Scarecrow gene

Details Scarecrow gene Scarecrow gene

1997-04-24

2002-08-27

Nelson, Amy J. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S069100, C435S320100, C435S419000, C435S468000, C536S023600, C800S298000

Reexamination Certificate

active

06441270

ABSTRACT:

TABLE OF CONTENTS
1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
2.1. Root Development
2.2. Genes Regulating Root Structure
2.3. Geortropism
3. SUMMARY OF THE INVENTION
3.1. Definitions
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. SCR Genes
5.1.1. Isolation of SCR Genes
5.1.2. Expression of SCR Gene Products
5.1.3. Antibodies to SCR Proteins and Polypeptides
5.1.4. SCR Gene of Gene Products as Markers for Qualitative Trait Loci
5.2. SCR Promoters
5.2.1. Cis-Regulatory Elements or SCR Promoters
5.2.2. SCR Promoter-Driven Expression Vectors
5.3. Production of Transgenic Plants and Plant Cells
5.3.1. Transgenic Plants that Ectopically Express SCR
5.3.2. Transgenic Plants that Suppress Endogenous SCR Expression
5.3.3. Transgenic Plants that Express a Transgene Controlled by the SCR Promoter
5.3.4. Screening of Transformed Plants for Those Having Desired Altered Traits
EXAMPLE 1: Arabidopsis SCR Gene
6.1 Material and Methods
6.1.1. Plant Culture
6.1.2. Genetic Analysis
6.1.3. Mapping
6.1.4. Phenotypic Analysis
6.1.5. Molecular Techniques
6.1.6. In Situ Hybridization
6.2. Results
6.2.1. Characterization of the SCR Phenotype.
6.2.2. Characterization of Cell Identify in SCR Roots
6.2.3. Molecular Cloning of the SCR Gene
6.2.4. The SCR Gene has Motifs that Indicate it is a Transcription Factor
6.2.5. SCR is a Member of a Novel Protein Family
6.2.6. SCR is Expressed in the Cortex/Endodermal Initials and in the Endodermis
6.3. Discussion
6.3.1. The SCR Gene Regulates an Asymmetric Division Required for Root Radial Organization
6.3.2. SCR Involvement in Cell Specification of Cell Division
6.3.3. A Role for SCR in Embryonic Development
6.3.4. Tissue-Specific Expression of SCR is Regulated at the Transcriptional Level
6.3.5. A new Family of Transcriptional Regulators
7. EXAMPLE 2: Enhancer Trap Analysis of Root Development
7.1. Material and Methods
7.1.1. Plant Growth Conditions
7.1.2. Histology and Gus Staining
7.1.3. Construction of Enhancer Trap Lines
7.2. Results
7.2.1. Differential in the LRP
7.2.2. Marker Lines
7.2.3. ET199 Provides Evidence for the Role of SCR in Plant Development
8. EXAMPLE 3: Activity of Arabidopsis SCR Promoter in Transgenic Roots
9. EXAMPLE 4: Isolation SCR Sequences Using PRC-Cloning Strategy
10. EXAMPLE 5: Expression Pattern of Maize ZCR Gene in Root Tissue
11. EXAMPLE 6: Expression Pattern of ZCR Gene in Soybean Roots and Root Nodules
12. EXAPMPLE 7: SCR Expression Affects Gravitropism of Aerial Structures
13. Deposit of Microorganisms
1. INTRODUCTION
The present invention generally relates to the SCARECROW (SCR) gene family and their promoters. The invention more particularly relates to ectopic expression of members of the SCARECROW gene family in transgenic plants to artificially modify plant structures. The invention also relates to utilization of SCARECROW promoter for tissue and organ specific expression of heterologous gene products.
2. BACKGROUND OF THE INVENTION
Asymmetric cell divisions, in which a cell divides to give two daughters with different fates, play an important role in the development of all multicellular organisms. In plants, because there is no cell migration, the regulation of asymmetric cell divisions is of heightened importance in determining organ morphology. In contrast to animal embryogenesis, most plant organs are not formed during embryogenesis. Rather, cells that form the apical meristems are set aside at the shoot and root poles. These reservoirs of stem cells are considered to be the source of all post-embryonic organ development in plants. A fundamental question in developmental biology is how meristems function to generate plant organs.
2.1. Root Development
Root organization is established during embryogenesis. This organization is propagated during postembryonic development by the root meristem. Following germination, the development of the postembryonic root is a continuous process, a series of initials or stem cells continuously divide to perpetuate the pattern established in the embryonic root (Steeves & Sussex, 1972,
