Patent Grant
7071380
This application incorporates by reference in its entirety the Sequence Listing contained in the accompanying two compact discs, one of which is a duplicate copy. Each CD contains a single file, name “1084Useqlist.txt”, the size of which is 1,203 KB, and which was created on Aug. 30, 2004 in IBM-PC MS-Windows format pursuant to 37 CFR §1.52 (e).
This application relates to control of systemic signaling processes in plants by phloem-regulatory molecules. In particular, this application is directed to the identification and isolation of phloem regulators of flowering in plants.
Flowering is an age (development)-dependent response which occurs following a period of vegetative growth in which the plant acquires the competence to flower. The timing of flowering is highly regulated to ensure effective pollination and that seed maturation occurs under favorable environmental conditions.
The earliest stage of flowering, floral evocation, is characterized by the normally irrevocable commitment of shoot apical meristem (SAM) to form an inflorescence or floral meristem. Floral evocation occurs prior to the appearance of detectable morphological or functional changes in the SAM. Subsequently, the floral primordium differentiates and forms floral organs, the flower matures and opens.
Environmental inputs are also involved in the onset of floral evocation. In certain species of plants, floral evocation will not occur in the absence of an appropriate environmental stimulus (e.g., daylength (photoperiodism), cold temperatures (vernalization) and/or other environmental stresses such as drought), while in other species, floral evocation will occur at a time that is predetermined genetically for that species but is susceptible to modulation by the environment. Genetic studies in Arabidopsis have provided information about the organization and integration of flower signaling pathways in this model system in which there exists three flower signaling pathways: an age-dependent, daylength-independent pathway which operates in the absence of promoting signals; an autonomous pathway which suppresses the vegetative-to-flowering transition; and a photoperiodic pathway, which in this species of plant, is triggered when the photoperiod exceeds a critical length (reviewed by Devlin and Kay, Science 288: 1600 (2000)).
Flowering control in Arabidosis is normally described as controlled by 4 pathways:
1) Autonomous promotion
2) vernalization promotion
3) gibberellic acid promotion
4) photoperiod promotion
Simpson, Gendall and Dean, Ann. Rev. Cell Dev. Biol. 99: 519–550, 1999.
An understanding of the flower control mechanisms that operate in agronomically and horticulturally important plants is a prerequisite for being able to control the occurrence, timing and extent of flower and seed production. The ability to override, or change, the genetic programs of flowering plants, to accelerate flowering or to permit flowering under environmental conditions different from those of normal, without altering the fertility of the plants or their desirable traits would be of tremendous importance in agriculture and horticulture. The acceleration of flowering is useful in plant breeding to increase the efficiency and speed of selection of improved traits, and in the ornamental flower industry. The ability to delay flowering is important in certain crop plants in which flowering overlaps with growth of harvested vegetation, such as onions, bulbs, lettuce, cabbage and pastures.
One of the most useful paradigms for studying the control of flowering is the photoperiodic response. In many plant species, mature leaves serve as photoreceptors for photoperiodic induction leading to floral initiation within the vegetative meristem. Although the identity of the transmitted flowering stimulus is presently unknown, grafting and defoliation experiments have provided irrefutable evidence that a flowering stimulus is produced in the leaves of plants exposed to a favorable photoperiod. Upon stimulation, a phloem-mobile agent, termed florigen, appears to be released from leaf cells for translocation and delivery into the terminal phloem cells within the apex of the plant. Relay of the florigenic signal from the phloem to the founder cells within the SAM then gives rise to the activation of a new developmental program that generates floral meristems and, eventually, flowers. Available evidence suggests that flower development in certain species of plants may be controlled by both positive and negative regulatory factors, the latter at times being produced in response to an unfavorable photoperiodic stimulus. See, e.g., Gibby and Salisbury, Plant Physiol. 47:784–789 (1971); Lang et al., Proc. Natl. Acad. Sci. USA 74:2412–2416 (1977).
Significant progress has been made in identifying structural and regulatory genes involved in pathways of flower development and in understanding the hierarchical controls that integrate multiple pathways. See, e.g., Levy and Dean, Plant Cell 10:1973–1989 (1998); Simpson et al., Annu. Rev. Cell Dev. Biol. 99:519–50 (1999). Multiple environmental and endogenous signals are integrated by floral integrator genes, which participate in the upregulation of floral meristem identity genes which is required for flowering.
Genetic analysis in Arabidopsis has revealed many of the genes involved in photoperiod-mediated flowering. In this pathway, the timing of the floral transition is coordinated with day-length, as sensed by the circadian oscillator. The transition is accelerated in response to long days and retarded in response to short days. CO (CONSTANS), a transcription factor, plays an important regulatory role in this process. The level of CO mRNA is circadian clock-regulated. Under long day length conditions (LD), CO protein accumulates late in the day and directly activates transcription of FT (FLOWERING LOCUS T), a floral integrator gene. The level of CO-activated FT expression in Arabidopsis is tightly regulated by TFL2 (TERMINAL FLOWER 2), a gene that is coexpressed with CO and FT in leaf vascular tissue (Takada and Goto, The Plant Cell 15, 2856–2865 (2003)).
FT encodes a RAF-kinase-inhibitor-like protein that promotes flowering. The Arabidopsis FT polynucleotide and polypeptide sequences and methods of use for accelerating or delaying flowering are disclosed in U.S. Pat. Nos. 6,225,530 and 6,713,663 respectively.
CO also activates LEAFY (LFY), a floral meristem identity gene. FT, together with LFY, promotes flowering. The loss of FT delays flowering, whereas overexpression of FT accelerates flowering by a CO- and photoperiod-independent process.
FT belongs to a gene family which is conserved in plants, mammals and fungi. Two known plant family members are CEN (Antirrhinum majus) and TFL1 (Arabidopsis thaliana). TFL1 is required for establishing and maintaining the inflorescence meristem. TFL1 and CEN are orthologous genes according to phylogenetic tree analysis of the protein sequences (see U.S. Pat. No. 6,225,530). FT, acting downstream of CO, promotes flowering, whereas TERMINAL FLOWER1 (TFL1) represses flowering.
Flowering promotion by FT requires FD, a bZIP transcription factor-encoding gene. FD is preferentially expressed in the shoot apex and activates transcription of the floral meristem identity genes AP1 and CAL. The overexpression of FD in seedlings upregulates AP1 expression only when FT is present. The interaction of FD with FT may require phosphorylation of the C-terminus of FD. (Daimon et al., Abstract T01–033, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
TFL1 interacts with FDP, a bZIP transcription factor which is closely related to FD. The interaction of FDP and TFL1 may be required to prevent inappropriate activation of flowering by FD and FT in the SAM (Wigge et al., Abstract T01–070, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
Reportedly, both TFL1 and FT protein move from cell to cell in the SAM. (Goto and Nakayama, Abstract T01–053, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
Unpublished studies from our laboratory have shown that ectopic expression of the Arabidopsis FT gene in the vasculature of Cucurbita moschata, grown under non-flowering photoperiod conditions, promotes flowering in the meristem. We used a viral vector system based on Zucchini yellow mosaic potyvirus (ZYMV) to express FT (ZYMV-FT) in C. moschata PI441726. In contrast to control plants inoculated with ZYMV-GFP, plants inoculated with ZYMV-FT formed floral buds and flowered within 23–35 days. These results were obtained in replicate independent experiments with four to six plants per experiment. Representative results are shown below in
Coupland and colleagues expressed CO::GUS promoter-reporter gene constructs in Arabidopsis and found expression of the reporter gene in both vascular tissue and the SAM. Mis-expression of CO from phloem-specific promoters, but not meristem-specific promoters, induced early flowering and complemented a late-flowering constans mutation. CO activates flowering through both FT-dependent and FT-independent processes (Laurent et al., Abstract T01–048, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
Micrografting experiments in Arabidopsis have shown that certain flowering time mutants can be rescued by long-distance signaling, and suggest that the expression of FT in the apex and leaf may be required (Turnbull and Justin, Abstract T01–099, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
The CEN, TFL1, FT and SP (SELF-PRUNING) genes are a closely related set of plant genes which are implicated as regulators of timing of switching of meristems from vegetative to reproductive growth (all except FT delay flowering) (Bradley et al., Science 275:80–83 (1997); Bradley et al., Nature 379:791–797 (1996); Pneuli et al., Development 125:1979–1989 (1998); Pneuli et al., The Plant Cell, 13:2687–2702 (2001). This gene family, recently named CETS (Pneuli et al., Ibid)), is comprised of plant homologs of the mammalian phosphatidylethanolamine binding proteins (PEBPs), which include serine protease inhibitors, Raf-1 kinase inhibitor (RKIP), and precursor for hippocampal neurostimulatory peptide (HCNP). Six CETS members have been identified in Arabidopsis and six in tomato.
Two-hybrid screens and in vitro binding assays have revealed the presence of SP-interacting proteins (SIPS) in tomato (Pneuli et al., 2001). These SIPs include: SPAK, a novel plant serine-threonine NIMA-like kinase; several isoforms of the 14-3-3 family of adapter proteins; and SPGB, a putative bZIP G-box binding transcription factor C-terminal sequence similarity to GBF4.
SIPs may function as components of an SP-dependent signaling network in tomato plants. SIPs form specific associations with one another and show overlapping spatio-temporal expression patterns in apical meristem, leaf and stem vasculature, floral primordial of the primary inflorescence, stamens and carpels in developing floral bud and vegetative meristem of the first sympodial segment (Pneuli et al., 2001, Ibid). Several of the binding interactions are phosphorylation-dependent. For example, the binding of SP to SPAK and the binding of SIP4 and SPAK to 14-3-3/74 requires phosphorylation of SPAK at Serine 406. Both CEN and TFL1 bind SPAK, 14-3-3/74 and SPGB proteins but not SIP4 (a novel 10 kDa protein). FT showed the same binding pattern as TFL1.
The biological functions of SP/14-3-3/SPAK interactions in tomato plants are not yet understood. Nevertheless, the finding that CETS proteins of Arabidopsis and Antirrhinum form specific complexes with tomato SIPs suggests that protein-protein interactions may be necessary for CETS protein function in plants with widely differing shoot and flowering architecture.
PCT Application No. WO 96/34088 discloses the isolation of the Id gene, which is thought to be important in regulating the transition to flowering in maize. The Id gene encodes a zinc-finger protein that is apparently transcribed in young leaves but not in the shoot apical meristem. The mechanism by which this gene produces its effects at the SAM has not yet been elucidated. Mutated forms of the gene are proposed for use in accelerating or delaying floral induction in a plant. PCT Application No. WO 97/25433 describes chimeric vectors comprising FPF (flowering promoting factor) genes from mustard, and homologous genes from other plants, and uses thereof for inducing early flowering or inhibiting flowering in various crop plants.
Progress has also been made in understanding how plants transport materials from cell-to-cell and systemically throughout the plant. These studies have provided evidence for a systemic communication network that comprises a phloem translocation system which is capable of transporting not only small phytohormones and nutrients but also macromolecules (such as peptides, proteins and nucleic acids) between spatially distant tissues and organs of the plant. The identification of specific transcripts and signaling molecules in phloem sap suggests that this communication network is involved in the coordination of growth and development, and may participate in systemic acquired resistance to pathogens (Narvaez-Vasquez et al., Planta 195:593–600 (1995), systemic gene silencing (Jorgensen et al., Science 279:1486–1487 (1988), review), biomass distribution, regulation of carbon metabolism and control of plant size (Lucas, PCT applications WO 97/06669 and WO 97/20470), and floral development (Ruiz-Medrano et al., Development 126:4405–4419 (1999) and references cited therein; Baulcombe, PCT application WO 99/15682). A systemic small RNA binding protein which may function in sRNA signaling has been isolated from several plant species and is disclosed in U.S. Application Ser. No. 60/487,358.
The broad outlines of florigen signaling have emerged from these and prior studies, but the signal(s) that initiate floral evocation and the manner in which the long-distance florigen signaling pathway is regulated are presently unknown. These objectives are addressed in the present application.
