Ph genes and their uses

Details Ph genes and their uses Ph genes and their uses

1996-02-16

1999-06-08

Carlson, Karen Cochrane

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters fat, fatty oil, ester-type wax, or...

800200, 800250, 800255, 800DIG67, 435468, 435469, 435470, 4353201, 435 6, 536 231, 536 236, 536 241, 536 245, A01H 104, A01H 502, C12N 1510, C12N 1563

Patent

active

059106271

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

The present invention relates to compositions and methods for controlling intra-cellular pH, e.g., vacuolar pH in plant cells. In particular, it relates to the isolation and characterization of Ph genes and their use in controlling vacuolar pH.
The vacuole is an important component of plant cells and often accounts for the majority of the cell volume. The vacuole contains water and a variety of organic and inorganic compounds such as sugars, organic acids, proteins, anthocyanin pigments and excretory products such as calcium oxalate and tannin compounds.
The vacuole is surrounded by a membrane (tonoplast membrane) which regulates the movement of materials into and out of the vacuole. Thus, the plant cell controls the contents of the fluid or cell sap within the vacuole. One of the properties of the cell sap which is regulated by the plant cell is proton concentration or pH of the solution.
A number of proteins can affect vacuolar pH. For instance, vacuolar ATPases generate pH and electrical gradients by hydrolyzing ATP and pumping protons across the tonoplast membrane. Nelson et al., Trends in Biochemical Science, 14:113-116 (1989). Another enzyme that affects vacuolar pH is inorganic pyrophosphatase. In Arabidopsis, this enzyme is a single 81 kDa protein that is encoded by a single gene (Sarafian et al., Proc. Natl. Acad. Sci. USA 89:1775-1779 (1992).
A group of genes in plants, referred to as Ph genes, encode proteins that also affect vacuolar pH. In Petunia hybrida (petunia) a number of Ph genes are known to play a role in determining vacuolar pH, although none has been reported as isolated or sequenced. de Vlaming et al. Theor. Appl. Genet. 66:271-278 (1983) and Wiering, et al., in Monographs on Theoretical and Applied Genetics 9: Petunia, K. C. Sink, ed. (Springer-Verlag, Berlin 1984), pp. 49-67, Gerats et al., Dev. Gen. 10:561-568 (1989) all of which are incorporated herein by reference.
In yeast, at least 17 different vph (vacuolar pH genes) required for acidification have been identified. For example, the vph1 mutation causes an elevation of vacuolar pH. Preston et al., Proc. Natl. Acad. Sci. USA 86:7027-7031 (1989). This mutant has been shown to lack ATPase activity as a consequence of a lesion in a gene encoding a 95kDa integral membrane subunit of the ATPase. Manolson et al. J. Biol. Chem. 267:14294-14303 (1992). A second vph mutant, vat2, is defective in the synthesis of the 60 kDa subunit of ATPase and also exhibits an elevated vacuolar pH. Nelson et al. Proc. Natl. Acad. Sci. USA 87:3503-3507 (1990).
Vacuolar pH is important in determining a number of plant traits. For instance, in petunia, certain forms of anthocyanins have been shown to appear red at low pH (e.g., pH 5.5) and blue at higher pH values (e.g., pH 5.9). Timberlake et al., in The Flavonoids, Harborne, et al. eds. (Academic Press, New York, 1975) pp. 214-266. Thus, flower color has been shown to change as the pH of the corolla cell vacuole is altered. In addition, the acidity of fruits such as tomatoes or citrus fruits depends upon the pH of the vacuolar contents. Other traits known to be affected by vacuolar pH include seed coat development, female fertility, protein transport.
There is currently a need for methods of producing new plant varieties with modified traits affected by vacuolar pH, such as flower color and fruit acidity. Control of the expression of genes encoding proteins affecting vacuolar pH provides a useful approach to this problem. The present invention addresses these and other needs.


SUMMARY OF THE INVENTION

The present invention provides isolated DNA constructs comprising a polynucleotide sequence from a Ph gene. As defined here, Ph genes encode proteins which regulate vacuolar pH. Preferably, the Ph gene regulates pH by controlling transcription of one or more genes (i.e., downstream genes) which in turn encode proteins which directly regulate vacuolar pH. The protein encoded by such Ph genes typically comprises a transcriptional activator sequence in particular, a helix-loop-

REFERENCES:
patent: 5534660 (1996-07-01), Chuck et al.
Chuck, George, et al. (1993) "Tagging and Cloning of a Petunia Flower Color Genewith the Maize Transposable Element Activator", The Plant Cell, 5:371-378.
Goodrich, Justin, et al. (1992) "A Common Gene Regulates Pigmentation Pattern in Diverse Plant Species", Cell, 68:955-964.
Lifschitz, E. (1992) "News and Views", Plant Molecular Biology, 19:iii-vi.
Lloyd, Alan M., et al. (1992) "Arabidopsis and Nicotiana Anthocyanin Production Activated by Maize Regulators R and C1", Science, 258:1773-1775.
Forkmann, G. (1991) "Flavonoids as Flower Pigments: The Formation of the Natural Spectrum and its Extension by Genetic Engineering", Plant Breeding, 106:1-26.
Dooner, Hugo K., et al. (1991) "Genetic and Development Control of Anthocyanin Biosynthesis", Annu. Rev. Genet., 25:173-199.
Mooney, M., et al. (1993) "Using the Regulatory Gene Delila to Investigate the Evolution of Plant Pigment Patterns", (Abstract) Journal of Cellular Biochemistry, Supplement, Keystone Symposia, 21.

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