Patterns in Plant Development,
Englewood Cliffs, N.J.: Prentice-Hall, Inc.).
Due to the organization of the Arabidopsis root it is possible to follow the fate of cells from the meristem to maturity and identify the progenitors of each cell type (Dolan et al., 1993, Development 119:71-84). The Arabidopsis root is a relatively simple and well characterized organ. The radial organization of the mature tissues in the Arabidopsis root has been likened to tree rings with the epidermis, cortex, endodermis and pericycle forming radially symmetric cell layers that surround the vascular cylinder (FIG.
1
A). See also Dolan et al., 1993, Development 20 119:71-84. These mature tissues are derived from four sets of stem cells or initials: i) the columella root cap initial; ii) the pericycle/vascular initial; iii) the epidermal/lateral root cap initial; and iv) the cortex/endodermal initial (Dolan et al., 1993, Development 119:71-84). It has been shown that these initials undergo asymmetric divisions (Scheres et al., 1995, Development 121:53-62). The cortex/endodermal initial, for example, first divides anticlinally (in a transverse orientation) (FIG.
1
B). This asymmetric division produces another initial and a daughter cell. The daughter cell, in turn, expands and then divides periclinally (in the longitudinal orientation) (FIG.
1
B). This second asymmetric division produces the progenitors of the endodermis and the cortex cell lineages (FIG.
1
B). 2.2. Genes Regulating Root Structure
Mutations that disrupt the asymmetric divisions of the cortex/endodermal initial have been identified and characterized (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62). short-root (shr) and scarecrow (scr) mutants are missing a cell layer between the epidermis and the pericycle. In both types of mutants the cortex/endodermal initial divides anticlinally, but the subsequent periclinal division that increases the number of cell layers does not take place (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62). The defect is first apparent in the embryo and it extends throughout the entire embryonic axis which includes the embryonic root and hypocotyl (Scheres et al., 1995, Development 121:53-62). This is also true for the other radial organization mutants characterized to date, suggesting that radial patterning that occurs during embryonic development may influence the post-embryonic pattern generated by the meristematic initials (Scheres et al., 1995, Development 121:53-62).
Characterization of the mutant cell layer in shr indicated that two endodermal-specific markers were absent (Benfey et al., 1993, Development 119:57-70). This provided evidence that the wild-type SHR gene may be involved in specification of endodermis identity.
2.3. Geotropism
In plants, the capacity for gravitropism has been correlated with the presence of amyloplast sedimentation. See, e.g., Volkmann and Sievers, 1979, Encyclopedia Plant Physiol., N.S. vol 7, pp. 573-600; Sack, 1991, Intern. Rev. Cytol. 127:193-252; Björkmann, 1992, Adv. Space Res. 12:195-201; Poff et al., in
The Physiology of Tropisms
, Meyerowitz & Somerville (eds); Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1994) pp. 639-664; Barlow, 1995, Plant Cell Environ. 18:951-962. Amyloplast sedimentation only occurs in cells in specific locations at distinct developmental stages. That is, when and where sedimentation occurs is precisely regulated (Sack, 1991, Intern. Rev. Cytol. 127:193-252). In roots, amyloplast sedimentation only occurs in the central (columella) cells of the rootcap; as these cells mature into peripheral cap cells, the amyloplasts no longer sediment (Sack & Kiss, 1989, Amer. J. Bot. 76:454-464; Sievers & Braun, in
The Root Cap: Structure and Function,
Wassail et al. (eds.), New York: M. Dekker (1996) pp. 31-49). In stems of many plants, including Arabidopsis, amyloplast sedimentation occurs in the starch sheath (endodermis) especially in elongating regions

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