Scarecrow gene, promoter and uses thereof Number:6,809,234 from the United States Patent and Trademark Office (PTO) owispatent

Title: Scarecrow gene, promoter and uses thereof

Abstract: The structure and function of a regulatory gene, SCARECROW (SCR), is described. The SCR gene is expressed specifically in root progenitor tissues of embryos, and in roots and stems of seedlings and plants. SCR expression controls cell division of certain cell types in roots and affects the organization of root and stem tissues, and affects gravitropism of aerial structures. The invention relates to the SCR gene, SCR-like genes, SCR gene products, (including but not limited to transcriptional products such as mRNAs, antisense, and ribozyme molecules, and translational products such the SCR protein, polypeptides, peptides and fusion proteins related thereto), antibodies to SCR gene products, SCR promoters and regulatory regions and the use of the foregoing to improve agronomically valuable plants.

Patent Number: 6,809,234 Issued on 10/26/2004 to Benfey,   et al.

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Inventors:	Benfey; Philip N. (New York, NY); Di Laurenzio; Laura (New York, NY); Wysocka-Diller; Joanna (New York, NY); Malamy; Jocelyn E. (New York, NY); Pysh; Leonard (New York, NY); Helariutta; Yrjo (New York, NY); Bruce; Wesley (Urbandale, IA); Lim; Jun (New York, NY)
Assignee:	New York University (New York, NY) Pioneer Hi-Bred International, Inc. (Johnston, IA)
Appl. No.:	09/265,585
Filed:	March 10, 1999

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	842445	Apr., 1997	6441270
	638617	Apr., 1996

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Current U.S. Class:	800/290 ; 435/320.1; 435/419; 435/468; 536/23.6; 800/278; 800/287
Field of Search:	800/287,290,278,298 435/320.1,419,468 536/23.1,23.6

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References Cited [Referenced By]

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U.S. Patent Documents

	5023179		June 1991		Lam et al.

Foreign Patent Documents

	WO 97/29123		Aug., 1997		WO
	WO 00/53723		Sep., 2000		WO

Other References

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Primary Examiner: Fox; David T.
Assistant Examiner: Baum; Stuart F.
Attorney, Agent or Firm: Nixon Peabody LLP

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Government Interests

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This invention was made with government support under grant number: GM43778 awarded by the National Institute of Health. The government may have certain rights in the invention.

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Parent Case Text

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This application is a continuation-in-part of application Ser. No. 08/842,445, filed Apr. 24, 1997, now U.S. Pat. No. 6,441,270 which is a continuation-in-part of application Ser. No. 08/638,617, filed Apr. 26, 1996, now abandoned, the disclosures of which are herein incorporated by reference in their entirety.

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Claims

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What is claimed is:

1. An isolated nucleic acid molecule wherein the nucleic acid molecule comprises SEQ ID NO: 95 or the complement thereof.

2. An isolated nucleic acid molecule comprising a nucleic acid sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 96.

3. A DNA vector containing the nucleic acid molecule of claim 1 or 2.

4. An expression vector containing the nucleic acid molecule of claim 1 or 2, operatively associated with a regulatory sequence containing transcriptional and translational regulatory elements that control expression of the nucleic acid in a host cell.

5. A genetically-engineered host cell containing the nucleic acid molecule of claim 1 or 2.

6. A genetically-engineered host cell containing the nucleic acid molecule of claim 1 or 2, operatively associated with a regulatory sequence containing transcriptional and translational regulatory elements that control expression of the nucleic acid in a host cell.

7. A genetically-engineered plant containing the nucleic acid molecule of claim 1 or 2.

8. A plant genetically-engineered to overexpress a SCARECROW protein or polypeptide said protein or polypeptide being encoded by the nucleic acid molecule of claim 1 or 2, wherein cell division in the plant is increased, resulting in a genetically-engineered plant that has thicker roots and/or is straighter than a non-genetically engineered plant.

9. A plant genetically-engineered to overexpress a SCARECROW protein or polypeptide comprising SEQ ID NO: 96, so that cell division is increased in roots, resulting in a genetically-engineered plant that has thicker roots than a non-genetically engineered plant.

10. A plant genetically-engineered to overexpress a SCARECROW protein or polypeptide comprising SEQ ID NO: 96, wherein the plant is straighter than a non-genetically engineered plant.

11. The plant of claim 10 wherein said plant is less susceptible to lodging than a non-genetically engineered plant.

12. A trangenic plant containing a transgene comprising the nucleic acid molecule of claim 1 or 2.

13. The transgenic plant of claim 12 wherein the nucleic acid molecule is operatively associated with a regulatory sequence containing transcriptional and translational regulatory elements that control expression of the nucleic acid in a transgenic plant cell.

14. A method for expressing a nucleic acid that encodes a SCARECROW protein or polypeptide comprising SEQ ID NO: 96 in a host cell, comprising: (a) culturing the genetically-engineered host cell of claim 5, and (b) inducing the transcriptional and translational regulatory elements that control expression of the nucleic acid.

15. A method for producing a transgenic plant, comprising transforming a plant cell with the nucleic acid molecule of claim 1 or 2 operatively associated with a regulatory sequence containing transcriptional and translational regulatory elements that control expression of the nucleic acid in the plant cell.

16. A method for expressing a nucleic acid that encodes a SCARECROW protein or polypeptide comprising SEQ ID NO: 96 in a host cell, comprising: (a) culturing the genetically-engineered host cell of claim 6, and (b) inducing the transcriptional and translational regulatory elements that control expression of the nucleic acid.

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Description

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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 the 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, wherein 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.).

2.1.1. Arabidopsis Root Development

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. 1A). See also Dolan et al., 1993, Development 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. 1B). 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. 1B). This second asymmetric division produces the progenitors of the endodermis and the cortex cell lineages (FIG. 1B).

Furthermore, root radial organization in Arabidopsis is produced by three distinct developmental strategies. First, primary roots employ stem cells, wherein initials undergo asymmetric divisions first to regenerate themselves and then to generate the cell lineages of the root (FIG. 1B). Second, in the embryo, sequential asymmetric divisions subdivide pre-existing tissue to form the cell layers of the embryonic root. Finally, lateral roots are formed by a strategy of cell proliferation that originates in differentiated tissues. Remarkably, within a given species, all three strategies result in roots with a nearly identical radial organization.