The present invention provides methods for (1) identifying the long-distance florigenic signaling component and/or signaling complex (LDFSC) and other genes in the florigenic signaling cascade; (2) modifying the occurrence, timing and extent of flower development by modulating the florigenic signaling pathway; and (3) screening for inducers, repressors and modulators of florigenic signaling. The invention further provides isolated polynucleotide sequences from plant species that encode putative regulators of long-distance florigenic signaling. These polynucleotide sequences are identified in the Sequence Listing as SEQ ID NOs: 1–25, 119–151, 271–298, 377–380, 561–638 (cucurbits); SEQ ID NOs: 51–75, 185–217 and 299–322, 405–482 (Arabidopsis orthologs), SEQ ID NO: 323 (Pinus radiata ortholog), and SEQ ID NOs: 717–794 (Festuca arundinacea). Their encoded polypeptide sequences are identified respectively as SEQ ID NOs. 26–50, 152–184, 324–351, 381, 382, 639–716 (cucurbits); SEQ ID NOs. 76–100, 218–250,352–375, and 483–560 (Arabidopsis orthologs), SEQ ID NO: 376 (Pinus radiata ortholog); and SEQ ID NOs: 795–872 (Festuca arundinacea).
SEQ ID NOs. 132 and 165 are respectively the polynucleotide and encoded polypeptide sequence of C. maxima FT; SEQ ID NOs: 198 and 231 are respectively the polynucleotide and encoded polypeptide sequence of A. thaliana FT, and SEQ ID NOs: 323 and 376 are respectively the polynucleotide and encoded polypeptide sequence of P. radiata FT.
Polynucleotide sequences with SEQ ID NOs. 271–322 encode regulatory molecules which include putative orthologs of SIPs described in Pneuli et al., The Plant Cell, 13, 2687–2702 (2001).
Putative sRNA phloem mobile regulators of flowering have been identified in Cucurbit phloem sap databases. These sequences are identified in the Sequence Listing as SEQ ID NOs. 251–263. The invention encompasses genetic constructs comprising these polynucleotides and plants comprising the constructs that display modified flowering responses as compared with unmodified native plants.
Previous studies on photoperiodic induction of flowering have shown that perception of a favorable photoperiod occurs in the leaf and involves phytochrome pigments (Blazquez J. Cell Science 113:3547–3548 (2000); Koorneef et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:345–370 (1998), reviews). Grafting studies provided evidence that a flower-inducing signal is produced in the stimulated leaf and is capable of moving through a graft junction to initiate flowering in the SAM of an unstimulated scion (recipient tissue) grafted to a stimulated stock (perception and production tissue). Genetic studies have identified transcription factors and their target gene pathways in the meristem cells that are involved in floral evocation. The mechanisms of cell-to-cell communication and the role of the phloem transport system in systemic transport of macromolecules between source and sink tissues are under active investigation by a number of laboratories. While it is certain that florigenic signaling is initiated by the perception of an appropriate stimulus by the leaf and culminates in the induction of the flowering response in the SAM, the identity of the florigenic signal(s) and the process by which a florigenic signal is communicated from the leaf to the meristematic target tissues (shoot apical meristem, lateral meristem) have not yet been elucidated.
The present invention describes a florigen signaling system, and methods of modulating the system components, involving a multistep pathway in which the companion cell (CC) of the phloem plays a central role. By analogy with a transmitter element in a communication network, the companion cell is responsible for converting the signal it receives from the leaf sensor cells to a transmissible form which can be propagated in a long-distance communication channel for arrival at an appropriate time and destination point, and in a manner that preserves the information content and intensity of the signal at its point of reception. In this conception, the companion cell is viewed both as the supplier of components required for long-distance systemic transmission of a flower-inducing stimulus from the leaf to the SAM and as a selectivity filter for ensuring that signal-to-noise ratio of the transmission is adequate.
As shown in
The transmissible long-distance flower-inducing stimulus is referred to herein as a “long-distance florigenic signaling component” or “LDFSC”. This term is intended to encompass regulatory molecules or complexes containing regulatory molecules which act directly or indirectly to induce the floral transition. The accessory components that function, for example, in the delivery of a florigenic signal from the companion cell to the plasmodesmal system, in stabilization of the florigenic signal during phloem transport, and in unloading of the florigenic signal from the terminal phloem for delivery to the apical meristem are considered to comprise LDFSC, but may also be involved in other types of long-distance signaling processes.
The LDFSC enters the functional, enucleate, sieve tube system (step E), which acts as the conduit for the delivery of the LDFSC to the shoot apex. The LDFSC is a protein/derivative, protein-hormone complex, nucleoprotein, peptide/derivative, a single-stranded or double-stranded nucleic acid molecule/derivative, or a combination of any of the foregoing in covalent or non-covalent linkage. The term “derivative” refers to an LDFSC that is structurally modified by biochemical, chemical, recombinant or genetic techniques.
The CC-SE complexes along the phloem translocation pathway provide a relay system for regenerating and amplifying the LDFSC during the translocation process (step E). The function and location of the regenerating and/or amplifying system in the CC-SE complex depends on the biochemical characteristics of the LDFSC. For example, if the LDFSC is an RNA molecule that serves as its own template for replication, the replicative machinery or part thereof may be cotransported with it in the phloem. For LDFSC that requires de novo transcription or translation, the relay mechanism is likely to be a highly regulated process involving the shuttling of the LDFSC back and forth between the phloem and the CC during translocation.
The egress of the LDFSC at the terminal phloem (sieve tubes) involves at least one of the following processes: (i) targeted transport through plasmodesmata into CCs or neighboring phloem parenchyma and (ii) activation of a secondary relay in vascular cells which propagates via plasmodesmata and/or the apoplasm to the SAM and initiates floral development (steps F—H); or (iii) direct transfer of the LDFSC through the apex via PD or the apoplasm into the SAM (step I) to initiate floral development. The mechanism is likely to depend on such factors as the molecular size and shape of the LDFSC in relation to the size exclusion limit (SEL) of plasmodesmata and/or movement via the apoplasm in postphloem tissues, whether specific signals are present for targeting the LDFSC to the meristem or retaining it in the SE-CC complex, and whether the LDFSC requires conversion into a different form for cell-to-cell transport or for activation of the vegetative to floral transition in the SAM. For a general review of phloem transport and unloading of macromolecules, see Oparka and Santa Cruz, Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 323–47 (2000) and Schobert, C., Lucas, W. J., Franceschi, V. R., and Frommer, W. B. (2000) Intercellular transport and phloem loading of sucrose, oligosaccharides and amino acids. Advances in Photosynthesis, Vol. 9, Photosynthesis: Physiology and Metabolism, eds. R. C. Leegood, T. D. Sharkey and S. von Caemmerer, pp. 249–274.
The present invention provides methods for (1) identifying the LDFSC and other genes in the florigenic signaling cascade; (2) modifying the occurrence, timing and extent of flower development by modulating the florigenic signaling pathway; and (3) screening for inducers, repressors and modulators of florigenic signaling.
In one of its aspects, the invention provides a method for identifying a candidate flowering control gene in the long-distance florigenic signaling pathway. The method comprises: comparing the gene transcripts or transcription products in the phloem sap of florally induced and noninduced plants and identifying a product that is differentially expressed during the floral transition.
In a related aspect, the invention provides a method of isolating an LDFSC, which comprises exposing a plant or heterograft to a flowering stimulus, identifying a gene transcript or transcription product that appears in the phloem sap, and ascertaining the ability of the gene product to affect floral induction in a plant.
The phrase “gene transcript or transcription product” refers to all products that are derived from the expression of a gene, including, for example, RNA and polypeptide molecules that are processed from precursor molecules or are otherwise modified after transcription and/or translation.
The invention further provides isolated polynucleotide sequences from cucurbits, Arabidopsis, Pinus and Festuca that encode putative regulators of long-distance florigenic signaling.
The word “polynucleotide(s),” as used herein, means a polymeric collection of nucleotides, and includes DNA and corresponding RNA molecules and both single and double stranded molecules, including HnRNA and mRNA molecules, sense and anti-sense strands of DNA and RNA molecules, and comprehends cDNA, genomic DNA, and wholly or partially synthesized polynucleotides.
A polynucleotide of the present invention may be an entire gene or any portion thereof. As used herein, a “gene” is a DNA sequence that codes for a functional protein or RNA molecule. Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of “polynucleotide” therefore includes all operable anti-sense fragments. Anti-sense polynucleotides and techniques involving anti-sense polynucleotides are well known in the art and are described, for example, in Robinson-Benion et al., Methods in Enzymol. 254(23): 363–375, 1995 and Kawasaki et al., Artific. Organs 20(8): 836–848, 1996.
Polynucleotides that comprise complements of such polynucleotide sequences, reverse complements of such polynucleotide sequences, or reverse sequences of such polynucleotide sequences, together with variants of such sequences, are also provided.
Table 1, below provides additional information about these sequences. These polynucleotide sequences are identified in the Sequence Listing as SEQ ID NOs: 1–25, 119–151, 271–298, 377–380, 561–638 (cucurbits); SEQ ID NOs: 51–75, 185–217 and 299–322, 405–482 (Arabidopsis orthologs), SEQ ID NO: 323 (Pinus radiata ortholog), and SEQ ID NOs: 717–794 (Festuca arundinacea). Their encoded polypeptide sequences are identified respectively as SEQ ID NOs. 26–50, 152–184, 324–351, 381, 382, 639–716 (cucurbits); SEQ ID NOs. 76–100, 218–250, 352–375, and 483–560 (Arabidopsis orthologs), SEQ ID NO: 376 (Pinus radiata ortholog); and SEQ ID NOs: 795–872 (Festuca arundinacea). SEQ ID NOs. 132 and 165 are respectively the polynucleotide and encoded polypeptide sequence of C. maxima; SEQ ID NOs: 198 and 231 are respectively the polynucleotide and encoded polypeptide sequence of A. thaliana FT, and SEQ ID NOs: 323 and 376 are respectively the polynucleotide and encoded polypeptide sequence of P. radiata FT.
As used herein, the term “polypeptide” encompasses amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The term “polypeptide encoded by a polynucleotide” as used herein, includes polypeptides encoded by a polynucleotide that comprises a partial isolated polynucleotide sequence provided herein.
Polypeptides of the present invention may be produced recombinantly by inserting a polynucleotide sequence of the present invention encoding the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a polynucleotide molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells. Preferably, the host cells employed are plant, E. coli, insect, yeast, or a mammalian cell line such as COS or CHO. The polynucleotide sequences expressed in this manner may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof. The expressed polypeptides may be used in various assays known in the art to determine their biological activity. Such polypeptides may also be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine levels of interacting proteins or other compounds.
Polynucleotide sequences with SEQ ID NOs. 271–322 encode regulatory molecules which include putative orthologs of SIPs described in Pneuli et al., Plant Cell 13:2687–2702 (2001). The following Table provides additional information about these sequences.
Cucumis sativus
Cucumis sativus
Cucurbita maxima
Cucurbita maxima
Sicyos angulatus
Cucumis sativus
Cucurbita maxima
Sicyos angulatus
A. thaliana
A. thaliana
A. thaliana
A. thaliana
A. thaliana
A. thaliana
A. thaliana
Genetic constructs comprising the polynucleotide sequences and host organisms comprising these constructs are also encompassed by the present invention. The term “genetic construct” refers to recombinant genetic constructs comprising, in the 5′–3′ direction, a gene promoter sequence; an open reading frame coding for at least a functional portion of a polypeptide of the present invention; and a gene termination sequence. The open reading frame may be orientated in either a sense or anti-sense direction. For applications where amplification of gene transcripts and transcription products is desired, the open reading frame may be inserted in the construct in a sense orientation, such that transformation of a target organism with the construct will lead to an increase in the number of copies of the gene and therefore an increase in the amount of gene transcripts and transcription products. When down-regulation of gene transcripts and transcription products is desired, the open reading frame may be inserted in the construct in an anti-sense orientation, such that the RNA produced by transcription of the polynucleotide is complementary to the endogenous mRNA sequence. This, in turn, will result in a decrease in the amount of gene transcription products. Alternatively, regulation may be achieved by inserting appropriate sequences or subsequences (e.g., DNA or RNA) in ribozyme constructs.
