Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-08-31
2002-12-03
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S286000, C800S298000, C800S295000, C800S317000, C800S320000, C800S306000, C435S069100, C435S468000, C435S419000, C536S023100, C536S023200, C536S023600, C536S024100, C536S024500
Reexamination Certificate
active
06489538
ABSTRACT:
FIELD OF INVENTION
The present method relates to the field of molecular biology and the regulation of protein synthesis through the introduction of foreign genes into plant genomes. More specifically, the method relates to the modification of plant lignin composition in a plant cell by the introduction of a foreign plant gene encoding an active ferulate-5-hydroxylase (F5H) enzyme. Plant transformants harboring the F5H gene demonstrate increased levels of syringyl monomer residues in their lignin, a trait that is thought to render the polymer more susceptible to delignification.
BACKGROUND
Lignin is one of the major products of the general phenylpropanoid pathway, and is one of the most abundant organic molecules in the biosphere (Crawford, (1981)
Lignin Biodegradation and Transformation,
New York: John Wiley and Sons). In nature, lignification provides rigidity to wood and is in large part responsible for the structural integrity of plant tracheary elements. Lignin is well suited to these capacities because of its physical characteristics and its resistance to biochemical degradation. Unfortunately, this same resistance to degradation has a significant impact on the utilization of lignocellulosic plant material (Whetten et al.,
Forest Ecol. Management
43, 301, (1991)).
The monomeric composition of lignin has significant effects on its chemical degradation during industrial pulping (Chiang et al.,
Tappi,
71, 173, (1988). The guaiacyl lignins (derived from ferulic acid) characteristic of softwoods such as pine, require substantially more alkali and longer incubations during pulping in comparison to the guaiacyl-syringyl lignins (derived from ferulic acid and sinapic acid) found in hardwoods such as oak. The reasons for the differences between these two lignin types has been explored by measuring the degradation of model compounds such as guaiacylglycerol-&bgr;-guaiacyl ether, syringylglycerol-&bgr;-guaiacyl ether, and syringylglycerol-&bgr;-(4-methylsyringyl) ether (Kondo et al.,
Holzforschung,
41, 83, (1987)) under conditions that mimic those used in the pulping process. In these experiments, the mono- and especially di-syringyl compounds were cleaved three to fifteen times faster than their corresponding diguaiacyl homologues. These model studies are in agreement with studies comparing the pulping of Douglas fir and sweetgum wood where the major differences in the rate of pulping occurred above 150° C. where arylglycerol-&bgr;-aryl ether linkages were cleaved (Chiang et al.,
Holzforschung,
44, 309, (1990)).
Another factor affecting chemical degradation of the two lignin forms may be the condensation of lignin-derived guaiacyl and syringyl residues to form diphenylmethane units. The presence of syringyl residues in hardwood lignins leads to the formation of syringyl-containing diphenylmethane derivatives that remain soluble during pulping, while the diphenylmethane units produced during softwood pulping are alkali-insoluble and thus remain associated with the cellulosic products (Chiang et al.,
Holzforschung,
44, 147, (1990); Chiang et al.,
Holzforschung,
44, 309, (1990)). Further, it is thought that the abundance of 5-5′-diaryl crosslinks that can occur between guaiacyl residues contributes to resistance to chemical degradation. This linkage is resistant to alkali cleavage and is much less common in lignin that is rich in syringyl residues because of the presence of the 5-O-methyl group in syringyl residues. The incorporation of syringyl residues results in what is known as “non-condensed lignin”, a material that is significantly easier to pulp than condensed lignin.
Similarly, lignin-composition and content in grasses is a major factor in determining the digestibility of lignocellulosic materials that are fed to livestock (Jung, H. G. & Deetz, D. A. (1993) Cell wall lignification and degradability in Forage Cell Wall Structure and Digestibility (H. G. Jung, D. R. Buxton, R. D. Hatfield, and J. Ralph eds.), ASA/CSSA/SSSA Press, Madison, Wis.). The incorporation of the lignin polymer into the plant cell wall prevents microbial enzymes from having access to the cell wall polysaccharides that make up the wall. As a result, these polysaccharides cannot be degraded and much of the valuable carbohydrates contained within animal feedstocks pass through the animals undigested. Thus, an increase in the dry matter of grasses over the growing season is counteracted by a decrease in digestibility caused principally by increased cell wall lignification. From these examples, it is clear that the modification of lignin monomer composition would be economically advantageous.