2.1.2. Maize Root Development

The root organization of Zea mays (maize), which is a very well characterized member of the grass family, is far more complex than the root organization in Arabidopsis. The root system of maize consists of primary, embryonic, lateral, seminal lateral and adventitious roots. Both primary and seminal lateral roots are formed during embryogenesis, wherein the primary root is the first root to emerge during germination, followed by the seminal lateral roots formed at the scutellar nodal region (Freeling, M. and Walbot, V. (1994), The Maize Handbook, (New York: Springer-Verlag); Hetz, W. et al., (1996), Plant J. 10:845-857). Both crown and prop roots which develop post-embryonically are shoot-borne roots, often termed "adventitious". However, since these roots are part of the normal development of the plant, they are not, strictly speaking, adventitious roots, which are typically formed as a result of injury or hormone treatment. Crown roots, representing the major roots of the mature plant, are formed at consecutive early nodes of the stem beginning with the coleoptilar nodes. Later in development, brace or prop roots emerge from nodes above the soil level (Freeling, M. and Walbot, V. (1994), The Maize Handbook, (New York: Springer-Verlag); Hetz, W. et al., (1996), Plant J. 10:845-857).

Currently, there are two notably different types of organization of root apical meristems: an open and a closed meristem. In an "open" meristem, the cell files of the mature tissues cannot be traced with much confidence to distinct initials, and the incipient tissues do not appear to have discrete boundary walls between the root proper and the root cap (Clowes, F. A. L., 1981, Ann. Bot. 48:761-767). Therefore, the interpretation of the organization of the open meristem has been problematic (Clowes, F. A. L., 1981, Ann. Bot. 48:761-767). In a "closed" meristem, however, since files of cells converge onto a pole at the root apex, it is easy to identify discrete layers in median longitudinal sections (Clowes, F. A. L., 1981, Ann. Bot. 48:761-767).

Both Arabidopsis and maize roots show characteristics of the closed meristem (FIGS. 23A-B). However, there are important differences. In maize roots, the root apical meristem consists of three independent layers of initials. One gives rise to the stele, the second gives rise to epidermis, cortex and endodermis and the third generates the root cap, whereas in the Arabidopsis root apical meristem, the epidermis shares a common initial with the lateral root cap (Esau, K., 1977, Anatomy of Seed Plants. 2nd ed. (New York: John Wiley & Sons); Esau, K., 1953, Plant Anatomy. (New York: John Wiley & Sons)).

Primary organization of the root apical meristem in maize occurs during embryogenesis, (Steeves, T. A. and Sussex, I. M., (1989), Patterns in plant development., 2nd ed. (Cambridge University Press)) as in Arabidopsis. There are three main phases in embryo development in maize (FIGS. 24A-B) (Freeling, M. and Walbot, V. (1994), The Maize Handbook, (New York: Springer-Verlag); Steeves, T. A. and Sussex, I. M. (1989), Patterns in plant development., 2nd ed., (Cambridge University Press); Sheridan, W. F. and Clark, J. K., (1993), Plant J. 3:347-358). As in Arabidops is, the very first division of the zygote establishes the initial asymmetry of the embryo (FIG. 24A). However, unlike Arabidopsis, embryonic development in maize is characterized by rather irregular cell divisions (Sheridan, W. F. and Clark, J. K., (1993), Plant J. 3:347-358). During the first phase, the apical-basal asymmetry of the embryo is established, and the embryo is regionalized into suspensor and embryo proper (FIGS. 24B-C). During the second phase, radial asymmetry appears and the embryonic axis and meristems are established (FIGS. 24D-E) (Clowes, F. A. L., (1978), New Phytol. 80:409-419). Finally, during the third phase, vegetative structures such as embryonic roots and leaves are elaborated (FIGS. 24F-G) (Sheridan, W. F. and Clark, J. K., (1993), Plant J. 3:347-358).

2.1.3. The Quiescent Center

The quiescent center (QC) of root apical meristems of angiosperms is a population of mitotically inactive cells. In the QC of the primary root of maize, for example, the average duration of a mitotic cycle is about 200 hours compared with only 12 hours in the cells just below the QC and 28 hours in the cells just above the QC (Clowes, F. A. L., (1961), J. Exp. Bot. 9:229-238). Moreover, there are also reductions in the rates of synthesis of DNA and protein, and corresponding reductions in the amounts of DNA and RNA per cell (Clowes, F. A. L., (1956), New Phytol. 55:29-34).

Although the precise role of the QC has remained speculative, it is generally accepted that cells within the QC are undifferentiated and, other than the anatomical pattern of cell files, lacking in radial pattern information. This theory has been supported by ablation studies performed in Arabidopsis, wherein, complete laser ablation of the four central cells in the Arabidopsis QC led to subsequent restoration of the QC by cells of the stele. Furthermore, laser ablation of only one or two cells in the QC resulted in differentiation of surrounding initial cells. Analysis of the hobbit mutants further supports these observations. In the hobbit mutants, there is no functional QC, leading all cortex initials to divide into cortex and endodermis during embryogenesis (van den Berg, C., et al., (1995), Nature 378:62-65). Taken together, it is suggested that the QC suppresses differentiation of surrounding initials in the range of a single cell (van den Berg, C., et al., (1995), Nature 378:62-65).

In maize, on which the contemporary view of the role of the QC is based (Feldman, L. J., (1984), Amer. J. Bot. 71:1308-1314; Freeling, M. and Walbot, V., (1994), The maize handbook (New York: Springer-Verlag)), surgical and tissue culture systems were developed to study the organization process of root apical meristems (Feldman, L. J., (1976), Planta 128:207-212). Following removal of the QC, the remaining root regenerates a new root tip. This process appears to involve de novo organization of the QC and the apical meristem (Feldman, L. J., (1976), Planta 128:207-212). In addition, the excised QC itself is capable of generating a new root (Feldman, L. J. and Torrey, J. G., (1976), Amer. J. Bot. 63:345-355). This suggests that there is indeed sufficient radial pattern information in the QC to allow the regeneration of more or less intact roots.