Genetic constructs comprising a non-coding region of a gene coding for a polypeptide of the present invention, or a nucleotide sequence complementary to a non-coding region, together with a gene promoter sequence and a gene termination sequence, are also provided. As used herein the term “non-coding region” includes both transcribed sequences that are not translated, and non-transcribed sequences within about 2000 base pairs 5′ or 3′ of the translated sequences or open reading frames. Examples of non-coding regions that may be usefully employed in the inventive constructs include introns and 5′-non-coding leader sequences. Transformation of a target plant with such a genetic construct may lead to a reduction in the amount of enzyme synthesized by the plant by the process of cosuppression, in a manner similar to that discussed, for example, by Napoli et al., Plant Cell 2:279–290, 1990; and de Carvalho Niebel et al., Plant Cell 7:347–358, 1995.
The genetic constructs of the present invention further comprise a gene promoter sequence and a gene termination sequence, operably linked to the polynucleotide to be transcribed, which control expression of the gene. The gene promoter sequence is generally positioned at the 5′ end of the polynucleotide to be transcribed, and is employed to initiate transcription of the polynucleotide. Gene promoter sequences are generally found in the 5′ non-coding region of a gene but they may exist in introns (Luehrsen, Mol. Gen. Genet. 225:81–93, 1991). When the construct includes an open reading frame in a sense orientation, the gene promoter sequence also initiates translation of the open reading frame. For genetic constructs comprising either an open reading frame in an anti-sense orientation or a non-coding region, the gene promoter sequence consists only of a transcription initiation site having a RNA polymerase binding site.
A variety of gene promoter sequences that may be usefully employed in the genetic constructs of the present invention are well known in the art. The promoter gene sequence, and also the gene termination sequence, may be endogenous to the host organism, e.g., target plant host, or may be exogenous, provided the promoter is functional in the target host. For example, the promoter and termination sequences may be from other plant species, plant viruses, bacterial plasmids and the like. Preferably, gene promoter and termination sequences are from the inventive sequences themselves.
Factors influencing the choice of promoter include the desired tissue specificity of the construct, and the timing of transcription and translation. For example, constitutive promoters, such as the 35S Cauliflower Mosaic Virus (CaMV 35S) promoter, will affect the activity of the enzyme in all parts of the plant. Use of a tissue specific promoter will result in production of the desired sense or anti-sense RNA only in the tissue of interest. With genetic constructs employing inducible gene promoter sequences, the rate of RNA polymerase binding and initiation can be modulated by external physical or chemical stimuli, such as light, heat, anaerobic stress, alteration in nutrient conditions and the like. Temporally regulated promoters can be employed to effect modulation of the rate of RNA polymerase binding and initiation at a specific time during development of a transformed cell.
The gene termination sequence, which is located 3′ to the polynucleotide to be transcribed, may come from the same gene as the gene promoter sequence or may be from a different gene. Many gene termination sequences known in the art may be usefully employed in the present invention, such as the 3′ end of the Agrobacterium tumefaciens nopaline synthase gene. However, preferred gene terminator sequences are those from the original gene or from the target species to be transformed.
Other regulatory sequences may be included in the construct, such as transcriptional and translational enhancers.
The genetic constructs of the present invention may also contain a selection marker that is effective in plant cells, to allow for the detection of transformed cells containing the inventive construct. Such markers, which are well known in the art, typically confer resistance to one or more toxins. One example of such a marker is the NPTII gene whose expression results in resistance to kanamycin or hygromycin, antibiotics which are usually toxic to plant cells at a moderate concentration (Rogers et al., in Weissbach A and H, eds., Methods for Plant Molecular Biology, Academic Press Inc.: San Diego, Calif., 1988). Alternatively, the presence of the desired construct in transformed cells can be determined by means of other techniques well known in the art, such as Southern and Western blots.
Techniques for operatively linking the components of the inventive genetic constructs are well known in the art and include the use of synthetic linkers containing one or more restriction endonuclease sites as described, for example, by Sambrook et al., Molecular cloning: a laboratory manual, CSHL Press: Cold Spring Harbor, N.Y., 1989. The genetic construct of the present invention may be linked to a vector having at least one replication system, for example, E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced and the correctness of the manipulation determined.
The expression of polynucleotide sequences in target cells may be controlled by choice of the promoter sequence, or by selecting the number of functional copies or the site of integration of the polynucleotides incorporated into the genome of the target organism. A target plant may be transformed with more than one construct of the present invention, thereby modulating flowering in the plant by affecting the activity of more than one polypeptide, affecting polypeptide activity in more than one tissue or affecting polypeptide activity at more than one expression time. Similarly, a construct may be assembled containing more than one open reading frame coding for a polypeptide encoded by a polynucleotide of the present invention or more than one non-coding region of a gene coding for such polypeptide. The polynucleotides of the present invention may also be employed in combination with other known sequences encoding polypeptides involved in various metabolic and biosynthetic pathways. In this manner, more than one pathway may be modulated to produce a plant having an altered flowering phenotype.
Techniques for stably incorporating DNA constructs into the genome of target plants are well known in the art and include Agrobacterium tumefaciens mediated introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction and the like. The choice of technique will depend upon the target plant to be transformed.
Plants which may be transformed using the inventive constructs include both monocotyledonous angiosperms (e.g., grasses, corn, grains, oat, wheat and barley) and dicotyledonous angiosperms (e.g., Arabidopsis, tobacco, legumes, alfalfa).
1. Identification of Genes Involved in the Long-Distance Florigenic Signaling Pathway
A. Characteristics of Florigen Signaling Genes
Genes involved in long-distance florigenic signaling may have one or more of the following characteristics:
Any plant that is developmentally competent to flower and undergoes flower induction under defined experimental conditions is potentially useful for identifying long distance flowering control molecules. Preferably, plants that are used are amenable to sampling of the phloem translocation stream, insofar as this permits the direct analysis of phloem-mobile gene transcripts/transcription products. Thus members of the cucurbit family (e.g., pumpkin and cucumber, Ruiz-Medrano, Development 126:4405–4419 (1999)), lupin, Ricinus communis (Sakuth et al., Planta 191:207–213 (1998)) and some trees species including Eucalyptus globulus (Pate et al., Oecologia 117:312–322 (1998)), Robinia and Tilia (Schobert et al., Planta 206:245–252 (1998)) are particularly useful in this regard. Phloem sap can be collected from the above plants by incision wounding, followed by bleeding, and can also be collected from most plant species by cutting the petiole/stem under a solution of EGTA.
In the process of identifying genes involved in the long-distance florigenic signaling pathway, it is useful to compare plants/plant systems that are induced to flower in different ways, for example, photoperiod-induced flowering, graft-induced flowering or flowering induced by other abiotic or biotic stimuli known to those skilled in the art. The term “abiotic stimulus” is used herein to refer to a non-living stimulus, e.g., light, photoperiod, pH, temperature, water, osmotic stress, and the like. The term “biotic stimulus” is used herein to refer to a living stimulus, e.g., pathogens, signaling molecules, factors, and others.
Exemplary plants for photoperiod-induced flowering include Sicyos angulatus and related species of Sicyoeae, e.g., Polakowskia spp. and Sechium spp., such as Sechium edule (choko) (Heywood, Flowering plants of the world, Oxford (1978)). These species are maintained in a non-flowering state under long day photoperiods and are induced to flower following a treatment of short day photoperiods (Takahashi et al., Plant Cell Physiol. 23:1–9 (1982)). The plant systems for identifying florigen candidates as described herein include Sicyos angulatus (Bur Cucumber), Cucurbita moschata (butternut), C. maxima (pumpkin) and Cucumis sativus (cucumber), N. tabacum cv Maryland mammoth, N. tabacum cv Samsun, N. sylvestris, N. benthamiana, and heterografts of C. sativus/C. maxima, C. sativus (stock)/Sicyos angulatus (scion), N. tabacum cv Samsun (stock)/N. tabacum cv Maryland mammoth (scion), Cucumis sativus cv Straight 8 (stock)/Cucurbita moschata PI441726 (scion), and early and late flowering accessions of C. moschata (Cucurbita moschata PI212011 and PI441726) respectively.
We have characterized and are using several different plant systems to study flowering control genes. For example, heterografts of naturally occurring early and late flowering variants of C. moschata plants are used to identify and isolate florigen candidate genes. Cucumber heterografts (Friedlander et al., Plant Cell Physiol. 18:1343–1350 (1977)) or pumpkin/cucumber heterografts are used to test for the presence of a graft transmissible florigenic signal or florigenic suppressor. Heterografts comprised of an early-flowering rootstock and late-flowering scion are useful for detecting signals that induce flowering, whereas heterografts comprising a vegetative rootstock and early flowering scion can be used to detect suppressors of flowering. Following confirmation that a graft-transmissible signal is produced in a particular plant heterograft system, phloem mobile gene products that correlate with floral induction or suppression can be identified (e.g., mRNA, peptides, proteins and miRNA). Additional details of the plant systems are given in Examples 1 and 2 below.
C. Bioinformatic Selection of Candidate Genes:
(1) Construction of Phloem Sap Libraries
The phloem sap is the port of entry of components of long-distance systemic signaling. The companion cells supply all components of the sieve tube system, and control the transport of phloem-mobile molecules into the phloem. Insofar as the methods for separating phloem (CC and SE) from surrounding tissues are not yet available, the phloem-containing tissues (i.e., roots, stems, petioles, leaf veins, and vascular strands) and preferably, the phloem sap, can be used to prepare cDNA libraries. Our studies thus far have shown phloem sap to be an excellent starting material for the isolation of putative regulators of long-distance florigenic signaling.
Phloem sap libraries were constructed as described below in Example 3. The EST sequences were databased and assembled into consensus sequences using Stackpack™, version 1.2 (Electric Genetics Corporation, Cape Town, South Africa). Prior to assembly of consensi, the database EST sequences were curated to remove 1) repetitive sequences at sequence read termini flanked by polyA or polyT of at least 20 basepairs; 2) polyA or polyT sequences longer than 20 basepairs (replaced by one A or T); and 3) those exact duplicate sequences that were found to disturb the assembly procedure. The masking file used in the assembly contained sequences known to disturb assembly, including commonly used vectors and various repeats, as well as selected plant ribosomal RNA sequences.
(2). In Silico Analysis of Differential Expression
The EST member status for consensi is used to develop an in silico expression profile for all databased consensus sequences. This provides the frequency for any consensus (gene) per 1000 EST runs. Knowledge of the tissue from which the consensus EST members were derived, e.g., phloem sap from flowering or non-flowering plants, provides the relative contribution of particular tissues to EST members for a given consensus sequence.
(3). Criteria for Selection of Flowering Control Candidate Genes for in Planta Analysis
Candidate flowering control genes are selected from cucurbit phloem sap and phloem tissue cDNA libraries for in planta analysis based on several criteria. Of particular interest, for example, are:
Consensi that satisfy more than one of the above criteria are judged to have an increased likelihood of being involved in phloem-mediated regulatory activities.
D. Other Analytical Approaches for Selection of Candidate Genes
(1). Real Time RT-PCR Analysis of Differential Expression
Real time PCR is used to evaluate the relative levels of candidate gene transcript accumulation in flowering and non-flowering tissue and provides a means of refining the candidate gene selections based on in silico expression analysis. cDNA templates for the assay are synthesized from total RNA extracted from tissue (leaves, apices or vascular strips from the stem) or phloem sap derived from flowering or non-flowering plants using random primers and Reverse Transcriptase Super Script III. Real time quantification is performed using the cDNA template with a specific set of oligonucleotide primers for each candidate. The primer pairs are designed based on the consensus coding sequence of each cucurbit candidate. The PCR amplification is monitored by fluorescence emitted by the SYBR Green dye included in the PCR mix. The SYBR Green dye emits an increased fluorescence when bound to double-stranded DNA. The fluorescence is measured at each cycle of amplification using the ABI PRISM® 7900HT Sequence Detection System.
