# Patent

## Title
Modulating developmental transitions in plants

## Abstract

The invention relates to a method to modulate plant growth or development. The invention provides a method for modulating a developmental transition of a plant comprising modulating expression of a RING-H2 protein or functional fragment thereof in said plant or parts thereof.

## Background
This application is the U.S. National Phase of International Application Number PCT/NL02/00026 filed on 15 Jan. 2002, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
The invention relates to a method to modulate plant growth or development.
Apart from short-term environmental factors, such as the daily rhythm of night and day and the immediate availability of water and nutrients, long-term environmental factors, such as periods with low temperature, (lack of) humidity, or little day length, have an important role in the life cycle of many plants. Where a short-term environmental factor such as ambient light has an immediate effect on day-to-day morphogenesis (e.g. seedlings grown in the light are characterised by short hypocotyls and expanded green cotyledons and seedlings grown in darkness are etiolated, with elongated hypocotyls and closed cotyledons) long-term environmental factors often determine or have effect on the transition of a plant from one phase to another phase in its development, and in particular effect plants that are in a developmental pause in their development.
For example, periods with low temperatures occurring in the temperate climate zone during winters effect many transitional aspects of plants. Typical transitional events are stem elongation (bolting) and the onset of flowering and the formation of fruit (fruiting), another is the event that is seen as the break-through event of dormant seed, germination.
Some plant species need a developmental pause comprising a prolonged period of low temperature and/or little light or day length to induce flowering, and, in a process called vernalisation, for many plants traditionally artificial methods (such as forcing) are in widespread use to provide for the premature bolting, flowering and/or fruiting, or germination of a plant, seed, seedling or bulb to obtain commercially attractive plants or plant parts (seedlings, flowers or flowering plants, fruit) in a season in which otherwise such a product would nor or not natively be available.
In some species flowering and stem elongation may be induced at the same time. Other species need a period of low temperature to induce the elongation of the stem, a process that we will further refer to as bolting. In these species the process of flower induction and formation precedes the required period of low temperature, so the two processes are temporally separated. Yet other processes influenced by long-term environmental factors (e.g. low temperatures or dryness) are quiescence, dormancy and germination of seeds. These long-term environmental factors or processes often synchronise a plant's development to the seasons and/or climatic conditions of the place of growth, and reflect evolutionary adaptations to various conditions under which a particular plant or species grows.
Tulip is an example of a species used as a model to study these various aspects of long-term environmentally induced changes in plant growth, via the study of the process of bolting [4]. Another process where low temperature is often involved is seed dormancy. Low temperature induced stem elongation can be viewed as a form of dormancy breaking, and stratification, vernalisation and dormancy breaking in tree buds are similar processes driven by low temperature. In germination of dormant seed in Arabidopsis, besides the chilling requirement to overcome dormancy, other similarities between bolting of tulips and the germination of the dormant seed are the fact that all organs are formed within a protective structure to enable rapid growth after dormancy has been removed, reserve substances are stored in specialised leaf like organs (for example in the form of starch in the cotyledons of Arabidopsis, and also in the form of starch in the bulb scales of tulip), and during dormancy there is a low water potential [5].
SUMMARY OF THE INVENTION
The invention provides a method to modulate a developmental transition of a plant, thereby for example providing a break-through in a developmental pause, said method essentially independent of a long-term environmental factor as discussed above, thereby providing a method to modulate transition of a plant from one developmental phase to another. In particular, a method for modulating developmental transitions in plants is provided wherein said transition comprises germination, bolting, the onset of flowering or fruiting. Herein we present the isolation of a low temperature induced expression product of a plant RING finger, subclass RING-H2 protein from Tulip with homology to other plant (such as Arabidopsis) proteins that interacts with developmental regulators. The implications of this are expression of said plant RING finger or RING-H2 protein, as shown herein for the low