Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
1998-01-13
2001-02-06
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C435S069700, C435S069800, C435S419000, C435S468000, C800S287000, C800S284000, C800S288000
Reexamination Certificate
active
06184440
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates generally to plants genetically engineered to display altered structure or morphology. The altered structure or morphology can be associated, for example, with greater biomass, yield, or growth, or larger plants or smaller plants. More particularly, the present invention relates to transgenic plants expressing a cell wall modulation transgene or gene construct that results in a transgenic plant having altered structure or morphology. The cell wall modulation transgene is a gene encoding a cellulose binding protein, a cellulose binding domain or a cell wall modifying protein or enzyme. The invention further relates to transgenic plants having altered structure or morphology expressing a transgene such as a gene encoding an endo-xyloglucan transferase, xyloglucan endotransglycosylase, cellulose synthase or a novel isolated endo-1,4-&bgr;-glucanase. The invention also relates to transgenic plants containing a gene construct encoding a secretion signal peptide with a cell wall modulation protein or polypeptide controlled by a constitutive or tissue specific promoter. In one embodiment, the tissue specific promoter is a novel elongating tissue specific promoter of
Arabidopsis thaliana,
i.e., the cel1 promoter. The invention also relates to a novel isolated endoglucanase gene, i.e., the
Arabidopsis thaliana
endo-1,4-&bgr;-glucanase gene (cel1), its promoter (cel1 promoter) and its encoded polypeptide (Cel1) and recombinant vectors containing the cel1 gene with or without a secretion signal peptide sequence and/or the cel1 promoter.
2. BACKGROUND OF THE INVENTION
2.1. PLANT ELONGATION AND GROWTH
The plant cell elongation mechanism is a fundamental process with primary importance in plant-tissue development. Cell elongation requires relaxation of the rigid primary cell wall (Carpita and Gibeaut, 1993, Plant J. 3:1-30; Cosgrove, 1993, Plant Physiol. 102:1-6; Fry, 1988,
The Growing Plant Cell Wall Chemical and Metabolic Analysis,
Lonoman Scientific & Technical, New York; Roberts, 1994, Curr. Opin. Cell Biol. 6:688-694). Several mechanisms for this relaxation have been suggested, including the activities of endo-xyloglucan transferase (Nishitani and Tominaga, 1992, J. Biol. Chem. 267:21058-21064), xyloglucan endotransglycosylase (Fry et al., 1992, Biochem. J. 282:821-828) and expansins (McQueen-Mason and Cosgrove, 1995, Plant Physiol. 107:87-100). Endo-1,4-&bgr;-glucanase (hereinafter, EGase) has been suggested to play an important role in the elongation process (Shoseyov and Dekel-Reichenbach, 1992, Acta Hort. 329:225-227; Verma et al., 1975, J. Biol. Chem.250:1019-1026).
Substantial evidence for the involvement of a 1,3-1,4-&bgr;-glucan-specific enzyme in cell elongation was found in monocotyledons (Hatfield and Nevins, 1987, Plant Physiol. 83:203-207; Hoson and Nevins, 1989, Plant Physiol. 90:1353-1358 1989; Inouhe and Nevins, 1991, Plant Physiol. 96:426-431). EGase has been implicated in xyloglucan degradation during vegetative growth and fruit ripening (Hayashi, 1989, Ann. Rev. Plant Physiol. 40:139-168; Hayashi et al., 1984, Plant Physiol. 25:605-610). The activity of this enzyme could affect the generation of oligosaccharins, signaling molecules that are involved, among other things, in plant development and cell elongation (see for review, Darvill et al., 1992, Glycobiology 2:181-198).
To date, most of the EGase genes isolated have been studied in relation to fruit ripening (Cass et al., 1990, Mol. Gen. Genet. 223:76-86; Fischer and Bennett, 1991, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:675-703; Lashbrook et al., 1994, Plant Cell 6:1485-1493; Tucker et al., 1987, Plant Mol. Biol. 9:197-203) and abscission zones (Kemmerer and Tucker, 1994, Plant Physiol. 104:557-562; Tucker and Milligan, 1991, Plant Physiol. 95:928-933; Tucker et al., 1988, Plant Physiol. 88:1257-1262).
More recently, Wu et al. (1996, Plant Physiol. 110:163-170) cloned the EGase gene from pea and showed its expression to be induced by auxin in elongating epicotyls.