The relative abundance of each target is calculated as follows. The Threshold Cycle (CT) for each candidate included in the reaction is measured as the fractional cycle number at which the fluorescence passes a fixed threshold. The delta CT value is then determined by subtracting the CT value of the endogenous control (e.g., PP1, WRKY or GAI) from the CT value of the candidate. The relative abundance of the candidate is then calculated by subtracting the delta CT value of the candidate in non-flowering tissue from the delta CT value of the same candidate in flowering tissue.
(2). Macroarray Analysis of Differential Expression
Expression profiling of flowering and non-flowering cucurbits is performed by macroarray analysis, as described below.
Plasmid DNA (10–100 ng) comprising cucurbit candidate genes are arrayed onto Hybond N+ membrane (Amersham), denatured and fixed by baking or UV cross-linking. Phloem and vascular tissue RNA is isolated from flowering and non-flowering plants, reverse-transcribed, and labeled. Probes with equal specific activity are hybridized to the plasmid DNA array overnight at 65° C. in a solution containing 0.5 M Na2HPO4, 1 mM EDTA, 1% BSA, and 7% SDS. Blots are washed twice for 10 min in 2×SSC, 0.1% SDS at 65° C. Signal is detected using a phosphorimager.
(3) Protein and Peptide Analysis
The phloem sap is a source of proteins, peptides, small RNA molecules, and protein-small RNA complexes that may be involved in flowering control. The phloem sap also contains proteins that are involved in cell-to-cell trafficking of mRNA and small RNA molecules. The proteins and peptides can be extracted and separated by chromatographic and electrophoretic techniques, and their profiles compared in plants in flowering and non-flowering states to identify products specific to floral induction. Useful approaches for identifying and analyzing changes in phloem-mobile proteins and peptides during the floral transition in cucurbits include 2-D Fluorescence Difference Gel Electrophoresis (Ettan™ DIGE, Amersham Biosciences), high pressure liquid chromatography (HPLC), gel filtration chromatography (GFC) and mass fingerprinting and fragmentation analysis (see, e.g., Shevchenko et al., Proc. Natl. Acad. Sci. USA 93: 14440–14445 (1996); Washburn et al., Nature Biotech. 19: 242–247 (2001)).
The temporal appearance and spatial distribution of phloem-mobile proteins and RNA of interest can be followed in plant tissues during the floral transition using both histological and microinjection techniques. (see General Methods below).
E. Small RNA (sRNA) Phloem Mobile Regulators of Flowering
Small RNA molecules are single stranded or double stranded noncoding RNA molecules generally in the range of 18–25 nt in length. They include microRNAs, which are single stranded and produced by the cleavage of short stem-loop precursors by Dicer-like enzymes, and small interfering RNAs (siRNAs), which are produced by the cleavage of long double-stranded RNA molecules.
The small RNAs in the phloem may play important roles in non-cell autonomous signaling events which occur between cells and over long distances in the plant, and are implicated in a broad spectrum of plant developmental, physiological and biochemical processes, e.g., flowering, coordination of plant growth and development, systemic resistance to pathogens, responses to environmental stresses, gene silencing related to viral defense, biomass distribution, regulation of carbon metabolism, control of plant size and developmental timing and patterning. Small RNA species produce epigenetic changes in gene expression patterns by processes as diverse as targeted degradation, chromatin methylation and arrest of translation.
Our analyses of phloem-mobile RNA populations of cucurbits demonstrated the presence of small RNA molecules in phloem sap and in vascular strands of cucurbits. These small RNA species were cloned (see Example 4), sequenced, and databased, and their size distribution and complexity was analyzed. The authenticity of the phloem small RNAs was confirmed by comparisons with cucurbit genomic sequences.
(1). Selection Criteria for sRNA Candidates
The identification of candidate sRNAs as flowering control regulators is based in part upon bioinformatic criteria that are applied to the databased sRNA sequences to distinguish sRNA from contaminating fragments of other noncoding RNAs. MicroRNA sequences (miRNAs) are annotated according to the following guidelines proposed by Ambros et al. (see Ambros et al., RNA 9:277–279 (2003)):
Small RNA ranging from 19–25 nt in length are subjected to FASTA analysis against Arabidopsis rRNA and tRNA. FASTA hits with up to 2 mismatches are discarded as probable structural RNA contamination. The remaining sequences and their reverse complements are subjected to FASTA analysis against Arabidopsis intergenic region sequences. All FASTA hits with up to 3 mismatches are annotated as putative regulatory sRNA candidates on the basis of phylogenetic conservation. Further FASTA analysis allowing for up to 3 mismatches is performed against rice, Brassica and Populus genome datasets to confirm conserved sRNA. Identified conserved sequences are further analyzed by an RNA-folding program, leading to identification of candidates with a potential fold-back precursor structure that contains the sRNA sequence within one arm of the hairpin.
sRNA candidates are then identified from the cucurbit sRNA database based on differential expression of sRNA across the floral transition, as determined by both in silico and transcriptional profiling of phloem sap sRNA by Northern/reverse Northern methods, carried out as described below.
sRNA candidates are also identified from small RNA libraries of other species, such as Arabidopsis. These candidates are then tested in plants for their effects on flowering time (see Example 5 below)
Northern Analysis
Low molecular weight RNA is extracted from phloem of vegetative and flowering plants as described in Example 4, resolved on denaturing 15% polyacrylamide gel, blotted onto Hybond XL membrane (Amersham) and UV cross-linked. DNA oligonucleotides complementary to sRNA sequences are labeled and hybridized to RNA blots overnight at 40–45° C. in a solution containing 0.5 M Na2HPO4, 1 mM EDTA, 1% BSA, and 7% SDS. Blots are washed twice for 10 min in 2×SSC, 0.1% SDS at 50–55° C. Sense DNA oligonucleotides are used as negative control.
Reverse Northern Analysis
DNA oligonucleotides (1–100 pmol) complementary to sRNA are arrayed onto Hybond N+ membrane (Amersham) and fixed by baking or UV cross-linking. Vegetative and flowering plant phloem sRNA fractions, isolated and recovered from the gel as described in Example 4 are labeled. Vegetative and flowering probes of equal specific activity are hybridized to oligo arrays to identify differentially expressed sRNAs. Array of sense DNA oligonucleotides is used as a control.
(2). Bioinformatics Identification of Small RNA Target Genes
FASTA analysis with conserved candidate small RNAs and reverse complement of conserved candidate small RNAs is performed against the following target datasets: Arabidopsis CDS, Arabidopsis 3′UTR, Arabidopsis 5′UTR, Rice CDS and Genesis plant consensi sets in SRS (Actinidia spp, Cucurbita spp, Eucalyptus spp, Festuca spp, Lolium spp, Malus domestica, Pinus radiata, PinusTaedaAll, Populus spp assembled ESTs, Sicyos angulatus and Vaccinium spp). Reverse complement hits with up to 3 mismatches are reported as putative targets. Targets conserved across taxonomic boundaries are confirmed using RNA ligase mediated 5′ RACE as described by Kasschau et al., Dev. Cell 4:205–217 (2003).
This approach is illustrated in the Table below for sequences identified herein as SEQ ID Nos. 251–263 and 383.
C. moschata phloem sap
Arabidopsis thaliana seedling
Table Legend:
miR319a: TTGGACTGAAGGGAGCTCCC (SEQ ID NO. 264)
Palatnik J F, Allen E, Wu X, Schommer C, Schwab R, Carrington J C, Weigel D. Control of leaf morphogenesis by microRNAs. Nature (2003) 425:257–263;
miR172: TTGGACTGAAGGGAGCTCCC (SEQ ID NO: 265)
Chen X. A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development. Science 303:2022–2025 (2004); Published online Jul. 31, 2003;
miR156: TGACAGAAGAGAGTGAGCAC (SEQ ID NO: 266)
miR167: TGAAGCTGCCAGCATGATCTA (SEQ ID NO: 267)
Reinhart B J, Weinstein E G, Rhoades M W, Bartel B, Bartel D P. MicroRNAs in plants. Genes Dev. (2002) 16:1616–1626. Erratum in: Genes Dev. (2002) 16:213; and Kasschau K D, Xie Z, Allen E, Llave C, Chapman E J, Krizan K A, Carrington J C. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and mRNA function. Dev. Cell (2003) 4:205–217.
miR159: TTTGGATTGAAGGGAGCTCTA (SEQ ID NO: 268)
miR169: CAGCCAAGGATGACTTGCCGA (SEQ ID NO: 269)
Reinhart B J, Weinstein E G, Rhoades M W, Bartel B, Bartel D P. MicroRNAs in plants.
Genes Dev. (2002) 16:1616–1626. Erratum in: Genes Dev. (2002) 16:2313; and
Rhoades M W, Reinhart B J, Lim L P, Burge C B, Bartel B, Bartel D P. Prediction of plant microRNA targets. Cell (2002) 110:513–520.
miR162: TCGATAAACCTCTGCATCCAG (SEQ ID NO: 270)
Reinhart B J, Weinstein E G, Rhoades M W, Bartel B, Bartel D P. MicroRNAs in plants.
Genes Dev. (2002) 16:1616–1626. Erratum in: Genes Dev. (2002) 16:2313; and
Xie Z, Kasschau K D, Carrington J C. Negative Feedback Regulation of Dicer-Likel in Arabidopsis by microRNA-Guided mRNA Degradation. Curr. Biol. (2003) 13:784–789.
miR158: TCCCAAATGTAGACAAAGCA (SEQ ID NO: 383)
Rhoades M W, Reinhart B J, Lim L P, Burge C B, Bartel B, Bartel D P. Prediction of plant microRNA targets. Cell 110:513–520, 2002.
(3). Use of Macroarrays to identify Small RNA Targets
This method utilizes an array containing DNA of putative sRNA targets (eg transcription factors, chromatin remodelling genes) identified in phloem sap or tissue cDNA libraries. Plasmid DNA (10–100 ng) is arrayed onto Hybond N+ membrane (Amersham), denatured and fixed by baking or UV cross-linking. The phloem sRNA fraction is isolated and recovered from the gel (Example 4) and is labeled. The labeled sRNA is hybridized to the plasmid DNA array overnight at 30° C. in a solution containing 0.5 M Na2HPO4, 1 mM EDTA, 1% BSA, and 7% SDS. Blots are washed twice for 10 min in 2×SSC, 0.1% SDS at 37° C. Higher stringency washes are used if required and signal is captured using a phosphorimager.
F. In Planta Candidate Gene Analysis
Once a candidate LDFSC is identified, the encoding polynucleotide sequence is analyzed for its effect on flowering by transforming a plant with a genetic construct comprising the isolated sequence operably linked to a promoter and terminator and expressing the sequence in the plant. See Examples 6–8 below. The test plant may be the same as or different from the plant that normally expresses the sequence. For example, a late flowering cucurbit may be used to test candidate LDFSCs from other cucurbits of the same or different species. If transgenic expression of a candidate LDFSC produces flowering under non-inductive conditions, it is reasonable to infer that the candidate molecule is a florigen, or a component thereof. Alternatively, an early flowering cucurbit may be used to test candidate LDFSCs from other cucurbits of the same or different species. If transgenic expression of a candidate LDFSC suppresses or delays flowering under inductive conditions, it is reasonable to infer that the candidate molecule comprises a component of flowering control.
The inventors have developed methods for transforming cucumber plants (e.g., C. sativus cv Straight Eight). GUS staining of F0 primary cucumber transformants was performed to confirm expression of the transgene. The seed from the primary transgenics was harvested and germinated. Following germination, histochemical analysis of seedlings revealed meiotic transmission of the transgene with the expected Mendelian ratio of 3:1.
Candidate genes are selected for in planta analysis both from cucurbits and from Arabidopsis. Arabidopsis orthologs of cucurbit genes are identified as follows.
