The composition of lignin in tobacco stems has been altered by genetic engineering. Antisense expression of sequences encoding cinnamyl alcohol dehydrogenase (CAD), the enzyme catalysing the final step in lignin precursor synthesis, leads to the production of a modified lignin in otherwise normal plants. Although Klason and acetyl bromide lignin determinations show little quantitative change in lignin deposition in CAD antisense plants, a number of qualitative changes have been identified. The lignin is altered in both composition and structure and is more susceptible to chemical extraction. Consistent with a block in CAD activity, antisense plants incorporate less cinnamyl alcohol monomers and more cinnamyl aidehyde monomers into lignin than corresponding control plants. Antisense plants with very low levels of CAD activity also show a novel phenotype with the appearance of a red‐brown colour in xylem tissues. A similar phenotype is correlated with altered lignification and improved digestibility in brownmidrib mutants of maize and sorghum. The improved chemical extractability of lignin in CAD antisense plants supports a role for this technology in improving the pulp and paper‐making value of forest trees while the similarity with brown‐midrib mutants suggests a route to more digestible forage crops.

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Craded washings of the starch granule, followed by release of polypeptides by gelatinization i n 2 % sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEll) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEll protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEll yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEll cDNA clones. These observations provide direct evidence that BEll exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes i n maize endosperm exhibits a differential propensity to associate with, or t o become irreversibly entrapped within, the starch granule.Starch has been the subject of much recent interest, with intensive efforts devoted toward improved understanding of its structure, function, biosynthesis, and degradation. Parameters such as the ratio of amylose to amylopectin,

Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions of Eucalyptus gunii Hook and partially characterized. They differ greatly in terms of both physical and biochemical properties, and can be separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The native molecular weight of of CAD 1 is 38 kDa as determined by gel-filtration chromatography on Superose 6, and this isoform is likely to be a monomer since it yields a polypeptide of 35 kDa upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It has a low substrate affinity for coniferyl and p-coumaryl alcohols and their corresponding aldehydes. No activity with sinapyl aldehyde and alcohol was detected. The more abundant isoform is CAD 2, which has a native molecular weight of 83 kDa and is a dinier composed of two subunits of slightly different molecular weights (42-43 kDa). These subunits show identical peptide patterns after digestion with N-chlorosuccinimide. The isoform, CAD 2, has a high substrate affinity for all the substrates tested. The two isoforms are immunologically distinct as polyclonal antibodies raised against CAD 2 do not cross-react with CAD 1. The characterization of two forms of CAD exhibiting such marked differences indicates their involvement in specific pathways of monolignol utilisation.

No abstract