Endogenous regulation of cell elongation appears to be dominated by cell wall mechanics. This process is a result of the interaction between internal turgor pressure and the mechanical strength of the cell wall (reviewed by Steer and Steer, 1989, New Phytol. 111:323-358). Unlike most plant cells, the growth of pollen tubes and root hairs is restricted to the tip zone (reviewed by Cresti and Tiezzi, 1992, “Pollen tube emission organization and tip growth,” in
Sexual Plant Reproduction,
pp. 89-97, eds. Cresti and Tiezzi, Springer-Verlag, Berlin). The growing region of pollen tubes consists of two distinct layers when fully mature. The inner layer consists mostly of callose-related molecules and the outer layer contains pectin, xyloglucan (XG), cellulose (at low levels and poor crystallinity) and other polysaccharides (reviewed by Steer and Steer, 1989, New Phytol. 111:323-358).
Xyloglucans (XGs) are linear chains of &bgr;-(1-4)-D-glucan, but unlike cellulose, they possess numerous xylosyl units added at regular sites to the 0-6 position of the glucosyl units of the chain (reviewed by Carpita and Gibeaut, 1993, Plant J. 3:1-30). XG can be extracted by alkaline treatment and then bound again in vitro to cellulose (Hayashi et al., 1994, Plant Cell Physiol. 35:1199-1205).
XG is bound to cellulose microfibrils in the cell walls of all dicotyledons and some monocotyledons (reviewed by Roberts, 1994, Curr. Opin. Cell Biol. 6:688-694). The XG bound to the cellulose microfibrils cross-links the cell-wall framework.
Plant-cell expansion, including elongation, requires the integration of local wall-loosening and the controlled deposition of new wall materials. Fry et al. (1992, Biochem J. 282:821-828) and Nishitani and Tominaga (1992, J. Biol. Chem 267:21058-21064) purified xyloglucan endo-transglycosylase (XET) and endo-xyloglucan transferase (EXT), respectively. These two enzymes were shown to be responsible for the transfer of intermicrofibrillar XG from one segment to another XG molecule and thus, suggested to be wall loosening-enzymes.
However, McQueen-Mason et al. (1993, Planta 190:327-331) showed that XET activity did not correlate with in vitro cell wall extension in cucumber hypocotyls.
The effect of XG on growing tissues has been extensively investigated. XG oligosaccharides, produced by partial digestion with &bgr;-(1-4)-D-glucanase and referred to as “oligosaccharins”, alter plant-cell growth (reviewed by Aldington and Fry, 1993, Advances in Botanical Research 19:1-101). One such oligosaccharin, XXFG (XG9), antagonizes the growth promotion induced in pea stem segments by the auxin 2,4-D at a concentration of about 1 nM (York et al., 1984, Plant Physiol. 75:295-297; McDougall and Fry, 1988, Planta 175:412-416). On the other hand, at high concentrations (e.g., 100 &mgr;M) oligosaccharins promote the elongation of etiolated pea stem segments (McDougall and Fry, 1990, Plant Physiol. 93:1042-1048). The mode of action of oligosaccharins is still unknown.
Another type of cell wall-loosening protein, termed “expansin”, was isolated by McQueen-Mason et al. (1992, The Plant Cell 4:1425-1433). Expansin does not exhibit hydrolytic activity with any of the cell-wall components. It binds at the interface between cellulose microfibrils and matrix polysaccharides in the cell wall, and is suggested to induce cell wall expansion by reversibly disrupting noncovalent bonds within this polymeric network (McQueen-Mason and Cosgrove, 1995, Plant Physiol. 107:87-100). Some cellulose-binding organic substances alter cell growth and cellulose-microfibril assembly in vivo. Direct dyes, carboxymethyl cellulose (CMC) and fluorescent brightening agents (FBAs, e.g., calcofluor white ST) prevent
Acetobacter xylinum
microfibril crystallization, thereby enhancing polymerization. These molecules bind to the polysaccharide chains immediately after their extrusion from the cell surface, preventing normal assembly of microfibrils and cell walls (Haigler, 1991, “Relationship between polymerization and crystallization in m
Shani Ziv
Shoseyov Oded
Shpigel Etai
Fox David T.
Mehta Ashwin D.
Pennie & Edmonds LLP
Yissum Research Development Company of the Hebrew University of
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