The phenylpropanoid pathway is responsible for the synthesis of a large range of natural products in plants, including flavonoids (pigments and UV protectants), the structural polymer lignin, and antimicrobial furanocoumarin and isoflavonoid phytoalexins (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995). Salicylic acid, which is involved in the establishment of both local and systemic plant defense responses, is also a product of this pathway (Klessig and Malamy, 1994). Although the importance of phenylpropanoid natural products makes the pathway an obvious target for plant improvement by metabolic engineering, little is known about the control of flux into the various branches of the pathway. Many phenylpropanoid

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...

PTL within the newly arising sepals is apparently prevented by the PINOID auxin-response gene. Surprisingly, PTL expression could not be detected in petals during the early stages of their development, so petal defects associated with PTL loss of function may be indirect, perhaps involving disruption to signalling processes caused by overgrowth in the region. PTL-driven reporter gene expression was also detected at later stages in the margins of expanding sepals, petals and stamens, and in the leaf margins; thus, PTL may redundantly dampen lateral outgrowth of these organs, helping define their final shape.

L6 is a nucleotide binding site-leucine rich repeat (NBS-LRR) gene that confers race-specific resistance in flax (Linum usitatissimum) to strains of flax rust (Melampsora lini) that carry avirulence alleles of the AvrL567 gene but not to rust strains that carry only the virulence allele. Several mutant and recombinant forms of L6 were made that altered either the methionine-histidine-aspartate (MHD) motif conserved in the NBS domain of resistance proteins or exchanged the short domain C-terminal to the LRR region that is highly variable among L allele products. In transgenic flax some of these alleles are autoactive; they cause a gene dosage-dependent dwarf phenotype and constitutive expression of genes that are markers for the plant defense response. Their effects and penetrance ranged from extreme to mild in their degree of plant stunting, survival, and reproduction. Dwarf plants were also resistant to flax rust strains virulent to wild-type L6 plants, and this nonspecific resistance was associated with a hypersensitive response (HR) at the site of rust infection. The strongest autoactive allele, expressed in Arabidopsis from an ethanol-inducible promoter, gave rise to plant death dependent on the enhanced disease susceptibility 1 (EDS1) gene, which indicates that the mutant flax (Linaceae) L6 gene can signal cell death through a defined disease-resistance pathway in a different plant family (Brassicaceae).

Dynamin-related proteins are large GTPases that deform and cause fission of membranes. The DRP1 family of Arabidopsis thaliana has five members of which DRP1A, DRP1C, and DRP1E are widely expressed. Likely functions of DRP1A were identified by studying rsw9, a null mutant of the Columbia ecotype that grows continuously but with altered morphology. Mutant roots and hypocotyls are short and swollen, features plausibly originating in their cellulose-deficient walls. The reduction in cellulose is specific since non-cellulosic polysaccharides in rsw9 have more arabinose, xylose, and galactose than those in wild type. Cell plates in rsw9 roots lack DRP1A but still retain DRP1E. Abnormally placed and often incomplete cell walls are preceded by abnormally curved cell plates. Notwithstanding these division abnormalities, roots and stems add new cells at wild-type rates and organ elongation slows because rsw9 cells do not grow as long as wild-type cells. Absence of DRP1A reduces endocytotic uptake of FM4-64 into the cytoplasm of root cells and the hypersensitivity of elongation and radial swelling in rsw9 to the trafficking inhibitor monensin suggests that impaired endocytosis may contribute to the development of shorter fatter roots, probably by reducing cellulose synthesis.

Tobacco was transformed with three different alleles (L2, L6, and L10) of the flax rust resistance gene L, a member of the toll interleukin-1 receptor, nucleotide-binding site, leucine-rich repeat (TIR-NBS-LRR) class of plant disease resistance genes. L6 transgenics had a stunted phenotype, expressed several defense response genes constitutively, and had increased resistance to the fungus Cercospora nicotianae and the oomycete Phytophthora parasitica pv. nicotianae. L2 and L10 transgenics, with one exception for L10, did not express these phenotypes, indicating that the activation of tobacco defense responses is L6 allele-specific. The phenotype of the exceptional L10 transgenic plant was associated with the presence of a truncated L10 gene resulting from an aberrant T-DNA integration. The truncated gene consisted of the promoter, the complete TIR region, and 39 codons of the NBS domain fused inframe to a tobacco retrotransposon-like sequence. A similar truncated L10 gene, constructed in vitro, was transiently expressed in tobacco leaves and gave rise to a strong localized necrotic reaction. Together, these results suggest that defense signaling properties of resistance genes can be expressed in an allele-specific and pathogen-independent manner when transferred between plant genera.

CesA1 and CesA3 are thought to occupy noninterchangeable sites in the cellulose synthase making primary wall cellulose in Arabidopsis (Arabidopsis thaliana L. Heynh). With domain swaps and deletions, we show that sites C terminal to transmembrane domain 2 give CesAs access to their individual sites and, from dominance and recessive behavior, deduce that certain CesA alleles exclude others from accessing each site. Constructs that swapped or deleted N-terminal domains were stably transformed into the wild type and into the temperature-sensitive mutants rsw1 (Ala-549Val in CesA1) and rsw5 (Pro-1056Ser in CesA3). Dominant-positive behavior was assayed as root elongation at the restrictive temperature and dominant-negative effects were observed at the permissive temperature. A protein with the catalytic and C-terminal domains of CesA1 and the N-terminal domain of CesA3 promoted growth only in rsw1 consistent with it accessing the CesA1 site even though it contained the CesA3 N-terminal domain. A protein having the CesA3 catalytic and C-terminal domains linked to the CesA1 N-terminal domain dramatically affected growth, but only in the CesA3 mutant. This is consistent with the operation of the same access rule taking this chimeric protein to the CesA3 site. In this case, however, the transgene behaved as a genotype-specific dominant negative, causing a 60% death rate in rsw5, but giving no visible phenotype in wild type or rsw1. We therefore hypothesize that possession of CesA3WT protects Columbia and rsw1 from the lethal effects of this chimeric protein, whereas the mutant protein (CesA3 rsw5 ) does not.

The impact of phenolics on a specialist herbivore, Manduca sexta, and a generalist herbivore, Heliothis virescens, was investigated using transgenic tobacco with differential expression of phenylalanine ammonia-lyase. Foliar phenolics such as chlorogenic acid, rutin, and total flavonoids differentially accumulated in the respective transgenic tobacco lines; the amount of chlorogenic acid ranged from 201 to 2202 μg g-1 of fresh leaf, that of total flavonoids from 211 to 500 μg g-1 of fresh leaf, and that of rutin from 73 to 172 μg g-1 of fresh leaf. However, the levels of the phenolics and larval growth of M. sexta or H. virescens were not significantly correlated. Likewise, phenolic levels were not correlated with larval survival of M. sexta. These results suggest that plant phenolics are not a determining factor for host plant resistance against these insects in this system. Keywords: Phenylalanine ammonia-lyase; phenolics; host plant resistance; Manduca sexta; Heliothis virescens