The problem to be overcome, therefore, is to develop a method for the creation of plants with increased levels of syringyl residues in their lignin to facilitate its chemical degradation. Modification of the enzyme pathway responsible for the production of lignin monomers provides one possible route to solving this problem.
The mechanism(s) by which plants control lignin monomer composition has been the subject of much speculation. As mentioned earlier, gymnosperms do not synthesize appreciable amounts of syringyl lignin. In angiosperms, syringyl lignin deposition is developmentally regulated: primary xylem contains guaiacyl lignin, while the lignin of secondary xylem and sclerenchyma is guaiacyl-syringyl lignin (Venverloo,
Holzforschung
25, 18 (1971); Chapple et al.,
Plant Cell
4, 1413, (1992)). No plants have been found to contain purely syringyl lignin. It is still not clear how this specificity is controlled; however, at least five possible enzmatic control sites exist, namely caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT), F5H, (hydroxy)cinnamoyl-CoA ligase (4CL), (hydroxy)cinnamoyl-CoA reductase (CCR), and (hydroxy)cinnamoyl alcohol dehydrogenase (CAD). For example, the substrate specificities of OMT (Shimada et al.,
Phytochemistry,
22, 2657, (1972); Shimada et al.,
Phytochemistry,
12, 2873, (1973); Gowri et al.,
Plant Physiol.,
97, 7, (1991); Bugos et al.,
Plant Mol. Biol.
17, 1203, (1992)) and CAD (Sarni et al.,
Eur. J. Biochem.,
139, 259, (1984); Goffner et al.,
Planta.,
188, 48, (1992); O'Malley et al.,
Plant Physiol.,
98, 1364, (1992)) are correlated with the differences in lignin monomer composition seen in gymnosperms and angiosperms, and the expression of 4CL isozymes (Grand et al.,
Physiol. Veg.
17, 433, (1979); Grand et al.,
Planta.,
158, 225, (1983)) has been suggested to be related to the tissue specificity of lignin monomer composition seen in angiosperms.
Although there are at least five possible enzyme targets that could be exploited, only OMT and CAD have been investigated in recent attempts to manipulate lignin monomer composition in transgenic plants (Dwivedi et al.,
Plant Mol. Biol.
26, 61, (1994); Halpin et al.,
Plant J.
6, 339, (1994); Ni et al.,
Transgen. Res.
3, 120 (1994); Atanassova et al.,
Plant J.
8, 465, (1995); Doorsselaere et al.,
Plant J.
8, 855, (1995)). Most of these studies have focused on sense and antisense suppression of OMT expression. This approach has met with variable results, probably owing to the degree of OMT suppression achieved in the various studies. The most dramatic effects were seen by using homologous OMT constructs to suppress OMT expression in tobacco (Atanassova et al., supra) and poplar (Doorsselaere et al., supra). Both of these studies found that as a result of transgene expression, there was a decrease in the content of syringyl lignin and a concomitant appearance of 5-hydroxyguaiacyl residues. As a result of these studies, Doorsselaere et al., (WO 9305160) disclose a method for the regulation of lignin biosynthesis through the genomic incorporation of an OMT gene in either the sense or anti-sense orientation. In contrast, Dixon et al. (WO 9423044) demonstrate the reduction of lignin content in plants transformed with an OMT gene, rather than a change in lignin monomer composition. Similar research has focused on the suppression of CAD expression. The conversion of coniferaldehyde and sin
Bui Phuong T.
Ibrahim Medina A.
Purdue Research Foundation
Woodard Emhardt Naughton Moriarty & McNett
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