BLASTX is performed using the cucurbit candidate consensus nucleotide sequence (using default BLASTX parameters) to identify the Arabidopsis ortholog from the TAIR Arabidopsis peptide database (May 2003). TBLASTN (using default parameters) is then performed using the suspected Arabidopsis ortholog peptide sequence to determine if the top reciprocal hit is the original cucurbit consensus sequence. A phylogenetic tree is assembled (using CLUSTALX version 1.8 software, with 1000 bootstrap trials) for each consensus sequence, and the cucurbit candidate peptide sequence and the putative Arabidopsis ortholog peptide sequence(s) are compared in the resulting tree. The phylogenetic tree is used in conjunction with visual inspection of the BLASTX sequence alignments to select the best matching putative ortholog or corresponding gene among the Arabidopsis genes.
(1). Overexpression of Arabidopsis Candidate Genes in Phloem Using a Companion Cell-Specific Promoter
The candidate cucurbit gene or Arabidopsis ortholog open reading frames (ATG to STOP codon) are amplified by the polymerase chain reaction from Arabidopsis cDNA or genomic DNA using gene specific primers with cloning sites (BamHI, ClaI, HindIII or SpeI). The amplified fragments are then restriction digested and cloned into a vector containing the Arabidopsis PP1 companion cell specific promoter and NOS terminator (An exemplary vector is shown in
Candidate binary constructs are transformed into Agrobacterium. Confirmed kanamycin resistant Agrobacterium are used to transform Arabidopsis thaliana Col. Plants by a floral dipping procedure. Transgenic T1 plants are selected based on kanamycin resistance and homozygous transgenic lines selected in the T2 generation. Homozygous lines are used to phenotype significant changes in flowering time under controlled long and short day light conditions.
(2). Promoter::GUS Mediated Analysis of Candidate Gene Expression Pattern in Arabidopsis
In order to provide information on the expression patterns of candidate genes in Arabidopsis, the corresponding promoter of the candidate gene (a 1.5 kb genomic region upstream of the ATG) is cloned and fused upstream of the GUS marker gene. The promoter::GUS construct is then transformed into Arabidopsis thaliana Col. plants and the GUS expression pattern determined in T2 homozygous plants at vegetative and flowering developmental stages. Correlative evidence supporting non-cell autonomous action of the candidate gene would include floral induction (or delay) and expression in the leaves but not the shoot apex.
(3). Analysis of Arabidopsis T-DNA Insertion Lines for Candidate Genes
To complement the candidate gene overexpression studies detailed above, downregulation in Arabidopsis is studied using SALK T-DNA insertion lines obtained from the NASC Center, Nottingham, UK. A T-DNA line is selected based on T-DNA insertion into the Arabidopsis candidate gene.
Segregating T3 seed lines obtained from the NASC seed stock center are then screened for significant changes in flowering time by germination of seed on Grodan rockwool blocks soaked in nutrient mix and pricking out of seedlings onto fresh blocks. Flowering time is compared to wild type and known flowering marker lines grown next to the candidate lines. Both candidate and marker lines are grown under controlled short and long day light conditions as detailed below in Example 1D.
(4). Analysis of Candidate Genes by Engineered Dominant Negative Forms and Analysis of Flowering Time in Arabidopsis Companion Cell Overexpression Transgenic Lines
Candidate transcription factors that demonstrate an alteration in flowering time following overexpression and/or downregulation in Arabidopsis or cucurbits will be engineered into dominant negative forms. In this instance, transcription factor candidates will be C-terminally translationally fused to the Arabidopsis thaliana EAR motif, a 12 amino acid repression domain that results in dominant repression of downstream target genes of the modified candidate (Hiratsu et at, (2003) Plant J. 34: 733–739). This dominant-negative approach can be used to achieve down regulation of gene families. In this way, the function of a putative flowering control transcription factor can be blocked and its effect on flowering time observed in transgenic plants overexpressing the fusion gene with a companion cell specific promoter, as described above.
In other instances, inspection of candidate sequences will suggest ways of manipulating these sequences into dominant negative forms, e.g., changes in essential amino acids, removal of RNA binding domains, and so on. These modified sequences will be tested for dominant negative effects.
(5). Analysis of Candidate Genes by Temporal Expression in Arabidopsis Companion Cell Overexpression Transgenic Lines
In addition to constitutive overexpression of candidate genes in Arabidopsis using a companion cell-specific promoter, it is possible to achieve temporal expression of a candidate gene using a chemically inducible gene switch system, such as the ethanol inducible alc gene switch system which has been characterized in Arabidopsis (see Roslan et al., (2001) Plant J. 28:225–235). Such a system would comprise an activation cassette consisting of a companion cell specific promoter driving expression of an inducible activator transcription factor and a reporter cassette consisting of promoter binding elements of the activation transcription factor fused to a minimal promoter driving expression of the candidate gene. Both activation and reporter cassettes would be cloned on a binary plasmid to enable Agrobacterium mediated plant transformation.
Transgenic Arabidopsis plants containing the inducible system are exposed to a chemical inducer at a particular stage of development, which causes a conformational change in the structure of the companion cell-expressed activation transcription factor that allows it to bind to its promoter binding elements in the reporter cassette thus driving the expression of the candidate gene. In this way, temporal companion cell-specific expression of the candidate gene can be achieved. Such induced transgenic Arabidopsis lines are grown under controlled short and long day light conditions and phenotyped for significant changes in the flowering response.
(6). Viral Vector Mediated in Planta Candidate Gene Analysis
In planta candidate gene analysis is performed, in part, utilizing a plant viral vector delivery system. The vector derived from Zucchini yellow mosaic potyvirus (ZYMV) is used to deliver and mediate expression of candidate genes in the phloem-associated cells of Sicyos angulatus or Cucurbita moschata PI441726 maintained under non-inductive conditions. The viral vector was originally developed by the lab of Shyi-Dong Yeh from the Department of Plant Pathology of the National Chung Hsing University in Taichung, Taiwan (Bot. Bull. Acad. Sin. 2002, 43:261).
The potyvirus group (including ZYMV) contain a single-stranded positive sense RNA molecule of 8–10 kb. The genome of the ZYMV is translated into a single polypeptide of 350 kDa which is subsequently cleaved by the viral protease into 10 proteins essential for the virus infectious cycle. To overexpress the candidate gene, the coding sequence of the candidate is introduced into the genome of the virus as a translational fusion bordered by the viral protease cleavage site. Two variants of ZYMV will be used for overexpression of candidate genes. In the first variant, the site of the candidate gene insertion is located between proteins 9 and 10 (Nib and CP), while in the second variant, the insertion is between protein 1 and 2 (P1 and HC-Pro).
In both vectors, the site of insertion is followed by sequences encoding the cleavage site recognized by the Nia viral protease (protein 8). The presence of an additional protease recognition site upstream ensures that the inserted protein is released from the polyprotein albeit with N-terminal and C-terminal amino acid extension by the Nia protease. During the infection process the expressed candidate sequence is released in the cytoplasm of the infected cell. The capacity of ZYMV to move cell to cell and systemically within the plant ensures release of the candidate protein in cells throughout the infection process. Using this viral vector delivery system, we have demonstrated that cucurbit CO and FT induce flowering when expressed in the Cucurbita moschata PI 441726 system (See
Other experiments in progress are designed to identify functional regions of the FT polypeptide that are involved in one or more aspects of floral control.
In these experiments, peptides of 3–27 amino acids are derived from the FT N-terminus and tested for their ability to induce flowering (
2. Methods of Modulating the Florigenic Signaling Pathway
In another of its aspects, the present invention provides methods for modulating the florigenic signaling pathway to modify the occurrence, timing and extent of flower development.
The phrase “modulating the florigenic signaling pathway” means altering one or more steps in the above-described florigenic signaling pathway to effect either positive or negative changes in flower induction. The one or more modulatable steps in the pathway include: the reception and transduction of a signal from a leaf sensor cell by a CC in the leaf; the process of rendering the signal transmissible; the controlled entry of the LDFCS into the phloem; the translocation of the LDFCS in the phloem (including regeneration and amplification steps); the delivery of the signal to the target meristem tissue (including any transformations of the signal that may be required for egress or propagation of the signal from the terminal phloem).
A. Modulation of Endogenous Gene Transcription Using Agonists or Antagonists
The transcription of genes, identified as described above, which are involved in the production or delivery of an LDFSC, can be modulated by an agonist or antagonist of a binding event between a signaling molecule and its cognate binding partner that initiates the florigenic signaling cascade in the CC cells. The agonist or antagonist acts at the binding step or downstream of it, e.g., at a site in the signal transduction pathway, to increase or decrease gene transcription. The modulatable binding event may occur anywhere in or on the cell. For example, the binding interaction may occur inside the cell or at its surface or beneath the cell surface, in the cytosol, in a subcellular membrane or organelle, e.g., the nucleus.
The binding molecule may act as a transcription factor for one or more genes in the florigenic signaling cascade. In this case, the agonist (or antagonist) may interact with the transcription factor and/or other endogenous transcription complex proteins, or with a polynucleotide regulatory sequence, to enhance (or inhibit) transcription. If transcription is normally repressed, the agonist may bind a repressor molecule or a cis-element of a regulatory region to relieve repression. Alternatively, the agonist (or antagonist) may be a polynucleotide sequence that is used as a decoy to bind (i.e., to titrate) a repressor (or transcription) factor.
The agonist or antagonist may be isolated from a natural source or produced by synthetic or recombinant techniques. Exemplary agonists/antagonists include proteins, peptides, RNAs, naturally-occurring or synthetic molecules/complexes, and combinations thereof. Further modification is also contemplated, e.g., post-translational modification of a protein or modification of one or more components of a complex of any of the aforementioned constituents. Antibodies or their derivatives (e.g., single chain antibody fragment(s) [ScFv], anti-idiotypic antibodies) that directly or indirectly modify the activity of the LDFSC or LDFSC component(s) are also useful agonists and antagonists and can be expressed in plants if desired.
Depending on the biochemical nature of the agonist or antagonist, the latter may be introduced into the plant by, for example, wick feeding directly into the vasculature, physical contact (e.g., rubbing) of agonist or antagonist in the presence of absence of compounds (e.g., surfactants) that facilitate penetration into plant tissues, use of an engineered RNA transporter (ERT) that is transported between cells by a defined protein machinery provided in trans to deliver a defined agonist or antagonist in the presence or absence of a virus (Lough, Emerson, Lee, Forster and Lucas, unpublished data)), use of a modified ERT that encodes both the transporting proteins and the transported agonist or antagonist, grafting techniques (e.g., bark grafting) for diffusible agonists or antagonists, Agrobacterium tumefaciens or biolistic bombardment mediated transient expression resulting in the local production of an agonist or antagonist, use of viral vectors (e.g., based on tobamoviruses, potexviruses or tobraviruses) to deliver the agonist or antagonist, and the like.
B. Modulation by Introducing a Recombinant Gene that Encodes an LDFSC or LDFSC Component
A genetic construct comprising one or more polynucleotide sequences that encode a candidate LDFSC or LDFSC accessory component can be introduced into a plant to determine the effect of expressing the sequence on floral induction. Ectopic expression can be used for validating the involvement of an endogenous gene in the florigenic signaling pathway and for determining the interchangeability of a candidate LDFSC or LDFSC component(s) that is derived from one plant species in the florigenic cascade of a different plant species.
The present invention contemplates the use of sense constructs to upregulate gene expression (either directly or by co-suppression of repressor genes). Genetic constructs comprising partial or full-length sequences can also be used to downregulate or silence the expression of repressor genes that affect the competency to flower, or to reduce the expression of florigenic signaling genes. RNAi methods and antisense technology are well known to those skilled in the art and can be used to selectively reduce or eliminate gene expression. RNAi reduces or eliminates specific gene functions by targeting for degradation the transcription products of genes with dsRNA homologous to the introduced sequence (reviewed by Sharp, Genes Dev. 13: 139–41 (1999)). RNAi can be produced by introducing double-stranded or single stranded RNA into plant cells, or by transforming the plants with RNAi expression constructs. The use of VIGS that can controllably and efficiently reduce gene expression in a systemic manner provides an alternative means for modulating the extent and timing of flowering. RNA virus vectors have also been described in connection with PTGS. Representative examples of RNAi silencing methods can be found in the following patent applications and publications: PCT Applications WO 99/49029, WO 98/36083, WO 99/15682, WO 98/53083, WO 99/53050, WO 00/49035, WO 01/77350, WO01/94603, WO02/00894, WO01/75164, and WO01/68836; Waterhouse et al., Proc. Natl. Acad. Sci. USA 95: 13959–13964 (1998); Smith et al., Nature 407: 319–320 (2000), Ruiz et al., Plant Cell 10: 937–946 (1998).
