Wilbur H. Campbell

MODIFICATION OF LIGNIN BIOSYNTHESIS IN TRANSGENIC NICOTIANA THROUGH EXPRESSION OF AN ANTISENSE O-METHYLTRANSFERASE GENE FROM POPULUS

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-l-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.

Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation

Spinach (Spinacia oleracea L.) leaf nitrate reductase (NADH:NR;NADH:nitrate oxidoreductase, EC 1.6.6.1) activity was found to rapidly change during light/dark transitions. The most rapid and dramatic changes were found in a form of NR which was sensitive to inhibition by millimolar concentrations of magnesium. This form of NR predominated in leaves in the dark, but was almost completely absent from leaves incubated in the light for only 30 min. When the leaves were returned to darkness, the NR rapidly became sensitive to Mg2+ inhibition. Modulation of the overall reaction involving NADH as electron donor was also found when reduced methyl viologen was the donor (MV:NR), indicating that electron transfer had been blocked, at least in part, at or near the terminal molybdenum cofactor site. Changes in activity appear to be the result of a covalent modification that affects sensitivity of NR to inhibition by magnesium, and our results suggest that protein phosphorylation may be involved. NR was phosphorylated in vivo after feeding excised leaves [32P]Pi. The NR subunit was labeled exclusively on seryl residues in both light and dark. Tryptic peptide mapping indicated three major 32P-labeled phosphopeptide (Pp) fragments. Labeling of two of the P-peptides (designated Pp1 and 3) was generally correlated with NR activity assayed in the presence of Mg2+. In vivo, partial dephosphorylation of these sites (and activation of NR assayed with Mg2+) occurred in response to light or feeding mannose in darkness. The light effect was blocked completely by feeding okadaic acid via the transpiration stream, indicating the involvement of type 1 and/or type 2A protein phosphatases in vivo. While more detailed analysis is required to establish a causal link between the phosphorylation status of NR and sensitivity to Mg2+ inhibition, the current results are highly suggestive of one. Thus, in addition to the molecular genetic mechanisms regulating this key enzyme of nitrate assimilation, NR activity may be controlled in leaves by phosphorylation/dephosphorylation of the enzyme protein resulting from metabolic changes taking place during light/dark transitions.

Functional domains of assimilatory nitrate reductases and nitrite reductases

Biochemical investigation of nitrate assimilation enzymes spans the past four decades. With the molecular cloning of genes for nitrate reductases and nitrite reductases, exciting new prospects are developing for the study of these enzymes. As large, complex enzymes with multiple redox centers, these two types of reductases should help us gain understanding of structural, functional and evolutionary relationships among the diverse group of multicenter redox enzymes.

cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen

A cDNA clone (Ptomt1) encoding a lignin-bispecific O-methyltransferase (OMT) was isolated by immunological screening of a λgt11 expression library prepared from mRNA of developing secondary xylem of aspen (Populus tremuloides). Nucleotide sequence analysis of Ptomt1 revealed an open reading frame of 1095 bp which encodes a polypeptide with a predicted molecular weight of 39 802, corresponding well with the size of the OMT polypeptide estimated by SDS-PAGE. Authenticity of Ptomt1 was demonstrated in part by detection of OMT activity and protein in extracts of Escherichia coli cultures transformed with a plasmid construct containing Ptomt1. In addition, peptides produced from a proteolytic digest of purified OMT and sequenced by automated Edman degradation matched to portions of the deduced amino acid sequence of Ptomt1. Comparison of this sequence to amino acid sequences of OMTs of diverse species identified regions of similarity which probably contribute to the binding site of S-adenosyl-L-methionine. Tissue-specific expression was demonstrated by northern analysis which showed that Ptomt1 hybridized to a 1.7 kb transcript from aspen developing secondary xylem and by tissue printing of aspen stems in which only the outer layer of xylem bound the antibody. A biphasic pattern of gene expression and enzyme activity for OMT was observed from xylem samples of aspen during the growing season which suggests linkage between gene expression for a monolignol biosynthetic enzyme and seasonal regulation of xylem differentiation in woody plants.

Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site

Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 Å. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis.

Glutamine Synthetase and Ferredoxin-Dependent Glutamate Synthase Expression in the Maize (Zea mays) Root Primary Response to Nitrate (Evidence for an Organ-Specific Response).

To define further the early, or primary, events that occur in maize (Zea mays) seedlings exposed to NO3-, accumulation of chloroplast glutamine synthetase (GS2; EC 6.3.1.2) and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), transcripts were examined in roots and leaves. In roots, NO3- treatment caused a rapid (within 30 min), transient, and cycloheximide-independent accumulation of GS2 and Fd-GOGAT transcripts. In addition, 10 [mu]M external NO3- was sufficient to cause transcript accumulation. The induction was NO3- specific, since NH4Cl treatment did not affect mRNA levels. GS2 and Fd-GOGAT mRNA accumulation in roots was similar to that observed for nitrate reductase (NR) mRNA. Therefore, the four genes involved in NO3- assimilation (NR, nitrite reductase, GS2, and Fd-GOGAT) are expressed in the root primary response to NO3-, suggesting that all four genes can respond to the same signal transduction system. In contrast, relatively high levels of GS2 and Fd-GOGAT mRNAs were present in untreated leaf tissue, and NO3- treatment had little or no influence on transcript accumulation. Rapid, transient, and cycloheximide-independent NR mRNA expression was seen in the NO3--treated leaves, demonstrating that NO3- was not limiting. The NO3--independent constitutive expression of GS2 and Fd-GOGAT is likely due to the requirement for reassimilation of photorespiratory NH4+ in these young leaves.

Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5å resolution: relationship to other flavoprotein reductases

BACKGROUND In the biological assimilation of nitrate in plants and microorganisms, nitrate is reduced to ammonium by transfer of eight electrons in a two-step process. The first step of the pathway, the reduction of nitrate to nitrite, is catalyzed by nitrate reductase, a multi-redox cofactor enzyme which belongs to the class of flavoprotein pyridine nucleotide cytochrome reductases. The enzyme can be divided into three functional fragments that bind the cofactors molybdopterin, heme-iron and flavin adenine dinucleotide (FAD)/nicotinamide adenine dinucleotide (NADH). RESULTS Here we describe the crystal structure of the recombinant cytochrome b reductase fragment of corn nitrate reductase, in complex with the cofactor FAD, determined to 2.5 A resolution. This catalytically competent fragment of nitrate reductase consists of two domains, the amino-terminal lobe, which binds FAD, and the carboxy-terminal lobe, which presumably binds NADH, connected by a linker region. CONCLUSIONS Nitrate reductase belongs to the class of flavoprotein pyridine nucleotide cytochrome reductases, a subgroup in the family of ferredoxin reductase-like flavoproteins. Comparison with other members of this family reveals that large structural differences are found in the relative orientation of the cofactor binding lobes. This indicates that conformational changes might be important for biological function.

Nitrate reductase transcript is expressed in the primary response of maize to environmental nitrate

The nitrate induction of NADH:nitrate reductase mRNA in maize roots, scutella and leaves was investigated in the presence and absence of inhibitors of protein synthesis. In the absence of inhibitors, nitrate treatment caused a fairly rapid (2 to 3 h) increase in the level of the nitrate reductase transcript in all tissues. When cytoplasmic protein synthesis was inhibited by cycloheximide, nitrate reductase mRNA was induced by nitrate in all tissues to levels equal to or greater than those found with nitrate treatment alone. Treatment of maize tissues with cycloheximide in the absence of nitrate had only a small effect on the accumulation of the nitrate reductase mRNA. Inhibition of organellar protein synthesis with chloramphenicol also had little or no effect on nitrate-induced nitrate reductase mRNA accumuiation in roots and scutella, but did appear to partially inhibit appearance of transcript in leaves. Excision of scutella in the absence of nitrate was sufficient to cause some accumulation of the nitrate reductase transcript. Since cytoplasmic protein synthesis was not required for expression of nitrate reductase transcripts, induction of these transcripts by nitrate is a primary response of maize to this environmental signal. Thus, it appears that the signal transduction system mediating this response is constitutively expressed in roots, scutella and leaves of maize.

Regulation of Maize Leaf Nitrate Reductase Activity Involves Both Gene Expression and Protein Phosphorylation

Nitrate reductase (NR; EC 1.6.6.1) activity increased at the beginning of the photoperiod in mature green maize (Zea mays L.) leaves as a result of increased enzyme protein level and protein dephosphorylation. In vitro experiments suggested that phosphorylation of maize leaf NR affected sensitivity to Mg2+ inhibition, as shown previously in spinach. When excised leaves were fed 32P-labeled inorganic phosphate, NR was phosphorylated on seryl residues in both the light and dark. Tryptic peptide mapping of NR labeled in vivo indicated three major 32P-phosphopeptide fragments, and labeling of all three was reduced when leaves were illuminated. Maize leaf NR mRNA levels that were low at the end of the dark period peaked within 2 h in the light and decreased thereafter, and NR activity generally remained high. It appears that light signals, rather than an endogenous rhythm, account primarily for diurnal variations in NR mRNA levels. Overall, regulation of NR activity in mature maize leaves in response to light signals appears to involve control of gene expression, enzyme protein synthesis, and reversible protein phosphorylation.

Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development

Seasonal expression of caffeoyl-CoA O-methyltransferase (EC 2.1.1.104) was analyzed in aspen developing secondary xylem in parallel with caffeate O-methyltransferase (EC 2.1.1.68). Enzyme activity and mRNA levels for both enzymes peaked in the middle of the growing season. These results strongly suggest that both forms of O-methyltransferase were actively participating in lignin precursor biosynthesis during the growing season. To determine the role of each enzyme form, xylem extracts from two days in the growing season were assayed with four substrates: caffeoyl-CoA, 5-hydroxyferuloyl-CoA, caffeate acid and 5-hydroxyferulic acid. Recombinant forms of caffeoyl-CoA and caffeate O-methyltransferase were also assayed with these substrates. The recombinant enzymes have different substrate specificity with the caffeoyl-CoA O-methyltransferase being essentially specific for CoA ester substrates with a preference for caffeoyl-CoA, while caffeate O-methyltransferase utilized all four substrates with a preference for the free acid forms. We suggest that caffeoyl-CoA O-methyltransferase is likely to be responsible for biosynthesis of lignin precursors in the guaiacyl pathway and may represent a more primitive enzyme form leftover from very early land plant evolution. Caffeate O-methyltransferase is more likely to be responsible for lignin precursor biosynthesis in the syringyl pathway, especially since it can catalyze methylation of 5-hydroxyferuloyl-CoA quite effectively. This latter enzyme form then may be considered a more recently evolved component of the lignin biosynthetic pathways of the evolutionarily advanced plants such as angiosperms.