peptidyl prolyl isomerase that possesses three FKBP-12-like domains, a putative tetratricopeptide motif, and a calmodulin-binding domain. Specific antibodies showed that the open reading frame encodes a heat-induced 77-kD protein, the wheat FKBP77 (wFKBP77), which exhibits 84% identity with the wFKBP73 and 42% identity with the human FKBP59. Because of the high similarity in sequence to wFKBP73, wFKBP77 was designated as the heat-induced isoform. The wFKBP77 mRNA steady-state level was 14-fold higher at 37°C than at 25°C. The wFKBP77 transcript abundance was the highest in mature embryos that had imbibed and 2-d-old green shoots exposed to 37°C, and decreased to 6% in 6-d-old green shoots. The transcript level returned to the level detected at 25°C after recovery of the embryos for 90 min at 25°C. We compared wFKBP73 and wFKBP77 with the heat-shock proteins having cognate and heat-stress-induced counterparts.

Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.

Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1

A major goal of plant biotechnology is the production of genetically engineered crops that express natural or foreign proteins at high levels. To enhance protein accumulation in transgenic plants, we developed a set of vectors that express proteins and peptides as C-terminal translational fusions with ubiquitin (UBQ). Studies of several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins can be readily expressed in plants as UBQ fusions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused protein moieties in free forms; (c) the synthesis of a protein as a UBQ fusion can significantly augment its accumulation; (d) proper processing and localization of a protein targeted to either the apoplast or the chloroplast is not affected by the N-terminal UBQ sequence; and (e) single amino acid substitutions surrounding the cleavage site can inhibit in vivo processing of the fusion by Ubps. Noncleavable UBQ fusions of β-glucuronidase became extensively modified, with additional UBQs in planta. Because multiubiquitinated proteins are the preferred substrates of the 26S proteasome, noncleavable fusions may be useful for decreasing protein half-life. Based on their ability to augment protein accumulation and the sequence specificity of Ubps, UBQ fusions offer a versatile way to express plant proteins.

Characterization of N-Glycans from Arabidopsis. Application to a Fucose-Deficient Mutant1

The structures of glycans N-linked to Arabidopsis proteins have been fully identified. From immuno- and affinodetections on blots, chromatography, nuclear magnetic resonance, and glycosidase sequencing data, we show that Arabidopsis proteins are N-glycosylated by high-mannose-type N-glycans from Man5GlcNAc2 to Man9GlcNAc2, and by xylose- and fucose (Fuc)-containing oligosaccharides. However, complex biantenary structures containing the terminal Lewis a epitope recently reported in the literature (A.-C. Fitchette-Lainé, V. Gomord, M. Cabanes, J.-C. Michalski, M. Saint Macary, B. Foucher, B. Cavalier, C. Hawes, P. Lerouge, and L. Faye [1997] Plant J 12: 1411–1417) were not detected. A similar study was done on the Arabidopsis mur1 mutant, which is affected in the biosynthesis of l-Fuc. In this mutant, one-third of the Fuc residues of the xyloglucan has been reported to be replaced by l-galactose (Gal) (E. Zablackis, W.S. York, M. Pauly, S. Hantus, W.D. Reiter, C.C.S. Chapple, P. Albersheim, and A. Darvill [1996] Science 272: 1808–1810). N-linked glycans from the mutant were identified and their structures were compared with those isolated from the wild-type plants. In about 95% of all N-linked glycans from the mur1 plant, l-Fuc residues were absent and were not replaced by another monosaccharide. However, in the remaining 5%, l-Fuc was found to be replaced by a hexose residue. From nuclear magnetic resonance and mass spectrometry data of the mur1 N-glycans, and by analogy with data reported on mur1 xyloglucan, this subpopulation of N-linked glycans was proposed to be l-Gal-containing N-glycans resulting from the replacement of l-Fuc by l-Gal.

Manipulation of in Vivo Sorbitol Production Alters Boron Uptake and Transport in Tobacco1

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched 10B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of 10B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied 10B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.

Changes of Mitochondrial Properties in Maize Seedlings Associated with Selection for Germination at Low Temperature. Fatty Acid Composition, Cytochrome c Oxidase, and Adenine Nucleotide Translocase Activities1

Mitochondria are affected by low temperature during seedling establishment in maize (Zea mays L.). We evaluated the associated changes in the mitochondrial properties of populations selected for high (C4-H) and low (C4-L) germination levels at 9.5°C. When seedlings of the two populations were grown at 14°C (near the lower growth limit), the mitochondrial inner membranes of C4-H showed a higher percentage of 18-carbon unsaturated fatty acids, a higher fluidity, and a higher activity of cytochrome c oxidase. We found a positive relationship between these properties and the activity of a mitochondrial peroxidase, allowing C4-H to reduce lipid peroxidation relative to C4-L. The specific activity of reconstituted ATP/ADP translocase was positively associated with this peroxidase activity, suggesting that translocase activity is also affected by chilling. The level of oxidative stress and defense mechanisms are differently expressed in tolerant and susceptible populations when seedlings are grown at a temperature near the lower growth limit. Thus, the interaction between membrane lipids and cytochrome c oxidase seems to play a key role in maize chilling tolerance. Furthermore, the divergent-recurrent selection procedure apparently affects the allelic frequencies of genes controlling such an interaction.

Differential Expression of Three Members of the 1-Aminocyclopropane-1-Carboxylate Synthase Gene Family in Carnation1

We investigated the expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes in carnation (Dianthus caryophyllus cv White Sim) under conditions previously shown to induce ethylene biosynthesis. These included treatment of flowers with 2,4-dichlorophenoxyacetic acid, ethylene, LiCl, cycloheximide, and natural and pollination-induced flower senescence. Accumulation of ACC synthase transcripts in leaves following mechanical wounding and treatment with 2,4-dichlorophenoxyacetic acid or LiCl was also determined by RNA gel-blot analysis. As in other species, the carnation ACC synthase genes were found to be differentially regulated in a tissue-specific manner. DCACS2 and DCACS3 were preferentially expressed in styles, whereas DCACS1 mRNA was most abundant in petals. Cycloheximide did not induce increased accumulation of ACC synthase transcripts in carnation flowers, whereas the expression of ACC synthase was up-regulated by auxin, ethylene, LiCl, pollination, and senescence in a floral-organ-specific manner. Expression of the three ACC synthases identified in carnation did not correspond to elevated ethylene biosynthesis from wounded or auxin-treated leaves, and there are likely additional members of the carnation ACC synthase gene family responsible for ACC synthase expression in vegetative tissues.