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 true also for 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 the 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; Bjorkmann, 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 of the stem (von Guttenberg, Die Physioloaischen Scheiden, Handbuch der Pflanzenanatomie; K. Linsbauer (ed.), Berlin: Gebruder Borntraeger, vol. 5 (1943) p. 217; Sack, 1987, Can. J. Bot. 65:1514-1519; Sack, 1991, Intern. Rev. Cytol. 127:193-252; Caspar & Pickard, 1989, Planta 177:185-197; Volkmann et al., 1993, J. Pl. Physiol. 142:710-6).

Gravitropic mutants have been studied for evidence that proves the role of amyloplast sedimentation in gravity sensing. However, many gravitropic mutations affect downstream events such as auxin sensitivity or metabolism (Masson, 1995, BioEssays 17:119-127). Other mutations seem to affect gene products that process information from gravity sensing. For example, the lazy mutants of higher plants and comparable mutants in mosses can clearly sense and respond to gravity, but the mutations reverse the normal polarity of the gravitropic response (Gaiser & Lomax, 1993, Plant Physiol. 102:339-344; Jenkins et al., 1986, Plant Cell Environ 9:637-644). Other mutations appear to affect gravitropism of specific organs. For example, sgr mutants have defective shoot gravitropism (Fukaki et al., 1996, Plant Physiol. 110:933-943; Fukaki et al., 1996, Plant Physiol. 110:945-955; Fukaki et al., 1996, Plant Res. 109:129-137).

Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The structure and function of a regulatory gene, SCARECROW (SCR), is described. The SCR gene is expressed specifically in root progenitor tissues of embryos, and in certain tissues of roots and stems. SCR expression controls cell division of certain cell types in roots, and affects the organization of root and stem. The present invention relates to the SCARECROW (SCR) gene (which encompasses the Arabidopsis SCR gene and its orthologs and paralogs), SCR-like genes, SCR gene products, (including, but not limited to, transcriptional products such as mRNAs, antisense and ribozyme molecules, and translational products such as the SCR protein, polypeptides, peptides and fusion proteins related thereto), antibodies to SCR gene products, SCR regulatory regions and the use of the foregoing to improve agronomically valuable plants.

The invention is based, in part, on the discovery, identification and cloning of the gene responsible for the scarecrow phenotype. In contrast to the prevailing view that the SCR gene was likely to be involved in the specification of endodermis, the inventors have determined that the mutant cell layer in roots of scr mutants has differentiated characteristics of both cortex and endodermis. This is consistent with a role for SCR in the regulation of asymmetric cell division rather than in specification of the identity of either cortex or endodermis. The inventors have determined also that SCR expression affects the gravitropism of plant aerial structures such as the stem.

One aspect of the invention relates to the heterologous expression of SCR genes and related nucleotide sequences, and specifically the Arabidopsis SCR and maize ZCARECROW (ZCR) genes, in stably transformed higher plant species. Modulation of SCR and ZCR expression levels can be used to advantageously modify root and aerial structures of transgenic plants and enhance the agronomic properties of such plants.

Another aspect of the invention relates to the use of promoters of SCR genes, and specifically the use of the Arabidopsis SCR and maize ZCR promoters to control the expression of protein and RNA products in plants. Plant SCR promoters have a variety of uses, including, but not limited to, expressing heterologous genes in the embryo, root, root nodule and stem of transformed plants.

The invention is illustrated by working examples, described infra, which demonstrate the isolation of the Arabidopsis SCR gene using insertion mutagenesis. More specifically, T-DNA tagging of genomic and cDNA clones of the Arabidopsis SCR gene are described. Other working examples include the isolation of SCR sequences from plant genomes using PCR amplification in combination with screening of genomic libraries, and heterologous gene expression in transgenic plants using SCR promoter expression constructs. Additional working examples describe the cloning and isolation of maize ZCR genes using probes derived from the Arabidopsis SCR gene on a maize genomic library. Still other working examples describe the characterization of the maize ZCR expression pattern in primary and embryonic roots, and during regeneration of the root tip following excision of the QC.

Structural analysis of the deduced amino acid sequence of Arabidopsis SCR protein indicates that SCR encodes a transcription factor. Northern analysis, in situ hybridization analysis and enhancer trap analysis show highly localized expression of Arabidopsis SCR and maize ZCR in embryos and roots. Genetic analysis shows SCR expression also affects gravitropism of aerial structures (e.g., stems and shoots). This indicates that SCR is also expressed in those structures.

Computer analysis of the deduced amino acid sequence of Arabidopsis SCR protein with those of Expressed Sequence Tag (EST) sequences and genomic sequences in GenBank reveals the existence of at least eighteen SCR genes in Arabidopsis, one SCR gene in maize, four SCR genes in rice, and one SCR gene in Brassica. A further aspect of the invention relates to the use of such EST sequences to obtain larger and/or complete clones of the corresponding SCR gene.

The various embodiments of the claimed invention presented herein are by way of illustration only and are in no manner intended to limit the scope of the invention.

3.1. Definitions

As used herein, the terms listed below will have the meanings indicated.

