Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
1999-07-07
2001-06-05
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C435S419000, C435S468000, C536S023600, C800S260000, C800S286000, C800S287000, C800S298000
Reexamination Certificate
active
06242668
ABSTRACT:
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
FIELD OF THE INVENTION
The present invention is directed to plant genetic engineering. In particular, it relates to modulation of expression of strawberry genes encoding endo-1,4-&bgr;-glucanases.
BACKGROUND OF THE INVENTION
Fruit ripening is a complex developmental program in which senescing tissues undergo programmed changes in firmness, texture, coloration, flavor and susceptibility to microbial infection. Changes in firmness and texture are largely attributed to alterations in the composition and structure of cell wall polysaccharides. As these modifications influence the post-harvest properties (i.e. storage time and expense, handling damage, desirability to the consumer) of important food crops and, consequently, are of great commercial importance, research in recent years has focused on identifying enzyme activities which are rate-limiting in the promotion of fruit deterioration. In the climacteric species, which are characterized by the autocatalytic production of the ripening hormone ethylene and a ripening-related transient burst in CO
2
evolution, the antisense suppression of ACC synthase (Oeller et al.,
Science
254:437-439 (1991)) and ACC oxidase (Picton et al.,
Plant J
3:469 (1993)) in tomato has provided fruit in which ripening and softening can be controlled by the application of ethylene. Similar approaches have been taken in efforts to diminish the activities of cell wall-associated hydrolases (Sheehy et al.,
Proc Natl Acad Sci USA
85:8805-8809 (1988); Smith et al.,
Nature
334:724-726 (1988)), which may play a central role in fruit cell wall breakdown during ripening (Brady C J,
Annu Rev Plant Physiol
38:155-178 (1987)).
In the non-climacteric species such as strawberry (
Fragaria x ananassa
), much less is known about the ripening process. As these plants lack a respiratory climacteric and ethylene appears to play little, if any role, in fruit ripening, there is growing interest in identifying the factor(s) which mediates ripening. Strawberry fruit exhibit low level ethylene production which is rather constant during ripening (Knee et al.,
J Exp Bot
28:377-39 (1977)), and there is no observable stimulation of ripening upon applying exogenous ethylene (Iwata et al.,
J Jap Soc Hort Sci
38:64-72 (1969)). Although there is no evidence for a ripening-related role for ethylene, strawberry fruit ripening has been shown to be negatively regulated by auxins which originate in the receptacle-born achenes (Given et al.,
Planta
174:402-406 (1988b); Manning K Planta 194:62-68 (1994)). As auxin levels decline, fruit exhibit a characteristic ripening profile, one of the major hallmarks of which is rapid deterioration once fruit achieve the red ripe stage. In general, strawberry fruit ripening is typified by the induction of enzyme markers for anthocyanin pigment biosynthesis (e.g. phenylalanine ammonia lyase), a concomitant decrease in chlorophyll and increase in anthocyanin pigments, and a progressive decrease in tissue firmness (Woodward JR
J Sci Food Agric
23:465-473 (1972); Given et al.,
J Plant Physiol
133:25-30 (1988
a
)).
Efforts to reveal the molecular basis of changes in firmness, which is a major contributing factor to fruit quality, have focused on cell wall-associated enzymes which are believed to mediate and/or contribute to cell wall breakdown. The most studied of these activities, endopolygalacturonase, is absent, or below the limit of detection, in ripening strawberry fruit (Neal G E
J Sci Food Agri
16:604-608 (1965); Barnes et al.
J Food Sci
41:1392-1395 (1976); Huber Dj
J Food Sci
49:1310-1315 (1984)). Although strawberry fruit is a rich source of pectin, this observation is consistent with cell wall studies which have shown that total extractable polyuronides remain constant as a proportion of cell wall material during ripening and do not show detectable depolymerization (Huber Dj
J Food Sci
49:1310-1315 (1984)). In contrast to these findings, however, the hemicellulosic fraction of cell walls prepared from ripening fruit demonstrates a progressive shift from high molecular weight to low molecular weight polymers (Huber Dj
J Food Sci
49:1310-1315 (1984)). While there is no discernible change in the neutral sugar composition of hemicelluloses isolated from stages ranging from small green to red ripe, the average net molecular weight change is quite dramatic; suggestive of an active, developmentally regulated endohydrolyase. Interestingly, this observed hemicellulose depolymerization correlates well with a soluble CMCase activity measured in extracts prepared from ripening strawberry fruit (Barnes et al.
J Food Sci
41:1392-1395 (1976)). In ripening avocado (HatCel11
d
and Nevins, 1986) and pepper (Harpster et al.,
Plant Mol Biol
33:47-59 (1997)) fruit, CMCase activity is attributed to an endo-1,4-&bgr;-glucanase (EGase; EC 3.2.1.4).
Although largely correlative, there is considerable evidence for the importance of EGases in a wide variety of physiological processes involving changes in cell wall architecture which range from cell wall expansion to disassembly (see review by Brummell et al., 1994). In abscission zone formation, for instance, the infusion of antiserum raised against an abscission zone-related EGase into explants which had been induced to abscise by ethylene was observed to inhibit cell separation (Sexton et al.,
Nature
283:8743-8744 (1980)). Furthermore, the induction of EGase gene expression in fruit of tomato (Lashbrook et al.,
Plant Cell
6:1485-1493 (1994)), avocado (Christoffersen et al.
Plant Mol Biol
3:385-391 (1984)) and pepper (Ferrarese et al.,
Plant Mol Biol
29:735-747 (1995); Harpster et al.,
Plant Mol Biol
33:47-59 (1997)) correlates well with the onset and development of ripening.
Control of the expression of genes associated with cell wall degradation is useful in controlling many plant processes, including fruit ripening. Characterization and cloning of such genes from many agronomically important plants is lacking in the prior art. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a strawberry Cel1 polynucleotide sequence. The polynucleotides of the invention can be identified by their ability to specifically hybridize to SEQ ID NO:1 under stringent conditions. The Cel1 polynucleotides of the invention are typically at least about 100 nucleotides in length. The Cel1 polynucleotides encode a polypeptide as shown in SEQ ID NO:2. A preferred Cel1 polynucleotide sequence is set forth in SEQ ID NO:1.
If transcription of the Cel1 polynucleotide is desired, the nucleic acid molecules of the invention may further comprise a plant promoter operably linked to the Cel1 polynucleotide. The plant promoter used is not critical to the invention and may be, for instance, a promoter that drives expression in fruit tissue. In some embodiments the Cel1 polynucleotide is linked to the promoter in an antisense orientation.
The invention also provides transgenic plants comprising an expression cassette containing a plant promoter operably linked to a heterologous Cel1 polynucleotides of the invention. The heterologous Cel1 polynucleotide may encode a Cel1 polypeptide, such as one shown in SEQ ID NO:2. Alternatively, the Cel1 polynucleotide may be linked to the promoter in an antisense orientation.
The invention further provides methods of modulating cell wall degradation in plants. The methods comprise introducing into the plant an expression cassette containing a plant promoter operably linked to a heterologous Cel1 polynucleotide. The heterologous Cel1 polynucleotide may be used to encode a Cel1 polypeptide or may be used to inhibit expression of an endogenous Cel1 polynucleotide. In these embodiments, the Cel1 polynucleotide may be linked to the promoter in a sense or antisense orientation. The expression cassette can be introduced into the plant using genetic engineering techniques or through a sexual cross.
Definitions
The phrase “nucle
Benzion Gary
DNA Plant Technology Corporation
Mehta Ashwin D.
Townsend and Townsend and Crew
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