Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
1999-07-21
2002-01-29
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S278000, C800S289000, C800S298000, C800S317400, C536S023600, C435S320100, C435S419000, C435S411000, C435S468000, C435S194000
Reexamination Certificate
active
06342656
ABSTRACT:
BACKGROUND OF THE INVENTION
Cells must sense their nutritional or environmental conditions and modify their metabolic activity appropriately. Yeast SNF1 (sucrose non-feirmenting) protein kinase and mammalian AMP-activated protein kinase (AMPK) are central components of kinase cascades that act as metabolic sensors of glucose availability and AMP:ATP levels respectively. Protein sequence and functional homology exists between the yeast and mammalian kinase subunits (SNF1/AMPK-&agr;), activation subunits (SNF4/AMPK-&ggr;) and the docking subunits (SIP/AMPK-&bgr;) that constitute the functional kinase complexes (Hardie, D., et al.,
Annu. Rev. Biochem
. 67:821-55 (1998)).
In yeast, the association of the SNF4 activating subunit with a regulatory region of the SNF1 protein is sensitive to glucose. When glucose concentration is low, the SNF4 protein associates with the regulatory domain of SNF1, and the activity of the catalytic kinase domain is increased, resulting in the derepression of genes required for the metabolism of alternative energy sources. When glucose concentration is high, the SNF1 kinase domain associates with its regulatory domain and kinase activity is inhibited. In mammals, the activation of AMPK, in response to increases in the AMP:ATP ratio, results in the switching on of ATP-producing pathways and the switching off of ATP-consuming pathways. For example, AMPK activation results in the phosphorylation and inactivation of acetyl coenzyme A carboxylase and 3-hydroxy-3-30 methylglutaryl coenzyme A reductase (HMGCoA reductase); but unlike in yeast, the specific functions of the &ggr;- and &bgr;-subunits are less well defined.
In plants, there is also evidence that carbohydrates control gene expression, growth, metabolism and differentiation. Jang and Shcen,
Trend's in Plant Sciences
, 2:208-214 (1997); Koch,
Annu. Rev. Plant. Physiol. Mol
., 2:509-540 (1996)). An extensivc family of SNF1 homologs and related kinases have been characterized and have been grouped into several subfamilies of SNF1-related kinases (SnRKs) (Halford, et al.,
Plant Mol. Biol
. 37:735-748 (1996)). In addition to exhibiting kinase activity on substrates common to the mammalian and yeast kinases and complementing yeast SNF1 mutants (Alderson, et al.
Proc. Natl. Acad. Sci. U.S.A
. 88:8602-05 (1991); Muranaka, et al.
Mol. Cell Biol
., 14:2958-65 (1994)), antisense suppression experiments suggest that plant SNF1 homologs may also be involved in regulation of carbon metabolism in planta. Purcell, et cal.,
Plant J
. 14:195 (1998). However, NPK5,a SNF1 homolog from tobacco was unable to complement an SNF4-deletion yeast mutant strain (&Dgr;-SNF4), suggesting that NPK5 may require an SNF4-like component for physiological activity in vivo (Muranaka et al.,
Mol Cell Biol
., 14:2958-65 (1994)).
No functional homolog of the SNF4 activating subunit has yet been demonstrated from plants, although a gene sequence (Pv42) isolated from developing bean seeds was reported to have predicted amino acid sequence similarity to SNF4. (Accession No. U40713.) Thus, there exist needs to identify and express plant homologs to yeast SNF4 proteins in order to understand how plants cope with metabolic and strcss conditions in the environment and to modulate these responses to engineer plants resistant to various environmental stresses. In addition, production of genetically engineered plants with improved carbon metabolism and source-sink relationships could be used to improve yields or qualities of harvested plant products. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides SNF4 homologs from plants. In particular, the present invention provides nucleic acid molecules which encode plant SNF4 polypeptides. The polypeptides of the invention comprise an amino acid sequence that has greater than about 70% identity to SEQ ID NO:3.
Also provided is the promoter sequence from SEQ ID NO:2. Promoters of the invention can be operably linked to heterologous nucleic acid sequences and used to drive expression of the heterologous sequences in desired plant tissues.
The present invention further provides SNF
1
polypeptides. The SNF1 polypeptides of the invention comprise an amino acid sequence that has greater than about 95% identity to the amino acid sequence of the polypeptide encoded by SEQ ID NO:4. An exemplary SNF
1
nucleic acid molecule from tomato is shown in SEQ ID NO:4 (LeSNF1). Preferably, the nucleic acid molecule can specifically hybridize to SEQ ID NO:4 or its complement.
The present invention fuirther provides recombinant expression vectors comprising the nucleic acid sequences of the invention. Preferably, the vectors comprise a plant promoter operably linked to the nucleic acid sequence. The promoter can be either a constitutive promoter, or an inducible promoter.
The present invention also provides for transgenic plants comprising a recombinant expression cassette of the invention. The recombinant expression cassettes are useful in methods of modulating source-sink relationships in plants and thereby enhancing yield or quality of harvested plant products, such as fruit. For example, the nucleic acids of the invention can be used to enhance sink activity and starch or lipid accumulation in seeds. Alternatively, the can be used to enhance sugar accumulation in fruit. The expression cassettes of the invention can also be used to enhance responsiveness to stress conditions in plants.
DEFINITIONS
The term “stress conditions” as used herein generally refers to nutritional and environmental stress that plants encounter in their life cycle. Examples of stress conditions are any nutritional or environmental changes that lead to changes in plant internal metabolic pathways and alterations in the plant's carbon reserves. Examples of environmental stresses include extreme temperature (e.g. excess heat or cold), high salt, flooding, anoxia, drought, toxic chemicals (e.g. herbicides, heavy metals) and the like.
The term “plant SNF4 polypeptide” refers to plant homologs of yeast SNF4. Without wishing to be bound by theory it is believed that the polypeptides of the invention are activating subunits in kinase cascades that act as metabolic sensors of carbohydrate availability and ATP levels in plant cells The proteins of the invention are a component in SNF1 related protein kinases which are composed of kinase subunits (SNF1), activation subunits (SNF4), and docking subunits (SIP). The tenn “LeSNF4” refers to plant SNF4 polypeptides derived from tomato (
Lycopersicon escuentum
).
Plant SNF4 polypeptides of the invention are typically from about 20 amino acids to about 400 amino acids in length, usually from about 100 to about 375, and often from about 200 to about 300 amino acids. A full length plant SNF4 polypeptide of the invention is typically about 375 amino acids.
The term “plant SNF1 polypeptide” refers to a plant homolog of the SNF1 subunit of the SNF1-related protein kinase. An example of a SNF1 nucleic acid is the LeSNF1 nucleic acid sequence as shown in SEQ ID NO.:4. An example of a SNF1 amino acid sequence is the LeSNF1 amino acid sequence as shown in SEQ ID NO.:5.
The phrase “nucleic acid sequence” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonuclcotidc bases read from the 5′ to the 3 end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
The term “promoter” refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Such a promoter can be derived from plant genes or from other organisms, such as viruses capable of infecting plant cells.
The term “plant” includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/str
Bradford Kent J.
Cooley Michael
Dahal Peetambar
Downie Bruce
Gee Oliver Henry
Fox David T.
Kubelik Anne R
The Regents of the University of California
Townsend and Townsend / and Crew LLP
LandOfFree
Regulation of source-sink relationships and responses to... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Regulation of source-sink relationships and responses to..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Regulation of source-sink relationships and responses to... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2867436