No abstract

A cDNA clone (Ptomt 1) encoding a lignin-bispecific O-methyltransferase (OMT) was isolated by immunological screening of a lambda gt11 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.

Violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the addition and removal of epoxide groups in carotenoids of the xanthophyll cycle in plants. The xanthophyll cycle is implicated in protecting the photosynthetic apparatus from excessive light. Two new sequences for violaxanthin de-epoxidase from tobacco and Arabidopsis are described. Although the mature proteins are well conserved, the transit peptides of these proteins are divergent, in contrast to transit peptides from other proteins targeted to the thylakoid lumen. Sequence analyses of both violaxanthin de-epoxidase and zeaxanthin epoxidase establish the xanthophyll cycle enzymes as members of the lipocalin family of proteins. The lipocalin family is a diverse group of proteins that bind small hydrophobic (lipophilic) molecules and share a conserved tertiary structure of eight ␤-strands forming a barrel configuration. This is the first reported identification of lipocalin proteins in plants.The xanthophyll cycle is comprised of de-epoxidation and epoxidation interconversions of three xanthophylls (violaxanthin, antheraxanthin, and zeaxanthin), catalyzed by two enzymes that are localized on opposite sides of the thylakoid membrane. Violaxanthin de-epoxidase (VDE) 1 is localized in the lumen of thylakoids and catalyzes de-epoxidation of violaxanthin in the presence of ascorbate and an acidic lumen, the latter formed by the light-induced proton pump (1-5). Zeaxanthin epoxidase is localized on the stromal side of the thylakoid membrane and catalyzes the epoxidation of zeaxanthin in the dark or under low light intensities (6, 7). The reaction is optimal near pH 7.5, and the enzyme utilizes oxygen, ferredoxin, and FAD as co-substrates (6 -10). A role for zeaxanthin was first described in 1987 by Demmig et al. (11) to protect the photosynthetic apparatus against the adverse effects of excessive light. Recent evidence demonstrates that accumulation of both antheraxanthin and zeaxanthin, along with the transthylakoid pH gradient, mediates the non-radiative dissipation of excess energy as heat (12)(13)(14)(15)(16)(17). The xanthophyll cycle is thought to have evolved early in the development of higher plants as it is present in all plants examined thus far (18). Pervaiz and Brew (19,20) first identified a group of proteins, based on sequence homology, that have a common role in binding and transport of small hydrophobic molecules. These proteins, designated the lipocalins, represent a diverse group of proteins from the animal kingdom (for review see Ref. 21) and recently from a prokaryote (22). These lipocalin proteins have a common tertiary structure of an eight-stranded anti-parallel ␤-barrel, and only one protein to date displays catalytic activity. We report that violaxanthin de-epoxidase and zeaxanthin epoxidase are members of the lipocalin family. To our knowledge, they are the first lipocalins described from plants and only the second reported to demonstrate enzymatic activity. The deduced polypeptide sequences of three VDE proteins are compared, a...

Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid ApH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.Light is essential for photosynthesis but potentially can damage the photosynthetic apparatus when the intensity exceeds photosynthetic capacity. Plants have evolved various mechanisms to cope with the excess-light conditions that, in fact, are experienced by most plants in natural environments. In higher plants, a highly conserved feedback mechanism, the zeaxanthin and antheraxanthin-dependent dissipation of energy in the light-harvesting complexes of photosystem II (PSII), is now generally accepted as a system that protects PSII by dynamically modulating energy transfer from the antennae to the reaction center, thus regulating the quantum efficiency of PSII (1-6). There is clear evidence from the effects of dark ATP-hydrolysis-induced acidification of thylakoids that this system is "energy dependent" and obviously independent of electron transport or associated redox changes induced by light (7). Under such conditions, a short-lifetime fluorescence component or putative quenching complex is formed (8).Zeaxanthin and antheraxanthin are formed from violaxanthin by violaxanthin de-epoxidase (VDE) activity in the socalled xanthophyll or violaxanthin cycle (9). De-epoxidationdependent energy dissipation is capable of responding to light environments over a wide dynamic range from sun-flecks to long-term growth conditions mainly due to the properties of the xanthophyll cycle (10, 11). The response range apparently gives higher plants the ability to adapt photosynthetically to diverse environments and, for this reason, may have be...

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.