35S = cauliflower mosaic virus promoter for the 35S transcript CDNA = complementary DNA cis-regulatory = A promoter sequence 5' upstream of the TATA element box that confers specific regulatory response to a promoter containing such an element. A promoter may contain one or more cis- regulatory elements, each responsible for a particular regulatory response coding = sequence that encodes a complete or partial sequence gene product (e.g., a complete protein or a fragment thereof) DNA = deoxyribonucleic acid EST = expressed sequence tag functional = a functional portion of a promoter is any portion portion of a promoter that is capable of causing transcription of a linked gene sequence, e.g., a truncated promoter gene = a gene construct comprising a promoter fusion operably linked to a heterologous gene, wherein said promoter controls the transcription of the heterologous gene gene = the RNA or protein encoded by a gene sequence product gene = sequence that encodes a complete gene product sequence (e.g., a complete protein) GUS = 1,3-.beta.-Glucuronidase gDNA genomic DNA heterologous = In the context of gene constructs, a gene heterologous gene means that the gene is linked to a promoter that said gene is not naturally linked to. The heterologous gene May or may not be from the organism contributing said promoter. The heterologous gene may encode messenger RNA (mRNA), antisense RNA or ribozymes homologous = a native promoter of a gene that selectively promoter hybridizes to the sequence of a SCR gene described herein mRNA = messenger RNA operably = A linkage between a promoter and gene sequence linked such that the transcription of said gene sequence is controlled by said promoter ortholog = related gene in a different plant (e.g., maize ZCARECROW gene is an ortholog of the Arabidopsis SCR gene) paralog = related gene in the same plant (e.g., Arabidopsis SCLa1 is a paralog of Arabidopsis SCR gene) RNA = ribonucleic acid RNase = ribonuclease SCR = SCARECROW gene or portion thereof, (italic) encompasses SCR and ZCR genes and their orthologs and paralogs SCR = SCARECROW protein scr = scarecrow mutant (e.g., scr1) (lower case) SCL = SCARECROW-like gene ZCR = maize ZCARECROW gene, an ortholog of, for example, the Arabidopsis SCR gene

SCR protein means a protein containing sequences or a domain substantially similar to one or more motifs (i.e., Motifs I-VI), preferably MOTIF III (VHIID), of the Arabidopsis SCR protein as shown in FIGS. 13A-F and FIGS. 15A-S. SCR proteins include SCR ortholog and paralog proteins having the structure and activities described herein.

SCR polypeptides and peptides include deleted or truncated forms of the SCR protein, and fragments corresponding to the SCR motifs described herein.

SCR fusion proteins encompass proteins in which the SCR protein or an SCR polypeptide or peptide is fused to a heterologous protein, polypeptide or peptide.

SCR gene, nucleotides or coding sequences mean nucleotides, e.g., gDNA or cDNA encoding SCR protein, SCR polypeptides, peptides or SCR fusion proteins.

SCR gene products include transcriptional products such as mRNAs, antisense and ribozyme molecules, as well as translational products of the SCR nucleotides described herein, including, but not limited to, the SCR protein, polypeptides, peptides and/or SCR fusion proteins.

SCR promoter means the regulatory region native to the SCR gene in a variety of species, which promotes the organ and tissue specific pattern of SCR expression described herein.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B. Schematic of Arabidopsis root anatomy. FIG. 1A. Transverse section showing the four tissues, epidermis, cortex, endodermis and pericycle that surround the vascular tissue. In the longitudinal section, the epidermal/lateral root cap initials and the cortex/endodermal initials are shown at the base of their respective cell files. FIG. 1B. Schematic of division pattern of the cortex/endodermal initial. The initial expands then divides anticlinally to reproduce itself and a daughter cell. The daughter then divides periclinally to produce the progenitors of the endodermis and cortex cell lineages. Abbreviations: C, cortex; Da, daughter cell; E, endodermis; In, initial.

FIGS. 2A-F. Phenotype of scr mutant plants. FIG. 2A. Shown left to right are 12-day scr-2, scr-1 and wild-type seedlings grown vertically on nutrient agar medium. FIG. 2B. 21-day scr-2 mutant plants in soil. FIG. 2C. Transverse section through primary root of 7-day scr-2. FIG. 2D. Transverse section through primary root of 7-day wild-type (WT). FIG. 2E. Transverse section through lateral root of 12-day scr-2 mutant seedling. FIG. 2F. Transverse section through root regenerated from scr-1 callus. Bar, 50 .mu.m. Abbreviations: C, cortex; En, endodermis; Ep, epidermis; M, mutant cell layer; P, pericycle; V, vascular tissue.

FIGS. 3A-F. Characterization of the cellular identity of the mutant cell layer. FIG. 3A. Endodermis-specific Casparian band staining of transverse sections through the primary root of 7-day scr-1 mutant. (Note: the histochemical stain also reveals xylem cells in the vascular cylinder.) FIG. 3B. Casparian band staining of transverse sections through the primary root of 7-day wild-type (WT). FIG. 3C. Immunostaining with the endodermis (and a subset of vascular tissue) specific JIM13 monoclonal antibodies on transverse root sections of scr-2 mutant. FIG. 3D. Immunostaining with JIM13 monoclonal antibodies on transverse root sections of WT. FIG. 3E. Immunostaining with the JIM7 monoclonal antibody that stains all cell walls on transverse root sections of scr-2 mutant. FIG. 3F. Immunostaining with JIM7 monoclonal antibodies on transverse root sections of WT. Bar, 25 .mu.m. Abbreviations are same as those for description of FIGS. 2A-2F and: Ca, casparian strip.

FIGS. 4A-F. Immunostaining. FIG. 4A. Immunostaining with the cortex (and epidermis) specific CCRC-M2 monoclonal antibodies on transverse root sections of scr-1 mutant. FIG. 4B. Immunostaining with CCRC-M2 antibodies on transverse root sections of scr-2 mutant. FIG. 3C. Immunostaining with CCRC-M2 antibodies on transverse root sections of wild-type (WT). FIG. 4D. Immunostaining with the CCRC-M1 monoclonal antibodies (specific to a cell wall epitope found on all cells) on transverse root sections of scr-1. FIG. 4E. Immunostaining with CCRC-M1 antibodies on transverse root sections of scr-2. FIG. 4F. Immunostaining with CCRC-M1 antibodies on transverse root sections of WT. Bar, 30 .mu.m. Abbreviations are same as those for description of FIGS. 2A-2F.

