Paul A. Howles

Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

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 L-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.

Overexpression of L-Phenylalanine Ammonia-Lyase in Transgenic Tobacco Plants Reveals Control Points for Flux into Phenylpropanoid Biosynthesis.

Transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the enzyme L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) were grown from seeds of a primary transformant containing the bean PAL2 gene, which had shown homology-dependent silencing of the endogenous tobacco PAL genes. Analysis of endogenous and transgene-encoded PAL transcripts and protein in the primary transformant (T0) and first-generation (T1) overexpressor plants indicated that the transgene-encoded PAL is the cause of the greater than wild-type levels of PAL activity (up to 5- and 2-fold greater in leaf and stem tissue, respectively) in the T1 plants. Leaves of PAL-overexpressing plants contained increased levels of the hydroxycinnamic acid ester chlorogenic acid but not of the flavonoid rutin, indicating that PAL is the key control point for flux into chlorogenic acid. In addition, levels of the glucoside of 4-coumaric acid increased in the overexpressing plants, suggesting that the 4-coumarate:coenzyme A ligase or coumarate hydroxylase reactions might have become limiting. These results help to define the regulatory architecture of the phenylpropanoid pathway and indicate the possibility of engineering-selective changes in this complex metabolic pathway by overexpression of a single early pathway gene.

Arabidopsis dynamin-like protein DRP1A: a null mutant with widespread defects in endocytosis, cellulose synthesis, cytokinesis, and cell expansion

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.

Chimeric Proteins Suggest That the Catalytic and/or C-Terminal Domains Give CesA1 and CesA3 Access to Their Specific Sites in the Cellulose Synthase of Primary Walls

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 (CesA3rsw5) does not.

In Trifolium subterraneum, chalcone synthase is encoded by a ultigene family

Chalcone synthase (CHS) catalyzes the first and key regulatory step in flavonoid biosynthesis. We report the existence and characterization of a CHS multigene family present in Trifolium subterraneum L. cultivar Karridale. The CHS family consists of at least four members, which are tightly clustered in a 15-kb region. The complete sequences of two of these genes (CHS1 and CHS2) are presented. The putative promoters of these genes have sequences which are homologous to those known, or implicated, in regulation of the expression of phenylpropanoid-encoding genes.

Prospects for the metabolic engineering of bioactive flavonoids and related phenylpropanoid compounds.

The successful engineering of complex metabolic pathways will require, in addition to availability of cloned genes and promoters, knowledge of the regulatory mechanisms that control metabolic flux into the pathway including post-translational phenomena such as metabolite channeling. We are interested in modifying pathways for the synthesis of isoflavonoids and other bioactive phenylpropanoid compounds in transgenic plants. We describe studies on flux control utilizing transgenic tobacco plants that under- and over-express key biosynthetic enzymes, and outline experimental approaches for the molecular dissection of potential metabolic channels in the synthesis of antimicrobial flavonoid derivatives in alfalfa and other species.

Nucleotide sequence of additional members of the gene family encoding chalcone synthase in Trifolium subterraneum.

CHS (EC 2.3.1.74) catalyzes the condensation of three molecules of malonyl-COA with one molecule of 4-coumaroyl COA to produce 2’,4,4’,6-tetrahydroxychalcone (Heller and Hahlbrock, 1980). The CHS genes have been isolated and characterized in a variety of plants. In the Leguminosae, CHS is a multiple gene family, with subterranean clover (Trifolium subterraneum) having at least nine copies (Arioli et al., 1994). We have recently shown that different copies within this gene family are induced by wounding and Rhizobium infection (Lawson et al., 1994). Screening of a T. subterraneum genomic library with the cDNA clone for CHSZ from bean (Ryder et al., 1987) isolated a number of clones; of these, CHSZ and CHS2 were fully characterized (Arioli et al., 1994). To permit identification of the promoters responsible for the expression of separate gene copies, we determined sequences for additional CHS genes. Sequence data for CHS3 and CHS4, which are linked to CHSZ and CHS2, and CHS5 and CHS6, which, together with a pseudogene CHS7, form a second linkage group, are presented here (Table I). An as-yet unlinked gene, CHS8, has been cloned and partially sequenced to confirm its homology to CHS; this sequence is not presented. The COOH-terminal portion of CHS4 is not located on any of the A clones characterized; thus, the sequence for this gene is truncated 236 bp prior to the end of the gene. Six hundred base pairs of upstream sequence is included in the data base listings for each of these genes. A11 of the CHS sequences within each linkage cluster are oriented in the same direction. The average homology of the six clover CHS genes is 93% at the DNA and 98% at the amino acid level. Curiously, greater homology is present between some members of the two linkage clusters than within each cluster. The phylogenetic relationships be-

Characterization of a phenylalanine ammonia-lyase multigene family in Trifolium subterraneum

The enzyme phenylalanine ammonia-lyase (PAL) was found to be encoded by a small gene family in the legume Trifolium subterraneum (subterranean clover). At least three of the family members are tightly clustered within approx. 20 kb of DNA. Sequencing of one of the genes established that it possesses two exons, the position of the single intron being identical to that found for PAL genes from other plants. The PAL protein consists of 725 amino acids, as deduced from the nucleotide sequence.

A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module

Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5′ and 3′ rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the proteins first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed.

Post-transcriptional regulation of phenylalanine ammonia-lyase expression in tobacco following recovery from gene silencing.

Abstract Introduction of a bean phenylalanine ammonia-lyase (PAL) transgene into tobacco plants results in epigenetic post-transcriptional gene silencing which is unstable, such that after self-pollination first generation progeny may become PAL over-expressors. The change from gene silencing to PAL over-expression is accompanied by a loss of cytosine methylation of the PAL transgene and reduced methylation of the endogenous tobacco PAL2 gene, but not the PAL1 gene. These changes are associated with the appearance of high levels of bean PAL and tobacco PAL2 transcripts in the total RNA fraction from PAL over-expressing plants. However, tobacco PAL2 transcripts are inefficiently recruited into polysomes, and tobacco PAL2 protein is not detected in leaves of PAL over-expressing or wild-type lines. Thus, in spite of the post-transcriptionally controlled increase in tobacco PAL2 transcripts in PAL over-expressors, the increased PAL activity is primarily the result of the increase in bean PAL transcripts and corresponding enzymatic activity. These results reveal a complex cross-talk between expression of the PAL transgene and the corresponding endogenous PAL genes at the levels of transcription, transcript stability and polysomal recruitment during sense transgene-mediated silencing and subsequent over-expresson of PAL in tobacco.