The above-described constructs utilize promoters that are expressed in many cell types (e.g., CaMV, 35S, superubiquitin), or promoters that are tissue specific and/or inducible. In a preferred embodiment of the invention, CC-specific promoters are used (e.g., the promoters for AtSut1, AtSUC2 (Genbank accession X79702), rolC, C. melo GAS1 (Genbank accession AF249912), AtPP16K (Genbank accession ATT22E16), AtPP1 (Genbank accession AL161544, AtPP2 (Genbank accession AL161551), AtLEAFY (Genbank accession M91208), and others.
Examples of inducible promoters include, for example, ethanol (Caddick et al., Nature Biotech. 16:177–180 (1998)), copper (Mett et al., Proc. Natl. Acad. Sci. USA 90: 4567–4571 (1993)), ecdysone (Martinez et al., Plant J. 19:97–106 (1999) and others. Many plant promoters are known that can be used for practicing this invention.
Stably transformed plants expressing an LDFSC or LDFSC component can be obtained using Agrobacterium tumefaciens or microprojectile bombardment transformation procedures. These and other plant transformation methods are disclosed in this application and are well known to plant biotechnologists.
C. Modulation of Flowering by Use of a Covalently Modified LDFSC
A florigenic signal that is taken up by, or produced within, the CC may require covalent modification(s) for entry into the phloem, for delivery to meristematic target tissue, and/or for activity at the target site. For example, a florigenic inducer that is produced elsewhere and taken up by the CC may contain additional amino acid residues/sequences (i.e. signal motifs/sequences) that direct its retention in CC cells. The cleavage of such sequences may be required for release from sites within the CC for subsequent transport into the SE. Alternatively, for entry into the phloem, a florigenic inducer may require modification, for instance, post-translational modification such as phosphorylation, glycosylation, or cleavage of amino acids, or by removal or addition of nucleotides as a result of alternative splicing (see, e.g., Lopato et al., Genes Dev. 13:987–1001 (1999)).
The identification of relevant modifications is made by correlating data from phloem sap analysis with sequence databases (prepared as described above). For example, phloem mobile peptides that originate from propeptide precursors or CC retention signals that require removal prior to translocation of a protein into the sieve tube system are identified by correlating mass fingerprinting and fragment analysis of phloem-mobile peptides and proteins with database sequences. Comparisons between phloem-mobile gene products predicted from the vascular gene database and phloem-mobile gene products defined by direct analysis are expected to yield information about potential targeting sequences, motifs and CC retention signals. In addition, comparison of phloem mobile RNAs, peptides and proteins may uncover common cis-acting zip code sequences and phloem-targeting sequences. Post-translational modifications may be detected by selective enrichment of post-translationally modified proteins from crude extracts using affinity-based methods (e.g., lectins for glycosylated proteins, high-affinity antibodies for phosphotyrosinyl residues, Pandey et al., Proc. Natl. Acad. Sci. USA 97:179–184 (2000)) or biotin affinity labels for phosphoseryl/phosphothreonyl residues (see Oda et al., Nature Biotech. 79:379–382 (2001)) combined with mass fingerprinting and fragmentation analyses (See, e.g., Zhou et al., Nature Biotech. 79:375–378 (2001)).
Putative retention signals, motifs or signal sequences identified by the above procedures can be translationally fused to a reporter molecule to ascertain the effect of the modification (e.g., selective retention or gain of function). The resulting information is useful in the design of fusion genes for modulating flowering when introduced into a plant.
The effect of other posttranslational modifications on a florigenic signaling protein, such as phosphorylation, isoprenylation, glycosylation, conjugation to small molecules and others, may be investigated, for example, by targeting specific cellular enzymes/enzyme cascades that catalyze the modifications, by changing the availability of substrates or cofactors, or by altering the amino acid sequence of a florigenic signaling protein.
D. Modulation of Delivery of an LDFSC to the Target Meristematic Tissue
The spatial and temporal characteristics of flowering can be modulated by affecting one or more of the following processes involved in the delivery of an LDFSC to the target meristem: the entry of the LDFSC into the phloem; the translocation of the LDFSC from the point of entry to the point of delivery in the terminal phloem; and the delivery of the signal to the target tissue.
The entry of the LDFSC into the phloem can be modulated by changing one or more of the following: the molecular dimensions of the LDFSC, the plasmodesmal SEL; or the translocation machinery within the plasmodesmata (PD).
Conformational changes in a protein or ribonucleoprotein LDFSC may involve a specific chaperone or receptor molecule within the PD that recognizes, binds and unfolds the protein. See, e.g., Kragler et al., EMBO J. 19:2856–2868 (2000). The binding of the chaperone to the LDFSC or to a binding site within the PD may be altered by modifying the binding properties of the chaperone directly, by changing its structure, or indirectly with agonists or antagonists of binding. Methods involving the use of viral and plant movement proteins to mediate cell-to-cell transport can be used in the practice of the present invention. Such methods are described in PCT/US96/13299 and PCT/US96/19260, which are incorporated by reference herein in their entirety.
Changes in the plasmodesmal. SEL, e.g., dilation or constriction of the PD aperture, may be produced by changes in the organization or contractility of filamentous actin, actomyosin or centrin brought about, e.g., by changes in calcium levels or phosphorylation-dephosphorylation. (See e.g., Zambryski and Crawford, Ann. Rev. Cell Dev. Biol. 16:393–421 (2000)). Modulation of calcium release and sequestration by Ca++ binding proteins in PD, of cell wall protein kinases and/or phosphatases, the introduction of actin-disrupting or stabilizing agents and/or modifications in myosin or centrin may alter the movement of materials through the PD.
Vectorial translocation of macromolecules through the PD may be effected by molecular motors (e.g., microtubule-based kinesin motors or actin-based myosin motors) (see Vale and Milligan, Science 288:88–95 (2000); Mermall et al., Science 279:527–533 (1998)), filament- and tubule-based springs, and by polymerization-induced growth (Mahadevan and Matsudaira, Science 288:95–99 (2000)). Changes in actomyosin and actin organization described above are expected to affect translocation as well.
The egress of the LDFSC from the terminal phloem may require additional modifications. For example, the removal of retention signals or a change in the size or conformation of LDFSC may be required to allow movement of the LDFSC from the phloem into the target site. If the LDFSC moves via symplastic transport, the extent and/or rate of movement may be influenced by the number and/or structure of functionally active PD at the apex (Ormenese et al., Planta 211:370–375 (2000)). As well, the delivery route may be dependent upon the presence of address signals on the LDFSC that can be recognized by the transporting channels. Alternatively, if the LDFSC is communicated by a relay mechanism, the relay may require regeneration of the original signal or its conversion to a different form that can be propagated to, and received and transduced by, target meristem cells. One or more of these processes may be susceptible to modification to modulate a flowering response.
In general, it is understood that modulators of the flowering response may be components of other pathways that use both local and systemic transport of signals, e.g., stress-activated responses, viral infection and immunity, etc. and that modulators may be interchangeable between different species of plants.
The ability to modulate the amount of an LDFSC produced in response to an external stimulus may also be useful, e.g., to determine the stability of an LDFSC, to determine the relationship between the amount of LDFSC produced and the rate of progression to flowering (see, e.g., Pouteau et al., Plant Physiol. 118:1191–1201 (1998)) and to identify cell types involved in the regeneration or amplification of LDFSC during systemic transport. Modulation may be effected by changing the duration of exposure of a plant to an external stimulus, or the total portion of the plant exposed to the stimulus (e.g., the number of induced leaves on the plant, Tooke and Battey, Plant Cell 12:1837–1848 (2000)).
The amount and position of flowers that form on a plant may be modulated by altering the responsiveness of axillary meristem to flower inducing signals, e.g., by interfering with repressors of flowering or by facilitating the transport of inducers to axillary meristems.
3. Methods of Screening for Modulators of the Florigenic Signaling Pathway
The present invention comprises various methods of screening for modulators of the florigenic signaling pathway. In the broadest aspect, these methods comprise the following steps:
exposing a plant (or a portion thereof) to a flowering stimulus;
contacting the plant (or portion thereof) with a candidate modulator; and
assessing the effect of the candidate modulator on florigenic signaling.
A “flowering stimulus” is any stimulus that is capable of inducing the florigenic signaling cascade, e.g., a photoperiod, a stress response, an artificial stimulus or a natural stimulus. A plant (or portion thereof) used for screening may be exposed to the signal directly or indirectly, e.g., via a heterograft that has been stimulated.
“Contacting” may be by any means, including direct surface application to an intact or cut portion of the plant, microinjection into a tissue or cell thereof, microbombardment, preferably under low pressure, etc.
The effect of the candidate modulator on florigenic signaling involves comparing a stimulus-dependent biochemical, functional, cytological or morphological change in the plant in the presence and absence of the candidate modulator. This change may involve, e.g., the production or inhibition of an inflorescence meristem or flower, a change in mitotic activity in shoot apical or lateral meristem cells, a change in symplastic transport of reporter-tagged macromolecules between cells in the meristem, the appearance of one or more unique transcripts or gene products in a portion of the plant involved in the generation, transmission or reception of a florigenic inducing signal (e.g., leaf cells or vasculature, stem vascular tissues, phloem sap, shoot apex), the activation of a genetic construct in a transformed plant cell, and other changes that are indicative of, or may correlate with florigenic signaling.
In vitro binding assays are also used according to the invention to identify agents that are capable of modulating the florigenic signaling pathway, e.g., by affecting the binding of an LDFSC to an LDFSC accessory component, an LDFSC to an LDFSC receptor in a target tissue or an LDFSC to a polynucleotide involved florigenic signaling.
Uses of this Invention:
The present invention provides methods for identifying an LDFSC and other components of the florigenic signaling pathway and for modulating floral development in a wide variety of plant species. These methods can be practiced by those of ordinary skill in the art, without undue experimentation, using currently available techniques that are routinely used in biotechnology and are referenced herein.
The identification of an LDFSC and other components of the florigenic signaling pathway is a prerequisite for the development of modified plants that display accelerated or delayed flowering responses compared with unmodified native plants. Such plant modifications have substantial practical uses, including for example: synchronization of floral induction in ornamental flowering plants and mechanical harvested crops such as peas to ensure that the produce obtained from all of the plants is uniform; suppression of florigenic signaling to delay or abolish flowering in instances where flowering lowers the value of the harvested crop (e.g., onion, celery, cabbage and other vegetables that bolt); selective induction of either male or female flowers when these flowers are normally derived from separate meristems; induction of male sterility by suppression of florigenic signaling that otherwise would lead to induction of male flowers or flower parts; acceleration of flowering time to lessen the time required to collect seed; acceleration of flowering time to enhance quality (e.g., in wine grapes); enhance reproductive to vegetative ratio in evergreen fruit crops such as kiwifruit and avocados; use of a florigenic activator to synchronize bud burst, e.g., in New Zealand kiwifruit (Actinidia spp.), table grapes, and for other applications where an agrichemical spray such as hydrogen cyanamide is presently used for this purpose.