FIGS. 5A-1, 5A-2, 5B, 5C, 5D, 5E-1, and 5E-2. Structure of the Arabidopsis SCARECROW gene. FIGS. 5A-1 and 5A-2. Nucleic acid sequence and deduced amino acid sequence of the Arabidopsis SCR genomic region (SEQ ID NO:1) and (SEQ ID NO:2), respectively. Regulatory sequences including: (i) TATA box, (ii) ATG start codon, and (iii) potential polyadenylation sequence are underlined. Within the deduced amino acid sequence, homopolymeric repeats are underlined. FIG. 5B. Schematic diagram of genomic clone indicating possible functional motifs, T-DNA insertion sites and subclones used as probes. Abbreviations: Q,S,P,T, region with homopolymeric repeats of these amino acids; b, region with similarity to the basic region of bZIP factors; I and II, regions with leucine beptad repeats; E, acidic region. FIG. 5C. Comparison of the charged region found in Arabidopsis SCR protein with that found in bZIP transcription factors, SCR bZIP-like domain (SEQ ID NO:3), GCN4 (SEQ ID NO:4), TGA1 (SEQ ID NO:5), C-Fos (SEQ ID NO:6), c-JUN (SEQ ID NO:7), CREB (SEQ ID NO:8), Opaque-2 (SEQ ID NO:9), OBF2 (SEQ ID NO:10), RAF-1 (SEQ ID NO:11). FIG. 5D. Translations of EST clones encoding putative peptide having similarities to the VHIID domain region of Arabidopsis SCR protein (SEQ ID NO:12), F13896 (SEQ ID NO:13), Z37192 (SEQ ID NO:14), and Z25645 (SEQ ID NO: 15) are from Arabidopsis, T18310 (SEQ ID NO:17) is from maize and D41474 (SEQ ID NO:16) is from rice. FIGS. 5E-1 and 5E-2. The deduced amino acid sequence of the Arabidopsis SCARECROW gene (SEQ ID NO:2).

FIGS. 6A-B. Expression of the Arabidopsis SCARECROW gene. FIG. 6A. Northern blot of total RNA from wild-type siliques (Si), roots (R), leaves (L) and whole seedlings (Sd) hybridized with Arabidopsis SCR probe a and with a probe from the Arabidopsis glutamine dehydrogenase (GDH) gene (Melo-Oliveira et al., 1996, Proc. Natl. Acad. Sci. USA 93:4718-4723) as a control for RNA integrity. (GDH expression is lower in siliques than in vegetative tissues.) The 1.6 kb band corresponds to the GDH gene and the approximately 2.5 kb band corresponds to SCR. Ribosomal RNA is shown as a loading control. FIG. 6B. Northern blot of Arabidopsis wild-type, scr-1 and scr-2 total RNA, probed with Arabidopsis SCR probe "a" corresponding to a cDNA sequence shown in FIG. 5B , and with the GDH probe. In scr-2 mutant additional bands of 4.1 kb and 5.0 kb were detected.

FIGS. 7A-G. In situ hybridization and enhancer trap analyses of Arabidopsis SCR expression. FIG. 7A. SCR RNA expression detected by in situ hybridization of SCR antisense probe to a longitudinal section through the root meristem. FIG. 7B. In situ hybridization of SCR antisense probe to a transverse section in the meristematic region. FIG. 7C. In situ hybridization of SCR antisense probe to late torpedo stage embryo. FIG. 7D. Negative control in situ hybridization using a SCR sense probe to a longitudinal section through the root meristem. FIG. 7E. GUS expression in a whole mount in the enhancer trap line, ET199 in primary root tip. FIG. 7F. GUS expression in the ET199 line in transverse root section in the meristematic region. FIG. 7G. GUS expression in ET199 detected in a section through the root meristem. GUS expression is observed in the cortex/endodermal initial, and in the first cell in the endodermal cell lineage but not in the first cell of the cortex lineage. Expression in two endodermal layers is observed higher up in the root because the section was not median at that point. Bar, 50 .mu.m. Abbreviations are same as those in the description of FIGS. 2A-2F.

FIGS. 8A-B. Partial nucleotide sequence (SEQ ID NO: 18) and deduced amino acid sequence (SEQ ID NO:19) of the Arabidopsis SCLa4 gene.

FIGS. 9A-B. Partial nucleotide sequence (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) of the Arabidopsis SCLa3 gene.

FIG. 10. Partial nucleotide sequence (SEQ ID NO:22) of the Arabidopsis SCLa1 gene.

FIG. 11A. Nucleotide sequence (SEQ ID NO:24) and deduced amino acid sequence (SEQ ID N0:25) of the maize Zm-Scl1 fragment.

FIGS. 11B1 and 11B2. Partial nucleotide sequence (SEQ ID NO:26) and deduced amino acid sequence (SEQ ID NO:27) of the maize SCLm1 gene (Zm-Scl2).

FIG. 12A-B. Nucleotide sequence of rice SCLo3 EST clone. FIG. 12A. Sequence of 5' end of EST clone (SEQ ID NO:28). FIG. 12B. Sequence of 3' end of EST clone (SEQ ID NO:29).

FIGS. 13A-F. Comparison of the amino acid sequence of members of the SCARECROW family of genes. Conserved Motifs I through VI are indicated by dashed line above the aligned sequences. Consensus sequences are shown in bold. See Table 1 for the identity and sequence identifier number of each of the sequences shown in this Figure.

FIG. 14. Restriction map of the approximately 8.8 kb EcoRI insert DNA of lambda clone, t643, containing the Arabidopsis SCR gene. The locations of the approximately 5.6 kb HindIII-SacI fragment subcloned in plasmid LIG1-3/SAC+MoB.sub.2 1SAC, and the SCR coding region are indicated below the restriction map. The location of the translational initiation site of the SCR gene is at the NcoI site at the left end of the indicated coding region. The SCR coding sequence begins at the translation initiation site and extends approximately 1955 nucleotides to its right. E. coli DH5.alpha. containing plasmid pLIG1-3/SAC+MoB.sub.2 1SAC, has the ATCC accession number 98031.

