Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-03-16
2004-02-24
Mehta, Ashwin (Department: 1638)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100
Reexamination Certificate
active
06696560
ABSTRACT:
BACKGROUND OF THE INVENTION
The ability to control the size of plants and plant tissues is an enormously valuable tool. For example, for many agricultural crops, increasing the size of a plant or of a specific tissues within a plant would be of obvious commercial value. Currently, most attempts to increase plant size or yield are accomplished through traditional or marker-assisted breeding programs. Such methods have, however, failed to provide methods to directly control the size of plants or plant tissues.
Most cell proliferation in plants occurs in tissues called meristematic tissue. Several types of meristematic tissue exist in plants, including the shoot apical meristem, which gives rise to all aerial parts of the plant, the root apical meristem, which establishes the root system, and the vascular meristem, which provides lateral growth of the plant. While several genes are known to alter meristem fate, and thereby plant development, the mechanism by which they function is poorly understood. The products of the CLAVATA (CLV) and SHOOT MERISTEMLESS (STM1) genes of Arabidopsis, for example, encoding a receptor-kinase and homeodomain protein, respectively, appear to work antagonistically in a shoot meristem maintenance pathway involved in the partitioning of the central-peripheral zone (CZ-PZ) of the meristem. Other genes, such as ZWILLE (ZLL) and WUSCHEL (WUS), function early in embryonic development to specify the stem cells which will be maintained in the central zone of the shoot apical meristem. Other genes such as MGOUN1(MGO1) and MGOUN2 (MGO2) appear to function in the partitioning of cells from the PZ of the shoot apical meristem to leaf primordia or the inflorescence, often resulting in a fasciated meristem phenotype.
Plants containing mutations in the genes described above are defective in specific stages of meristem function and have well-characterized developmental phenotypes. As such, these genes are likely involved in the differentiation of meristematic cells, and are thus unlikely, by themselves, to provide tools to increase the size of plants or of plant tissues. Instead, it would be desirable to manipulate both the differentiation of meristematic cells as well as their growth and proliferation.
One potential method to alter the growth and/or proliferation of plant cells would be to modulate the activity of genes controlling these processes. For example, several groups have reported the cloning of at least a fragment of a Retinoblastoma-related protein in maize. See, e.g. Ach et al. (1997) Mol. Cell. Biol. 17:5077; Huntley et al. (1998)
Plant Mol. Biol
. 37:155; Grafi et al. (1996)
PNAS
93:8962; Shen et al. (1994) Plant Mol. Biol. 26:1085; Xie et al. (1996) EMBO J 15:4900; and WO 97/47745. None of these studies, however, has investigated the function of RRB in proliferating, virus-free cells. Further, no studies have heretofore addressed the role of RRB in an intact plant. As well known to those of skill, only by examining the role of a protein in its normal environment, in an intact organism, can its true activity and/or function be determined.
Thus, the art lacks a good understanding of the function of RRB in plant cells and/or intact plants. Without this understanding, its use to control plant growth in an efficient manner is difficult if not impossible. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
This invention provides methods and compositions for altering the growth and differentiation of plant tissues. The invention is based on the discovery that, in plants, genetically altering the levels of Retinoblastoma-related gene (RRB) activity produces dramatic effects on the growth, proliferation, and differentiation of plant meristem. Altering the level of RRB activity in a plant tissue, therefore, can be used to specifically control the growth and/or differentiation of plant meristem, thereby controlling, e.g. the relative size and distribution of individual tissues in a plant.
In certain embodiments, this invention provides polynucleotides and polypeptides with plant RRB function. In one embodiment, the polynucleotide is as shown in SEQ ID NO:1 or SEQ ID NO: 9. In one embodiment, the polynucleotide encodes the polypeptide shown as SEQ ID NO:2, or fragments thereof. In a preferred embodiment, the polynucleotide encodes a fill-length RRB protein. However, truncated forms of RRB proteins can be used as well. In addition, mutated forms of the RRB proteins can be used, e.g. as dominant negative forms.
This invention also provides transgenic plants comprising RRB polynucleotides. In preferred embodiments, the RRB polynucleotides are operably linked to a promoter, such as an inducible or tissue-specific promoter.
This invention also provides methods for inhibiting or enhancing the growth of plant cells, plant tissues, or entire plants. In preferred embodiments, RRB activity is enhanced or inhibited in a plant tissue by expressing a wild type,.mutant, or truncated form of an RRB polynucleotide, or by expressing an inhibitor of RRB activity, e.g. a peptide that competitively binds RRB, thereby preventing its normal interaction with intracellular substrates.
The methods provided herein can also be used to alter the differentiation of a plant tissue. In preferred embodiments, the differentiation of a meristem is altered. For example, the present invention provides methods for modulating the RRB activity in an apical shoot meristem, thereby altering the size, organization, and/or differentiation of the meristem and, as a result, affecting the structure and/or number of, e.g., a leaf primordium or an inflorescence bolt. Increasing or decreasing RRB activity can be effected in a plant, a plant tissue, or a plant cell by expressing a wild type, mutant, or truncated form of an RRB polynucleotide, or by expressing a peptide inhibitor of RRB activity. Such RRB polynucleotides are preferably linked to promoters such as a tissue-specific or an inducible promoter.
DEFINITIONS
A “nucleic acid” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. The term includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role. With respect to a naturally occurring nucleic acid that is “isolated” from its natural environment, the nucleic acid is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment. However, an “isolated” nucleic acid can refer to a recombinantly or synthetically produced nucleic acid, that is identical or altered from the naturally occurring nucleic acid sequence. In addition, an “isolated nucleic acid” can comprise naturally occurring nucleotides or can comprise any nucleotide derivative or analog, e.g. labeled nucleotides, that can be incorporated into a polynucleotide chain. Any aspect of the polynucleotide chain can be altered, such as the base, sugar, or phosphate backbone.
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 that works in plants, e.g. CaMV 35S. A “tissue-specific promoter” is a promoter capable of initiating transcription in a certain tissue of a plant. A “tissue specific promoters” can comprise a naturally occurring promoter that drives the expression of a gene in one or more specific tissues, or can comprise modified, truncated, or otherwise modified derivatives of naturally occurring promoters, or can comprise a synthetic promoter with the desired properties. A “tissue specific promoter” can drive the expression of a gene in one or more tissues, and throughout the entire tissue or only in a subset of the tissue. In addition, a “tissue-specific promoter” can drive gene expression in a tissue throughout the life of a plant, or transiently at one or more times during the life of the plant.
The te
Durfee Tim
Feiler Heidi
Gruissem Wilhelm
Jenkins Susan
Roe Judith
Chang Randall W. C.
Daubenspeck William C.
Gottlieb Paul A.
Mehta Ashwin
The United States of America as represented by the United States
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