Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanold synthesis. The role of PAL In pathway regulation was investigated by measurement of product accumulation as a function of enzyme activity in a coilection of near-isogenic transgenic tobacco plants exhibiting a range of PAL levels from wild type to 0.2% of wild type. In leaf tissue, PAL level is the dominant factor regulating accumulation ofthe major product chlorogenic acid and overall flux into the pathway. In stems, PAL at wild-type levels contributes, together with downstream steps, in the regulation of lguin deposition and becomes the dominant, rate-determining step at levels 3-to 4-fold below wild type. The metabolic impact of elevated PAL levels was investigated in transgenic leaf callus that overexpressed PAL. Accumulation of the flavonoid rutin, the major product in wild-type callus, was not increased, but several other products accumulated to similarly high levels. These data indicate that PAL is a key step in the regulation of overall flux into the pathway and, hence, accumulation of major phenylpropanoid products, with the regulatory architecture of the pathway poised so that downstream steps control partitioning into different branch pathways.

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry. Similar reduction of lignin levels by down-regulation of i-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.There is currently intense interest in modifying the content and / or composition of the cell wall structural polymer lignin as a means of improving the efficiency of the paper pulping process for forest trees or of increasing digestibility of forages for ruminant animals (Whetten and Sederoff, 1991; Boudet and Grima-Pettenati, 1996; Campbell and Sederoff, 1996).Recent studies have concentrated on attempts to downregulate the levels of enzymes involved in the reactions specific for lignin monomer synthesis by expression of homologous or heterologous antisense genes in transgenic plants (Dwivedi et al., 1994;Halpin et al., 1994;Ni et al., 1994; Atanassova et al., 1995;Van Doorsselaere et al., 1995;Sewalt et al., 1997). Although the biosynthetic pathway to lignin monomers is relatively well understood, involving consecutive hydroxylation and O-methylation reactions leading from p-coumaric acid via ferulic acid (the monomethoxylated precursor of the G residues of lignin) to sinapic acid (the dimethoxylated precursor of the S residues of lignin), it has recently been suggested that parallel pathways of monomer hydroxylation and methylation could occur at the level of the COA thioesters (Ye et al., 1994) or even at the level of the aldehydes formed after the first reduction of the COA thioesters (Matsui et al., 1994; Fig. 1).The existence of a metabolic grid for the O-methylation of monolignols would complicate the interpretation of experiments in which a single enzyme of the pathway was down-regulated. Indeed, severa1 reports of the effects of antisense inhibition of enzymes involved in the late reactions of monolignol biosynthesis have presented unpredicted and sometimes contradictory results. Ni et al. (1994) reported that modest down-regulation of COMT activity in transgenic tobacco (Nicofiana fabacum) leads to a small reduction in lignin content with no significant change in lignin composition. However, other groups have shown that strong down-regulation of COMT in tobacco or poplar (Populu...

It has been proposed that natural products synthesized by plants contribute to their resistance to pests and pathogens. We show here that transgenic tobacco plants with suppressed levels of the phenylpropanoid biosynthetic enzyme phenylalanine ammonia-lyase (L-phenylalanine ammonialyase, EC 4.3