Key floral integrators coordinate the initiation of flowering. We have shown that both the Arabidopsis Flowering locus T (FT) and the pumpkin ortholog of FT promoted flowering via the phloem. FT and other flowering control genes acting in the phloem will serve as targets for the rational design of “drugs”. Crystallographic data and molecular modeling will be used to design small molecule “drugs” to specifically interact with identified targets. The drugs are then synthesized and tested for their ability to interact with the target, using binding assays. Preferably, these drugs pass through the barriers of the leaf, e.g. the cuticle, when sprayed onto the leaf, and act systemically to modify and control the flowering response of the plant. The candidate drugs are then tested in plants for the ability to act systemically and modify flowering. The capacity of a spray to promote flowering will be tested using Arabidopsis maintained under non-inductive short days. Positive controls will include transgenic plants ectopically expressing FT or plants photoperiodically induced to flower. Quantifying the effect will be achieved by recording the number of rosette leaves formed at time of bolting.
The capacity of the spray to suppress flowering will be tested using Arabidopsis induced to flower by long day treatment. Controls will include plants maintained under short day photoperiods. Quantifying the effect will be achieved by recording the number of rosette leaves formed at time of bolting. Additional evaluation systems are envisaged. For instance spray testing of lettuce plants maintained under inductive conditions (in this case >30° C.).
The following examples are presented solely to illustrate the practice of the invention, and not to limit the scope of the invention as claimed.
This Example describes methods of controlling floral induction in plant systems used for identification and isolation of candidate flowering control genes from Cucurbit species and Arabidopsis.
A. Sicyos angulatus
Sicyos angulatus is grown in controlled environment chambers. Surface sterilized, imbibed, decoated seeds are germinated on a heated pad at 27° C. in vermiculite under constant light. Seedlings are transferred to Grodan rockwool blocks soaked in nutrient mix (recipe, CF values and pH available on request) and maintained as follows: 1) 26° C.; 2) 50% humidity and 3) an 8 hr light period with 16 hr dark period interspersed with red light night breaks. Currently these night breaks are of 5 min duration and occur every 60 min. Plants maintained in this manner remain vegetative without visible signs of floral bud development. When the plants are established and have grown ˜8–10 nodes (usually 10–14 days) the temperature is altered and maintained at 15° C. until time for tissue/phloem sap collection. Flowering is induced by removal of the night breaks while maintaining an 8 hr light period.
Stages of flowering were assessed phenotypically as described by Takahashi et al., Plant Cell Physiol. 23:1–9 (1982). Flower initiation, sexuality and development of flowers on each node of the main shoot were observed. Many floral buds produced in the present experiments stopped development at various floral stages, and the final stages reached were recorded for each bud. Floral stages were determined by the longitudinal length for pistillate flowers and by the width for staminate flowers according to the following classification: 0, vegetative; 1, inflorescence primordium; 2, floral bud not exceeding 0.5 mm; 3, 0.6–1.5 mm; 4, 1.6–2.5 mm; 5, above 2.6 mm; 6, flower in anthesis.
B. Cucurbita moschata
100 different accessions of Cucurbita moschata were obtained from the seed stock center. These were phenotyped in a greenhouse to identify early and late flowering accessions. Cucurbita moschata PI212011 was identified as an accession that flowered readily under both long days and short days. We selected this as an early flowering accession and seed stocks were bulked up in the greenhouse. Cucurbita moschata PI441726 was identified as late flowering, and we have shown that this accession can be maintained in a vegetative state under long days. There is a temperature component to floral induction with this accession, as long day repression appears to be relaxed as the temperature rises. Phloem sap obtained is used for comparing the phloem mobile population of molecules in flowering and non-flowering plants.
C. Heterograft
We have determined that Cucumis sativus cv Straight 8 is an excellent source of floral stimulus, by showing that:
The Cucumis sativus cv Straight 8 (stock)/Cucurbita moschata P1441726 (scion) heterografted plants are maintained in a greenhouse. Cucumis sativus cv Straight 8 plants are germinated at 27° C. on a heated pad, then transferred to Grodan rockwool blocks and grown hydroponically. When the plants are 8–15 nodes in height they are grafted with a Cucurbita moschata PI441726 scion using a reciprocally cut V-shaped wedge graft. The graft union is physically held using aluminum foil, and humidity is maintained by placing a plastic bag over the top. The bag is removed 7–10 days later when the graft shows signs of active growth. As the plants continue to grow, ˜100% of grafted scion show signs of floral induction. Buds appear and eventually flowers go to full anthesis. To maximize transport of phloem into the scion, and hence floral induction, we remove the leaves and wrap the scion in black paper, thereby manipulating the source to sink ratio. Phloem sap is collected from the scion either by removing the scion above the graft union and bleeding from the cut end or nicking the scion stem while still attached and collecting the exuding droplets.
D. Arabidopsis
Floral induction can be induced and synchronized by first vernalizing plants and then growing plants under short day conditions prior to a single long day floral induction. These procedures have been described and refined by the group of George Bernier (Corbesier et al., (1996), Plant J. 9:947–952; Jacqmard et al., (2003), Ann. Bot. (Lond). 91:571–576; Tocquin et al., (2003), BMC Plant Biol. 3:2). Plants are first vernalized for 42 days at 4° C. and then maintained under non-inductive short day conditions for 40–60 days. A single long day (e.g., a single intervening dark period is replaced with continuous light) is sufficient to induce synchronized flowering.
Candidate genes derived from the Genesis cucurbit phloem sap database (whether Arabidopsis orthologs or full length cucurbit candidate genes) are introduced into Arabidopsis Col. by a floral dip procedure under the control of a constitutive promoter (e.g. superubuiquitin promoter), a companion cell-specific promoter (e.g., the Arabidopsis PP1 promoter), another tissue specific promoter or an inducible promoter. Selection of primary transformants is performed using seeds sown on plates or directly onto rockwool containing kanamycin. Alternatively, homozygous T2 or T3 seed is pre-screened and collected. Seedlings are vernalized for 1–6 weeks at 4° C. in the dark and then transferred to SD growing condition (8 h light/16 h dark, 22° C., 60–65% humidity, and light levels of 50–200 μmol m−2s−1 for growth and synchronized induction. Floral induction is initiated using an uninterrupted long day light period.
The above procedure is used to determine the ability of flowering control candidate genes to induce Arabidopsis to flower under non-inductive SD conditions or to suppress or otherwise modify synchronized floral induction.
This Example illustrates the use of C. sativus (scion)/C. maxima (rootstock) to identify phloem-mobile pumpkin sequences represented in libraries constructed from RNA of heterografted plants.
1. Grafting Protocols
The side-grafting technique of Tiedemann, Plant Physiol. 134:427–440 (1989) was employed, with some modifications, to generate heterografts between scions cut from 4-week-old cucumber (Cucumis sativus cv. Straight Eight) plants (vegetative apex to the second expanded leaf) and stocks provided by equivalent-aged pumpkin plants. Each excised cucumber scion (approx. 5–10 cm in length) was carefully inserted into an incision made in a pumpkin (or cucumber for autografts) stem at a location 10 cm back from the vegetative apex. The graft site was fastened with tape or aluminium foil and sealed with a ziplock clear plastic bag. Grafted plants were grown in a greenhouse for 2–3 weeks. These conditions were optimized for the formation of functional graft unions. Plants were employed for phloem sap analysis 3–4 weeks after being grafted.
2. Phloem Sap Collection and RNA Extraction
Phloem sap was collected essentially as described by Ruiz-Medrano et al., Development 126:4405–4419 (1999) from Cucurbita maxima, Cucurbita moschata and Cucumis sativus plants and the grafted scions of Cucumis sativus. Stems or petioles were excised from the plant and blotted, twice, for several seconds onto sterile filter paper (#3 MM; Whatman, Maidstone, UK). Phloem sap exuded thereafter was collected using sterile micropipette tips (200 ml) and immediately mixed with an equal volume of 8 M guanidinium buffer (Logemann et al., Anal. Biochem. 163:16–20 (1987)) or phenol. Proteins in this sap were then extracted, twice, with a 25:24:1 phenol:chloroform:isoamyl alcohol mixture in the case of guanidinium buffer or chloroform in the case of phenol. The remaining RNA was then precipitated with 1.0 volume of isopropanol, 0.1 volumes of 3 M sodium acetate, pH 5.2 and 20 μg of a carrier such as linear acrylamide or glycogen, centrifuged at 4° C. for 45 minutes, washed with 70% ethanol, centrifuged at 4° C. for 30 minutes and then resuspended in sterilized deionized water.
Typically, RNA was extracted from 5–15 ml phloem sap of Cucurbita maxima, Cucurbita moschata, Cucumis sativus or heterografted plants. cDNAs were cloned from phloem sap RNA as described below in Examples 3 and 4. Cucurbita maxima (or Cucurbita moschata) and Cucumis sativus are taxonomically distinct species (Jobst et al., Mol. Phylogenetics Evolution 8:204–219 (1998)). This is reflected in sequence divergence and, as a consequence, comparative bioinformatic analysis of Cucurbita, Cucumis and heterograft phloem sap libraries provides a means of identifying phloem mobile pumpkin sequences represented in libraries constructed from RNA of heterografted plants.
PolyA+ RNA was isolated from phloem sap using methods described by Ruiz-Medrano, Development 126: 4405–4419 (1999).
One methodology, APAC (Arbitrarily Primed Amplified cDNA) was developed to clone ESTs from cucurbit phloem sap. The APAC method relies on PCR and is used with up to 40 cycles of amplification.
APAC Method
This method is illustrated in
Small RNA Collection from Phloem Sap
Phloem sap was collected according to methods described by Ruiz-Medrano, Development 126:4405–4419 (1999) and Yoo et al., J. Biol. Chem. 277:15325 (2002).
Purification of High and Low Molecular Weight RNA
High and low molecular weight fractions of RNA were prepared from total RNA using RNAqueous™ kit (Ambion, Austin Tex.). High molecular weight RNA was recovered from the glass fibre filter to which it binds in concentrated chaotropic salt solutions. Low molecular weight RNA was recovered from the flow-through and separated by electrophoresis through a denaturing 15% acrylamide gel. RNA in the 19–25 nt range (small RNA or sRNA) was excised from the gel and eluted with 0.5 M Na acetate, 10 mM EDTA at 4° C. overnight. The sRNA fraction was recovered by ethanol precipitation with 40 μg of glycogen and cloned.
Library Construction
Cloning Protocol 1 (Adapted from Elbashir et al., (2001) EMBO J. 20:6877–6888).
The sRNA sample, prepared as described above, was treated with Shrimp Alkaline Phosphate (SAP, Roche Diagnostics, Indianapolis Ind.), extracted with phenol/chloroform, and precipitated with ethanol. The dephosphorylated sRNA sample was ligated to the 3′ RNA/DNA adapter (PO4-UUUAACCGCATCCTTCTC-fluorescein), where UUU are ribonucleotides, (SEQ ID NO: 111) in 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 5% PEG 8000, 0.1 mg/ml BSA, with 50 U T4 RNA ligase at room temperature for 2–4 hours or at 5° C. overnight. The ligation product is recovered from the gel as described above and is 5′ phosphorylated in 70 mM Tris-HCl pH 7.6, 10 mM MgCl2, 100 mM KCl, 1 mM 2-mercaptoethanol, 1 mM ATP, with 10 U T4 polynucleotide kinase at 37° C. for 10 minutes. The phosphorylation reaction is stopped by phenol/chloroform extraction and RNA is recovered by ethanol precipitation. The 5′ DNA/RNA adapter (Cy5-TACTAATACGACTCACTAAA), where AAA are ribonucleotides, (SEQ ID NO: 112) is then ligated to the phosphorylated ligation product as described above. A fluorescent 3′ end block on the 3′ adaptor and Cy5 on the 5′ adaptor are useful for ease of monitoring the products dating synthesis. The new ligation product is extracted with phenol/chloroform, and precipitated with ethanol. This is followed by RT-PCR with 3′ and 5′ DNA oligonucleotides, respectively (GACTAGCTGGAATTCAAGGATGCGGTTAAA) (SEQ ID NO: 113) and CAGCCAACGGAATTCATACGACTCACTAAA (SEQ ID NO: 114) (bold, EcoRI site), using SuperScript One-Step™ RT-PCR with Platinum Taq (Invitrogen, Carlsbad, Calif.). Alternatively, reverse transcription is performed, as described below, with 3′ DNA oligonucleotide using SuperScriptII™ reverse transcriptase, followed by PCR with Advantage2 Polymerase (Clontech Laboratories, Palo Alto Calif.). The PCR product is recovered from the gel as described above, precipitated with ethanol and cloned into pCR4-TOPO (Invitrogen).
