Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-06-09
2002-12-31
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S320100, C435S419000, C536S023200, C536S023600, C800S278000, C800S290000
Reexamination Certificate
active
06501008
ABSTRACT:
BACKGROUND OF THE INVENTION
Growth in plants is controlled by the mechanical properties of the cell wall, a structure that otherwise constrains cells and restricts protoplast expansion (Masuda 1990, Sakurai 1991, for review). Changes in the cell wall mechanical properties that impart “loosening” might be achieved through the breakdown and reconstitution of cross-linked polymers in the cell walls. In fact, substantial changes in cell wall components related to cell wall loosening have been reported in many plants (Taiz 1984, Sakurai 1991, Hoson 1993, for reviews). As a parallel to documented changes in cell wall components, specific enzymes capable of degrading cell wall polysaccharides have been identified from the apoplastic compartments of mono- and dicotyledonous plants (Greve and Ordin 1971, Huber and Nevins 1980, 1981a, Labrador and Nevins 1990, Dopico et al. 1990, Nishitani and Tominaga 1992, Hayashi and Ohsumi 1994).
Cereal coleoptile segments, which have served as a model for numerous growth and hormone investigations, undergo molecular changes in non-cellulosic &bgr;-glucans coinciding with the initiation of growth. Physical displacement of molecular structures within the wall matrix in response to structural change in the wall is visualized as a means to accommodate pressure driven elongation governed by auxin (Masuda, 1990).
In cereals, most of the effort designed to describe the molecular events in growth has focused on disclosing the role of non-cellulosic &bgr;-glucans in auxin-induced growth of coleoptile segments. Auxin causes a specific decrease in the quantity of glucans in the cell walls in vivo, a process coupled with cell elongation of coleoptile segments in oat, barley, rice and maize (Loescher and Nevins 1972, Sakurai and Masuda 1978a, Zarra and Masuda 1979b, Inouhe and Nevins 1991 a). The decrease in the wall glucan content appears to reflect an obligatory chemical basis for cell wall loosening necessary for cell elongation (Sakurai and Masuda 1978b, Sakurai et al. 1979).
Cell wall autohydrolysis, an approach used to disclose the consequences of metabolism mediated by constitutive components, has been employed to identify pertinent enzymes and their substrates. It has been reported that cell walls isolated from maize coleoptiles possess a high autohydrolytic activity (autolysis) specifically directed toward degradation of non-cellulosic &bgr;-glucans eventually producing glucose (Huber and Nevins 1979). This process is mediated by wall associated exoglucanase (EC 1.2.3.58, Huber and Nevins 1982) and endoglucanase (Huber and Nevins 1981a, Hat field and Nevins 1986, 1987). The two enzymic activities account for 90% of all the recovered glucanase from maize coleoptile cell walls (Inouhe and Nevins 1991b). The endoglucanase converts the non-cellulosic &bgr;-glucans to polymers of an average molecular size of 1-1.5×10
4
(degree of polymerization of 60-70) and indicates that the endo-glucanase cleaves widely-spaced sites. Auxin is capable of enhancing glucan autolysis in maize coleoptile cell walls (Inouhe and Nevins 1991 a). In addition, polyclonal antibodies specific for cell wall glucanases inhibit the glucan autolysis and auxin-induced growth of maize coleoptile segments (Inouhe and Nevins 1991b). These observations provide evidence to support the idea that the cell wall glucanases have an important role in auxin-induced cell elongation in coleoptile segments.
Notable changes in cell wall glucan content have also been reported in coleoptile tissues developing in intact seedlings of maize (Luttenegger and Nevins 1985), barley (Sakurai and Masuda 1978b), and rice (Zarra and Masuda 1979a). In maize coleoptiles, the non-cellulosic glucans are rapidly synthesized and incorporated into cell walls during early developmental stage but subsequently are degrated to substantially diminished levels after completion of elongation (Luttenegger and Nevins 1985, Inouhe and Nevins 1997a). These data imply that the glucan metabolism mediated by cell wall glucanases is an important phase in coleoptile growth in the intact plant system. However, little is known about changes in cell wall glucanases during coleoptile development, although the timing and spatial distribution of glucanases have been investigated in detail in germinating seeds or leaves of barley seedlings (Mundy and Fincher 1986, Stuart et al. 1986, Slakeski and Fincher 1992a, b).
Diverse glucanases have been isolated from plant sources (Simmons, 1994) and some genes have been identified (Hoj and Fincher, 1995). Information on the precise characteristics of those gene responsible for glucanases in coleoptiles with putative roles in cell extension is, however, not available. Specifically, exo- and endoglucanases from maize have not been isolated or purified until the present invention.
The composition of plant stem material has a strong influence on the feeding quality of major forage crops. Forages contain significant portions of plant cell wall material. From the standpoint of a forage user, the amount and type of plant cell wall is extremely important because it greatly influences how a particular forage will be utilized by animals to produce meat or milk. In silages such as whole plant corn, alfalfa, and the like, the digestibility of the silage is important to ensure availability of the fiber, and/or providing more nutrients per amount of silage at a faster rate. Therefore cell wall constituents are very important in the feeding of animals to produce meat or milk.
SUMMARY OF THE INVENTION
In the present invention, both exo- and endoglucanases were purified and extracted from maize coleoptile cell walls. New maize endoglucanase polynucleotides and related polypeptides were identified. Now by altering expression or modulation of the newly identified maize endoglucanases, one is able to alter the composition of cell walls thereby facilitating cell elongation or expansion and thus altering the growth of a plant or improving kernel growth rates. In addition, alterations in cell wall composition can enhancing silage or forage crop digestibility.
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to endoglucanases. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention. It is another object of the present invention to provide methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide amplified from a
Zea mays
nucleic acid library using primers designed from the polynucleotides of the present invention; (c) a polynucleotide comprising at least 20 contiguous bases of the polynucleotides of the present invention; (d) a polynucleotide having at least 80% sequence identity to the polynucleotides of the present invention, where sequence identity is determined by Blast 2.0 under default parameters; (e) a polynucleotide comprising at least 25 nucleotide in length which hybridizes under low stringency conditions to the polynucleotides of the present invention; (f) a polynucleotide comprising the sequence set forth in SEQ ID NO: 1; and (g) a polynucleotide complementary to a polynucleotide of (a) through (f). The isolated nucleic acid can be DNA. The isolated nucleic acid can also be RNA.
In another aspect, the present invention relates to vectors comprising the polynucleotides of the present invention. Also the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.
In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.
In yet another aspect, the present invention relates to a transgenic plant or plant cell comprising a recombinant expression cassette wi
Nevins Donald J.
Simmons Carl R.
Kallis Russell
McElwain Elizabeth F.
The Regents of the University of California
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