FIGS. 15A-S. Comparison of the partial and complete amino acid sequences of several plant members of the SCARECROW family of genes. The amino acid sequences are aligned in a manner that maximizes amino acid sequence similarity and identity among SCR family members. Each sequence shown is continuous except where noted otherwise; the dots are inserted between two sequence segments in order (2 to align homologous segments. "X" in the middle of a sequence indicates ambiguity in the corresponding nucleotide sequence and, possible termination of the ORF at the "X" residue site. "X" at the end of a sequence indicates termination of the ORF at the "X" residue site. The numbering of the amino acid residues is shown at the bottom of each figure and is based on the Arabidopsis SCR amino acid sequence. Conserved Motifs I through VI are indicated by the various dashed lines above the figures. The new and old names of the family members are shown in FIG. 15A. The sequences of SCR, Tf1 and Tf4 are of the complete SCR protein. The sequence identifier numbers are as follows: SCR (SEQ ID NO: 2); 3989 (SEQ ID NO: 36); 12398 (SEQ ID NO: 52); 4871 (SEQ ID NO: 46); 11846 (SEQ ID NO: 59); 2504 (SEQ ID NO: 44); 3935 (SEQ ID NO: 21); 11261 (SEQ ID NO: 50); 713 (SEQ ID NO: 43); 10964 (SEQ ID NO: 48); 23196 (SEQ ID NO: 58); Tf1 (SEQ ID NO: 34); Tf4 (SEQ ID NO: 35); 18310 (SEQ ID NO: 37); 18652 (SEQ ID NO: 54); 4818 (SEQ ID NO: 19); 21729 (SEQ ID NO: 151); 1110 (SEQ ID NO: 23); 174 (SEQ ID NO: 42); and 33/08 (SEQ ID NO: 41).

FIGS. 16A-M. The partial nucleotide sequences of several plant members of the SCARECROW family of genes. "N" indicates an unknown base. See Table 1 for the identity and the sequence identifier number of each sequence shown in these figures.

FIGS. 17A-1 and 17A-2. The partial nucleotide sequence (SEQ ID NO:66) of the maize ZCR gene.

FIG. 17B. The partial amino acid sequence (SEQ ID NO:67) of the maize ZCR gene. The underlined sequence shares approximately 80% sequence identity with a corresponding sequence of Arabidopsis SCR protein.

FIG. 18. Comparison of the partial amino acid sequences of several SCR ortholog sequences amplified from the genomes of carrot, soybean and spruce. The SCLd1 and SCLp1 sequences each were obtained by PCR amplification using a combination of IF and 1R primers. The SCLg1 sequence was obtained by PCR amplification using a combination of 1F and WP primers. See, for example, Section 5.1.1., infra. The amino acid sequences are aligned in a manner that maximizes amino acid sequence identity and similarity amongst these sequences. Each sequence shown is continuous except where noted otherwise; the dashes are inserted between two sequence segments in order to allow alignment of homologous segments. "x" in the middle of a sequence indicates ambiguity in the corresponding nucleotide sequence and, possible termination of the ORF or existence of an intron at the "x" residue site. See Table 1 for the identity and the sequence identifier number of each sequence shown in this figure.

FIGS. 19A-G. Comparison of promoter activities in transgenic lines and roots. FIG. 19A. A stably transformed line containing four copies of the B2 subdomain of the 35S promoter of CaMV upstream of GUS (Benfey et al., 1990). GUS is expressed in the root tip. FIG. 19B. Roots emerging from callus transformed with four copies of the B2 subdomain of the 35S promoter fused to GUS. GUS expression can be seen in the emerging root tips (arrows). FIG. 19C We. Higher magnification of a root emerging from the callus in FIG. 19C. GUS is clearly restricted to the root tip. The morphology of roots regenerated from calli often appears abnormal. FIG. 19D. A transgenic plant regenerated from the calli and roots shown in FIG. 19B. GUS expression in this plant appears to be similar to that of the original line shown in FIG. 19A. FIG. 19E. ET199, a stably transformed line that contains an enhancer trapping construct with a minimal promoter fused to the GUS coding region inserted 1 kb upstream from the SCR coding region. GUS expression is primarily in the endodermal layer of the root. FIG. 19F. Roots emerging from calli transformed with the SCR promoter::GUS construct. Expression of the GUS gene appears to be limited to an internal layer (arrows). FIG. 19G. SCR promoter::GUS transformed root in liquid culture. Roots shown in FIG. 19F were excised and transferred to liquid cultures. GUS expression is primarily found in the endodermal layer as in ET199. The expression of GUS in the quiescent center, as seen here, is also sometimes observed in ET199. Bar, 50 .mu.m.

FIGS. 20A-B. Analysis of SCR promoter activity in the scr mutant background. FIG. 20A. Roots emerging from scr calli transformed with the SCR promoter::GUS construct. Roots regenerated from scr calli are very short. GUS expression appears to be limited to an internal layer of the root (arrows). FIG. 20B. Root regenerated from transformed scr calli and transferred to liquid culture. The scr phenotype, a single layer between the epidermis and pericycle, is easily seen. GUS expression is limited to this mutant layer. E, Epidermis. M, Mutant Layer. P, Pericycle. Bar, 50 .mu.m.

FIGS. 21A-F. Molecular Complementation of the scr mutant. FIGS. 21A, 21B, 21C, and 21E. scr transformed with the SCR promoter::GUS construct. FIGS. 21B, 21D, and 21F. scr transformed with the SCR promoter::SCR coding region construct. FIGS. 21A, 21B. Roots emerging from scr calli. Arrows point to several very short roots among many fine root hairs in the scr calli transformed with the SCR promoter::GUS construct. In contrast, roots from scr calli transformed with the SCR promoter::SCR coding region construct appeared to be wild-type in length, suggesting molecular complementation by the transgene. FIGS. 21C and 21D. Transgenic roots in liquid culture. The scr roots transformed with the SCR promoter::GUS construct appeared short, while those transformed with the SCR promoter::SCR coding region construct appeared of wild-type length. FIGS. 21E and 21F. Transverse sections through roots emerging from calli. Whereas there is only a single cell layer between the epidermis and stele in the SCR promoter::GUS transformed root, the radial organization of the root transformed with the SCR promoter::SCR coding region appeared identical to wild-type, with both cortex and endodermal layers. E, epidermis. M, mutant layer. C, cortex. En, Endodermis. P, Pericycle. Bar, 50 .mu.m.