Cloning Protocol 2 (Based on Clontech SMART Technology)
The miRNA sample is polyadenylated in 1 mM ATP, 20 mM Tris-HCl, pH 7.0, 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 μg/ml BSA, 10% glycerol and 500 U poly(A) Polymerase at 30° C. for 20 min. Excess ATP is removed using a ProbeQuant™ column (Amersham Pharmacia, Uppsala, Sweden) and the polyadenylated RNA is precipitated with ethanol. This sample is then used for reverse transcription using an anchored 3′(SMART) oligonucleotide,
(ATTCGAATTCCGAGGCGGCCGACATG(T)12VN) (SEQ ID NO: 115), where V=G, C or A and N=G, A or T; (bold, EcoRI site);
a 5′(SMART) oligonucleotide,
(AAGCAGTGGTATCAACGCAGAGTGAATTCTACGGCCGGG), (SEQ ID NO: 116),
and PowerScript™ reverse transcriptase (Clontech). Reverse transcription is followed by PCR using 5′ and 3′ PCR primers, respectively (AAGCAGTGGTATCAACGCAGAGT) (SEQ ID NO: 117) and (ATTCGAATTCCGAGGCGGCCGACATG) (SEQ ID NO: 118) (bold, EcoRI site). The PCR product is purified with phenol/chloroform extraction, precipitated with ethanol, digested with EcoRI and concatamerized using T4 DNA ligase. Concatamers of a size >200 bp are cloned into pBluescriptSK.
The polynucleotide sequence of mRNA158 was amplified from A. thaliana genomic DNA using primers given in SEQ ID NOS: 385 and 386 (see Table below). The amplified fragment, given in SEQ ID NO: 384 which contained the mature mRNA (SEQ ID NO: 383) (Rhoades et al., Cell 110:513–520, 2002) was cloned into the plasmid pART27 under control of a constitutive promoter (SEQ ID NO: 2 from U.S. Pat. No. 6,380,459). The construct was transformed using standard Agrobacterium-mediated techniques into A. thaliana Columbia and the plants were maintained under non-inductive short day conditions (8 hours light).
Five control sets of plants were transformed with DNA derived from five separate regions of A. thaliana Columbia genomic DNA
For each of these control sets, a fragment of A thaliana Columbia genomic DNA was amplified using the primer pairs given in SEQ ID NOS: 389 and 390; 392 and 393; 395 and 396; 399 and 400; 403 and 404, respectively. The primers were derived from the miRNA sequences listed in the Table below, and the sequences of the amplified miRNA precursors are given in SEQ ID NOS: 388, 391, 394, 398, and 402 respectively. The sequences of the respective mature miRNAs are given in SEQ ID NO: 387, 267, 268, 397, and 401. Each amplified fragment was cloned into pART27 under the control of a constitutive promoter (SEQ ID NO: 2 from U.S. Pat. No. 6,380,459) and transformed into A. thaliana Columbia. These control plants were similarly maintained under non-inductive short day conditions (8 hours light).
At 91 dps, 23 test plants were analyzed for development of flowers. Of the plants analyzed, one did not flower, six plants had bolts of less than 2 cm, and eight had bolts of less than 5 cm. Control plants from each of the five control plant groups (20–24 plants per control group) were all flowering at 91 dps with bolts longer than 10 cm (
A. thaliana
References: mi39 and mi2: Llave, C., Kasschau, K. D., Rector, M. A. and Carrington, J. C. Endogenous and Silencing-Associated Small RNAs in Plants. Plant Cell 14:1605–1619, 2002.
Llave, C. Xie, Z., Kasschau, K. D, Carrington, J. C. Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA. Science 297:2053–2056, 2002.
To test the effect of FT on induction of flowering, the coding sequence of A. thaliana FT (AtFT; given in SEQ ID NO: 198) was cloned into the ZYMV vector using standard cloning techniques. Three independent experiments were performed. In each experiment, four to six Cucurbita moschata PI441726 plants were inoculated with the ZYMV/AtFT construct and maintained under non-inductive conditions. An empty ZYMV vector was used as control. Results from these experiments showed that experimental plants containing the ZYMV/AtFT construct formed floral buds and flowered within 25–35 days (
Similar results were obtained when Cucurbita moschata PI441726 plants was inoculated at different ages with the ZYMV/AtFT (
These results showed that AtFT and AtCO promoted flowering in Cucurbita moschata PI441726 plants maintained under a non-inductive photoperiod.
To test the effect of two DNA-binding proteins isolated from cucurbit phloem libraries on induction of flowering, the coding sequences given in SEQ ID NOS: 378 and 380 were cloned into the ZYMV vector using standard cloning techniques. A description of the genes is given in Table 1. The ZYMV vector was used to express these genes in six Cucurbita moschata PI441726 plants that were maintained under non-inductive conditions. An empty ZYMV vector was used as control. Up to 50% of experimental plants flowered within 45 days of inoculation with ZYMV expressing either of the DNA binding proteins (SEQ ID NOS: 378 or 380). No flowering was observed in the control plants under these conditions.
The polynucleotide encoding the FT ortholog from Cucurbita maxima (given in SEQ ID NO: 132) was cloned into the plant transformation vector pART27 under control of Suc2, a companion cell-specific promoter (Imlau et al., Plant Cell 11:209–322, 1999). This construct was transformed into 35 A. thaliana plants using standard Agrobacterium transformation techniques. Sixteen control plants were transformed with an empty vector under control of 35S promoter without a reporter gene. An additional set of twenty control plants were transformed with the companion cell specific promoter PP1 (Clark et al., Plant J. 12:49–61, 1997) with a GUS reporter. All plants were maintained under non-inductive short day conditions of 8 hours light. Companion cell mediated expression of FT was analyzed 34 days post sowing (dps) and flowering was observed in 25 of the 35 experimental plants (
General Methods:
Plant material is grown in controlled environment chambers as described by Xoconostle-Cazares, Plant J. 24:1–16 (2000).
Alternatively plant material is grown in a greenhouse under the following conditions: a minimum of 22° C. (night) and 22–28° C. (day); supplemental lighting (daylength is approximately 10 h/day in mid-winter and approximately 15 h/day in mid summer) to extend daylength to 18 hrs, ambient humidity (normally 50–100% in Auckland, New Zealand), automatic watering and nutrient addition (an equal dose of solution A [calcium nitrate, 85 kg; ammonium nitrate, 7.8 kg; potassium nitrate, 9.6 kg; iron chelate, 0.675 kg; per 1000 l] and solution B [potassium nitrate, 56.2 kg; monopotassium phosphate, 20.4 kg; magnesium sulphate, 30.8 kg; manganese sulphate, 0.17 kg; zinc sulphate, 0.145 kg; borax, 0.240 kg; copper sulphate 0.019 kg; sodium molybdate, 0.012 kg; per 1000 l] were provided to plants such that the conductivity ranged between 23–30 and pH 5.5–6.5 when delivered to the plant pot).
Analysis of Proteins and Peptides
Polyacrylamide gel electrophoresis (1D and 2D) is performed using standard procedures as described in Sambrook et al., Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, and Matsudaira et al., A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press, 1989).
Mass finger printing and fragment analysis is performed by LC-MS/MS. A general review of mass spectrometry techniques in protein identification can be found in Roepstorff, Curr. Op. Biotech. 8:6–13 (1997). Prior to mass spectroscopy, samples of phloem sap proteins and peptides are partially purified by chromatography to remove ions and non-protein contaminants.
Interpro Analysis
The InterPro motif database is available on the Internet.
The software InterProScan Release v3.1 was used to scan the predicted proteins from the cucurbit Stackpack assembled consensus DNA sequences. The InterProScan optional parameters used in running the software were as follows:
Interproscan.pl:
+ipr+go+scr
The Database used was InterPro as of 3 Oct. 2002, distributed together with the InterProScan vs 3.1, and the following component databases were included in the motif searches:
PFAM, PRINTS, PRODOM, PROSITE, SMART and TIGRFAM.
Isolation, Cloning and Sequencing of RNA from Phloem Sap
All cloning and molecular biology techniques, including RNA isolation, construction of cDNA libraries and mass sequencing of cDNA libraries can be performed according to standard procedures (Sambrook et al., Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
PolyA+ RNA is isolated from phloem sap and plant tissues using methods described by Ruiz-Medrano, Development 126: 4405–4419 (1999).
Mass sequencing of cDNA libraries is performed using an ABI sequencing machine according to manufacturers instructions (Applied Biosystems Inc, Foster City, Calif.).
Photoperiodic Induction of Flowering
Experiments are performed in either controlled environment chambers or in a greenhouse. Manipulation of the day-length in a greenhouse is carried out essentially as described by Takahashi et al., Plant Cell Physiol. 23:1–9 (1982).
Localization of RNA in Sectioned Tissue
Localization of RNA in paraffin-embedded sectioned tissues is performed essentially as described by Eshed et al., Cell 99:199–209 (1999) and references therein. Localization of RNA in fresh cut sections is performed essentially as described by Xoconostle-Cazares, et al., Science 283:94–98 (1999), Ruiz-Medrano et al., Development 126:4405–4419 (1999) and Koltai and Bird, Plant Physiol. 123:1203–1212 (2000).
Localization of Protein in Sectioned Tissue
Localization of protein in paraffin- or resin-embedded sectioned tissues is performed essentially as described by Busse and Evert, Int. J. Plant Sci. 160:1–13 (1999); Lucas et al., Science 270:1980–1983 (1995) and Sutherland et al., Int. J. Plant Sci. 160: 1099–1109 (1999).
Plant Transformation
Plant polynucleotide sequences are subcloned into a binary vector (e.g., pART27 (Gleave, Plant Mol. Biol. 20:1203–1207 (1992)) and introduced into a plant species (e.g., a cucurbit species) by Agrobacterium tumefaciens-mediated leaf-disc transformation (Horsch et al., Science 227:1229–1231 (1985). Standard floral dip procedures may be used, as described for Arabidopsis thaliana (Clough and Bent, Plant J. 16:735–743 (1998)).
Epifluorescence Microscopy and Confocal Laser Scanning Imaging
Analysis of the spatial distribution of the reporter protein, GFP, in plant tissues is performed using epifluorescence microscopy, according to standard procedures (see, e.g. Lough et al., Mol. Plant Microbe Interact. 13:962–974 (2000)). The Leica MZFLIII stereomicroscope equipped with a DC200 digital camera for imaging is useful for this purpose. Image analysis, display (adjustments in contrast, brightness, etc.) and preparation for plates can be carried out with Adobe Photoshop software (Adobe Systems, Inc., Mountain View, Calif.).
Alternatively, all permanent images are obtained with the Leica CLSM, using a long-working distance 16× multimedia objective or a 25× oil immersion lens. A low intensity laser (75 mW RYB Krypton/Argon laser [Omnichrome 643-75RYB]) is used to image the spatial distribution of GFP. Two-channel recordings are made using the following filter sets: GFP, 488 nm excitation, 525 nm emission (BPFITC filter). Image analysis, display (adjustments in contrast, brightness, etc.) and preparation for plates can be carried out using Adobe Photoshop software (Adobe Systems, Inc., Mountain View, Calif.).
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, method step or steps, for use in practicing the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
All of the publications, patent applications and patents cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
This application claims priority to U.S. Provisional Patent Application No. 60/498,940, filed Aug. 29, 2003, and U.S. Provisional Patent Application No. 60/509,440, filed Oct. 7, 2003, and U.S. Provisional Patent Application No. 60/587,881, filed Jul. 14, 2004.
| Number | Date | Country | |
|---|---|---|---|
| 60587881 | Jul 2004 | US | |
| 60509440 | Oct 2003 | US | |
| 60498940 | Aug 2003 | US |