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In higher plants, three subfamilies of sucrose nonfermenting-1 (Snf1)-related protein kinases have evolved. While the Snf1-related protein kinase 1 (SnRK1) subfamily has been shown to share pivotal roles with the orthologous yeast Snf1 and mammalian AMP-activated protein kinase in modulating energy and metabolic homeostasis, the functional significance of the two plant-specific subfamilies SnRK2 and SnRK3 in these critical processes is poorly understood. We show here that SnRK2.6, previously identified as crucial in the control of stomatal aperture by abscisic acid (ABA), has a broad expression pattern and participates in the regulation of plant primary metabolism. Inactivation of this gene reduced oil synthesis in Arabidopsis (Arabidopsis thaliana) seeds, whereas its overexpression increased Suc synthesis and fatty acid desaturation in the leaves. Notably, the metabolic alterations in the SnRK2.6 overexpressors were accompanied by amelioration of those physiological processes that require high levels of carbon and energy input, such as plant growth and seed production. However, the mechanisms underlying these functionalities could not be solely attributed to the role of SnRK2.6 as a positive regulator of ABA signaling, although we demonstrate that this kinase confers ABA hypersensitivity during seedling growth. Collectively, our results suggest that SnRK2.6 mediates hormonal and metabolic regulation of plant growth and development and that, besides the SnRK1 kinases, SnRK2.6 is also implicated in the regulation of metabolic homeostasis in plants.Plants are constantly confronted by biotic and abiotic stresses and nutrient deprivation that disrupt metabolic and energy homeostasis or diminish carbon and energy availability for maintaining cell vitality, growth, and proliferation. It is believed that maintaining energy balance and availability at the cellular and organism levels is critical for optimizing plant growth and development. This underscores the cellular and physiological importance of energy sensors that control energy balance through regulating fundamental metabolic pathways in response to nutritional and environmental stresses.At present, a prevailing view is that energy sensors are evolutionarily conserved in eukaryotes, which are represented by Snf1 (for sucrose nonfermenting-1) in yeast, AMPK (for AMP-activated protein kinase) in

Lignin is a major structural polymer of secondarily thickened plant vascular tissue and fibres, imparting mechanical strength to stems and trunks and hydrophobicity to conducting vessels. Constitutive expression of a lucerne caffeic acid 3-O-methyltransferase antisense RNA in transgenic tobacco leads to a significant reduction in lignin content, particularly in the younger parts of the stems, without apparent alterations in lignin monomer composition. These observations open up the possibility of genetically manipulating plants with reduced lignin for improved processing and biomass digestibility.

Cell wall digestibility, lignin content, and lignin composition were analyzed in transgenic tobacco altered in the expression of the phenylpropanoid biosynthetic enzymes caffeic acid 3-O-methyltransferase (COMT) and L-phenylalanine ammonia-lyase (PAL). Reduction of COMT activity by antisense technology resulted in reduced lignin content accompanied by an increased syringyl (S)/ guaiacyl (G) monomer ratio, as determined by pyrolysis/GC/MS and measurement of lignin methoxyl content by wet chemistry. These results resemble those obtained by reduction of flux of lignin precursors into the phenylpropanoid pathway by PAL suppression, which results in drastically reduced lignin with sharply increased methoxyl content. Enzymatic digestibility of cell walls from stem internodes was improved in the transgenic lines and was highly negatively correlated with lignin concentration (r ) -0.97). Although lignin composition was also affected, lignin concentration was the overriding factor influencing cell wall digestibility. The results provide a basis for new strategies for lignin modification to improve digestibility of forages.

Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ϳ1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ϳ30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.Xenorhabdus and Photorhabdus spp. are two bacterial genera belonging to the family Enterobacteriaceae, known to be associated with entomopathogenic nematodes (1-4) These bacteria represent potential sources for new genes encoding potent insecticidal toxins that could be put into plants as alternatives to Bacillus thuringiensis genes (5). Gene sequence analysis of Xenorhabdus and Photorhabdus bacteria show that these organisms contain a family of related toxin complex (tc) 2 genes located at different loci (6 -9). The toxin complexes are composed of three different classes of protein components, which, according to ffrench-Constant et al. (10,11), can be categorized as class A, B, and C proteins based upon sequence similarity and size. Class A proteins are very large, having a molecular mass of ϳ280 kDa. Class B proteins are ϳ170 kDa, and class C proteins are ϳ110 kDa. There are many different varieties of class A, B, and C proteins in both Gram-negative and Gram-positive bacteria (12-15).From earlier studies, it has been suggested that class A proteins harbor the cytotoxic effects of the Tc toxins, whereas class B and C proteins modulate and enhance the toxicity of class A proteins (16). However, recently, we elucidated the molecular mechanism of the Photorhabdus luminescens Tc complex, which consists of the class A protein TcdA1, the class B protein TcdB2, and the class C protein TccC3 or TccC5 (17). These studies revealed that the class C proteins harbor the biological activity. It was shown that TccC3 and TccC5 are ADP-ribosyltransferases, which target the actin cytoskeleton by modification of actin and Rho GTPases, respectively (17). Moreover, these studies su...