FIGS. 22A-F. Expression of ZCR in maize root tips. FIG. 22A. Expression of ZCR is in the endodermal layer and extends down through the region of the quiescent center. FIGS. 22B-C. Higher magnification showing expression in a single cell layer through the quiescent center. FIG. 22D. Expression of ZCR in the maize embryonic root. FIG. 22E. Higher magnification showing expression in the embryonic root. FIG. 22F. Expression of ZCR in the maize lateral root.

FIGS. 23A-B. Root apical meristems of maize and Arabidopsis. Both show a type of a closed meristem in which all files of cells converge onto a pole at the root apex, making the boundary between the root proper and the root cap discrete. FIG. 23A. A schematic representation of the monocotyledonous closed-type of root apical meristem of maize. FIG. 23B. A schematic representation of the dicotyledonous closed-type of root apical meristem of Arabidopsis.

FIGS. 24A-G. Embryo development in Maize. FIG. 24A. Three-celled embryo establishing the initial asymmetry and showing the first division of a terminal cell. FIGS. 24B-C. Embryos showing embryo proper and suspensor. FIGS. 24D-E. Embryos showing radial asymmetry and the initial development of shoot and root apical meristems. FIGS. 24F-G. Embryos showing the elaborate organization of shoot and root apical meristems.

FIGS. 25A-C. Maize Scarecrow gene. The nucleotide (SEQ ID NO:95) and deduced amino acid sequence (SEQ ID NO:96) of the maize scarecrow gene (ZCR) is shown (SEQ ID NO:95-98). The amino acid numbers are shown on the right, while the nucleotides are numbered on the left.

FIGS. 26A-1 and 26A-2. Amino acid sequence alignment of maize ZCR (SEQ ID NO: 96) and Arabidopsis SCR (SEQ ID NO:2). Identical residues are marked by asterisks. In addition, three copies of an LXXLL motif are underlined.

FIGS. 27A-H. Maize Scarecrow gene expression during regeneration of the root apex following excision of the QC. FIGS. 27A-B. Immediately after removal of the root cap and excision of the QC, no significant alteration in the expression pattern was observed. FIGS. 27C-D. Maize expression pattern 24 hours following excision of the QC. These figures show isolated expression of the gene between cell files. FIG. 27E. Expression 48 hours following excision of the QC. This figure shows that the root tip has regained much of its normal shape, although the cell files have not organized into the converging files seen in normal roots. FIG. 27F. Expression 72 hours following excision of the QC. At this stage, the expression pattern resembles that found in the unexcised root. FIG. 27G. Expression 96 hours following excission of the QC. At this stage, the expression pattern is similar to that seen in the primary root.

FIGS. 28 and 28A-1 to 28A-33. The partial nucleotide and amino acid sequences (SEQ ID NOS:68-94) of Arabidopsis EST's that encode members of the SCARECROW-like (SCL) gene family (SEQ ID NOS: 68-94,23, 21, 19, 46, 50, 54, and 58 respectively). "N" indicates an unknown base.

FIGS. 29A-D. Alignment of the Arabidopsis GRAS gene products (SCL3 (SEQ ID NO: 21), SCL11 (SEQ ID NO: 50), SCL9 (SEQ ID NO: 113), SCL14 (SEQ ID NO: 58), SCL16 (SEQ ID NO: 126), SCL13 (SEQ ID NO: 54), SCL5 (SEQ ID NO: 128), aceh SCL1 (SEQ ID NO: 23), SCL8 (SEQ ID NO: 116), SCL4 (SEQ ID NO: 117), SCL7 (SEQ ID NO: 52), SCL6 (SEQ ID NO: 46; residues 21-378), SCL15 (SEQ ID NO 119), SCL18 (SEQ ID NO: 120), GAI (SEQ ID NO: 150), RGA (SEQ ID NO: 149), RGAL (SEQ ID NO: 123), SCL19 (SEQ ID NO: 130 and SCR (SEQ ID NO: 2)). The highly conserved region of the GRAS products can be divided into five recognizable motifs, indicated in the figure. See also, for example, Section 5.1.5., infra. The absolutely conerved residues within the VHIID (SEQ ID NO: 145) and SAW (SEQ ID NO: 146) motifs are highlighted in bold, as are the hydrophobic residues of the leucine heptads, the P-F-Y-R-E residues of the PFYRE motif (SEQ ID NO: 147), and the two short sequences that define the end of the VHIID motif (SEQ ID NO: 145) and the beginning of the PFYRE motif (SEQ ID NO: 147). The @ symbol in the alignment indicates the location of an apparent insertion in the SCL3 gene (SEQ ID NO: 148). The deduced amino acid sequence of the insertion is shown at the bottom of the figure.

FIG. 30. RNA Gel Blot. mRNA from siliques (Si) and 14 day old shoots (Sh) and roots (R) was isolated and analyzed by RNA gel blot hybridization with specific antisense digoxygenin-labeled probes. The SCLs analyzed are all expressed within the roots, and many of them are expressed in all of the organs tested. As the amount of mRNA loaded on the gels and the exposure times for all of these blots varied, direct comparisons of the levels of expression are not possible. Detection of SCL1, however, required significantly shorter exposures than the others, and SCL6, SCL7 and SCL9 required significantly longer exposures and more mRNA. A representative ethidium bromide-stained RNA gel is shown below as a loading control.

FIGS. 31A-D. In situ Hybridizations with SCR and SCL3. Transverse sections (FIGS. 31A, 31B, and 31D) and a longitudinal section (FIG. 31C) of 7 day old roots were hybridized with either an antisense SCR riboprobe (FIG. 31A), an antisense SCL3 riboprobe (FIGS. 31B and 31C) or a sense SCL3 riboprobe (FIG. 31D). Strong signal is observed in the endodermis with the antisense SCR probe and the antisense SCL3 probe, but not with the sense SCL3 probe. Scale bars in FIGS. 31A and 31C are both 25 mn. The magnification is the same in FIGS. 31A, 31B and 31D.

FIG. 32. RNA Blot Analysis. An RNA blot analysis in which either total RNA or poly-A selected RNA from roots (R) and shoots (S) were probed with the full-length ZCR cDNA. The hybridizing band is approximately 2.6 kilobases.

FIGS. 33A-B. CBPBTT44 Partial cDNA (SEQ ID NO: 104) and Amino Acid Sequence (SEQ ID NO: 105). The partial nucleotide and amino acid sequence of CBPBTT44, a closely related gene to the maize ZCR gene.

FIG. 34. Alignment of the Arabidopsis SCR (SEQ ID NO: 2, positions 364-653), the maize ZCR (SEQ ID NO: 101) and the CBPBTT44 (SEQ ID NO: 102) amino acid sequence. As shown in bold, all three genes contain the leucine heptad repeats. The alignment further shows that all three genes share a high degree of homology.

FIG. 35. Southern Blot Analysis. A Southern of maize genomic DNA probed with (left) the maize ZCR cDNA, wherein the "H" lane represents DNA digested with HindIII and the "RV" lane represents DNA digested with EcoRV restriction enzymes; (right) gene-specific probes (A) maize ZCR cDNA for comparison; (B) maize ZCR gene-specific probe and (C) CBPBTT44 gene-specific probe. The results demonstrate that CBPBTT44 is the source of the other hybridizing bands picked up by the maize ZCR cDNA.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the SCARECROW (SCR) gene; SCARECROW-like (SCL) genes, SCR gene products, including, but not limited to, transcriptional products such as mRNAs, antisense and ribozyme molecules, translational products such as the SCR protein, polypeptides, peptides and fusion proteins related thereto; antibodies to SCR gene products; SCR regulatory regions; and the use of the foregoing to improve agronomically valuable plants.

In summary, the data described herein show the identification of SCR, a gene involved in the regulation of a specific asymmetric division, in controlling gravitropic response in aerial structures, and in controlling pattern formation in roots. Sequence analysis shows that the SCR protein has many hallmarks of transcription factors. In situ and marker line expression studies show that SCR is expressed in the cortex/endodermal initial of roots before asymmetric division occurs, and in the quiescent center of regenerating roots. Together, these findings indicate that the SCR gene regulates key events that establish the asymmetric division that generates separate cortex and endodermal cell lineages, and that affect tissue organization of roots. The establishment of these lineages is not required for cell differentiation to occur, because in the absence of division, the resulting cell acquires mature characteristics of both cortex and endodermal cells. However, it is possible that SCR functions to establish the polarity of the initial before cell division, or that it is involved in generating an external polarity that has an effect on asymmetric cell division.

Genetic analysis indicates that SCR expression affects gravitropism of plant stems, hypocotyls and shoots. This indicates that SCR is expressed also in these aerial structures of plants.

The SCR genes and promoters of the present invention have a number of important agricultural uses. The SCR promoters of the invention may be used in expression constructs to express desired heterologous gene products in the embryo, root, root nodule, and starch sheath layer in the stem of transgenic plants transformed with such constructs. For example, SCR promoters may be used to express disease resistance genes such as lysozymes, cecropins, maganins or thionins for anti-bacterial protection, or the pathogenesis-related (PR) proteins such as glucanases and chitinases for anti-fungal protection. SCR promoters also may be used to express a variety of pest resistance genes in the aforementioned plant structures and tissues. Examples of useful gene products for controlling nematodes or insects include Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, chitinase, glucanases, lectins and glycosidases.

Gene constructs that express or ectopically express SCR, and the SCR-suppression constructs of the invention, may be used to alter the root and/or stem structure, and the gravitropism of aerial structures of transgenic plants. Since SCR regulates root cell divisions, overexpression of SCR can be used to increase division of certain cells in roots and thereby form thicker and stronger roots. Thicker and stronger roots are beneficial in preventing plant lodging. Conversely, suppression of SCR expression can be used to decrease cell division in roots and thereby form thinner roots. Thinner roots are more efficient in uptake of soil nutrients. Since SCR affects gravitropism of aerial structures, overexpression of SCR may be used to develop "straighter" transgenic plants that are less susceptible to lodging.

Further, the SCR gene sequence may be used as a molecular marker for a quantitative trait, e.g., a root or gravitropism trait, in molecular breeding of crop plants.

For purposes of clarity and not by way of limitation, the invention is described in the subsections below in terms of (a) SCR genes and nucleotides; (b) SCR gene products; (c) antibodies to SCR gene products; (d) SCR promoters and promoter elements; (e) transgenic plants which ectopically express SCR; (f) transgenic plants in which endogenous SCR expression is suppressed; and (g) transgenic plants in which expression of a transgene of interest is controlled by the SCR promoter.

5.1. SCR Genes

The SCARECROW genes and nucleotide sequences of the invention include: (a) a gene listed below in Tables 1 or 2 (hereinafter, a gene comprising any one of the nucleotide sequences shown in FIG. 5A-1, FIG. 5A-2, FIGS. 8A-B, FIGS. 9A-B, FIG. 10, FIG. 11A, FIG. 11B1, FIG. 11B2, FIGS. 12A-B, FIGS. 16A-E, FIG. 16F-1, FIG. 16F-2, FIGS. 16G-J, FIG. 16J-1, FIG. 16J-2, and FIGS. 16K-M, segment of such nucleotide sequences), or as contained in the clones described herein and deposited with the ATCC (see Section 13, infra); (b) a nucleotide sequence that encodes a protein comprising any one of the amino acid sequences shown in FIG. 5A-1, FIG. 5A-2, FIG. 5D, FIG. 5E-1, FIG. 5E-2, FIGS. 8A-B, FIGS. 9A-B, FIG. 11A, FIG. 11B1, FIG. 11B2, FIGS. 13A-F. FIGS. 15A-S, FIG. 17B, FIG. 18, or FIGS. 25A-C, or a segment of such amino acid sequences, or that is encoded by any one of the genes and/or nucleotide sequences listed by their sequence identifier numbers in Tables 1 or 2, or any segment of such genes and/or nucleotide sequences, or contained in any one of the clones described herein and deposited with the ATCC (see section 13, infra); (c) any gene comprising a nucleotide sequence that hybridizes to the complement of any one of the genes and/or nucleotide sequences listed by their sequence identifier numbers in Tables 1 or 2, or any segment of such genes and/or nucleotide sequences, or as contained in any one of the clones described herein and deposited with the ATCC, under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3) and that encodes a gen