temperature induced TGRING1 expression, targets a plant's homologue of ABI3 for proteolysis, and thereby ends a developmental pause, e.g. the state of dormancy or quiescence, allowing the plant to break-through (for example transcend) from one developmental phase to another and grow towards its subsequent developmental state. Although the fact that RING or RING-H2 proteins have been recognized as involved in ubiquitination or proteolysis before, the involvement of these proteins in developmental changes in plants has not been demonstrated ear er This is especially true for the developmental changes such as those relating to germination, stratification, bolting, the onset of flowering or fruiting, low temperature induced stem elongation, dormancy breaking, bulb induction, timing of flowering. For example, Stoop-Myer et al [17] show that an N-terminal fragment of COP1 retains part of it's functionality. They indicate this fragment to have at least two functional domains, the RING and coiled-coil domains. But, importantly they don't assign the retained functionality to the RING domain, and neither do they propose a function for this domain alone. Also, McNellis et al [18] show that an N-terminal fragment of COP1 retains part of it's functionality. Again they indicate this fragment to have at least two functional domains, the RING and coiled-coil domains. Again, they don't assign the retained functionality to the RING domain. McNellis et al [19] show by overexpression of COP1 it's role in photomorphogenesis. They indicate COP1 to have at least three functional domains, the RING domain, the coiled-coil domain, and the WD-40 repeat domain. But, importantly they don't assign the functionality of COP1 to the RING domain, and neither do they propose a function for this domain alone. Kurup et al [16] show in their article that AIP2 has an interaction with the arabidopsis developmental regulator ABI3. They fail to recognize the role of AIP2 and other RING-H2 proteins in developmental transitions, on the contrary, they propose a role for AIP2 as a transcriptional activator. The article by Freemont [20] is the first published article that proposes a general role for RING and RING-H2 domains in ubiquitination and proteolytic targeting. His article focuses mainly on RING proteins found in animal systems with one exception: PRT1 [21]. The arabidopsis PRT1 protein is apparently involved in a particular kind of pathway, the ‘N-end rule’ ubiquitination pathway. However, neither in this article nor in the article by Potuschak et al, [21] is there any mention of using RING finger or RING-H2 proteins for manipulating the protein level of developmental regulators in order to obtain a developmental change in plants. Although the fact that RING or RING-H2 proteins have been recognized as involved in ubiquitination or proteolysis before, the involvement of these proteins in developmental changes in plants has not been demonstrated earlier This is especially true for the developmental changes such as those relating to germination, stratification, bolting, the onset of flowering or fruiting, low temperature induced stem elongation, dormancy breaking, bulb induction, timing of flowering. Tamminen [22] illustrates that ectopic expression of ABI3 results in changes in freezing tolerance. The present invention provides the use of RING or RING-H2 proteins for developmental transitions of plants by manipulating the protein level of ABI3 or other developmental regulators by targeting them for proteolysis by ubiquitination. The article by Torii et al [23] shows that the arabidopsis protein CIP8 interacts with the arabidopsis photomorphogenic regulator COP1. The interaction is ascribed to the RING domain of COP1 and the RING-H2 domain of CIP8. However, the article does not indicate or propose a functional role for the RING or RING-H2 domain in ubiquitination or proteolysis. It only proposes a role as a protein-protein interaction domain.
Typical plant proteins having the desired RING finger or RING-H2 motif can be found in FIG. 1A and B, and others may be found by simple alignment as shown herein. In a preferred embodiment the invention provides a method for modulating a developmental transition of a plant comprising modulating expression of a RING finger or RING-H2 protein or functional fragment thereof in said plant or parts thereof, in particular wherein said transition comprises germination, stratification, bolting, the onset of flowering or fruiting. Such a RING finger or RING-H2 protein, as herein is disclosed, provides ubiquination and/or proteolysis of a target protein (e.g. developmental regulatory protein) related to the transition from one developmental phase to another, thereby for example providing for proteasome-mediated degradation and removal of the target protein (e.g. regulatory protein) involved. A target protein as used herein is a protein capable of modulating a developmental transition of a plant. Particular regulatory proteins to be mentioned here comprise for example COP1, HY5, FLC, FRIGIDA and LD. Preferred herein is modulation of a regulatory protein that in Arabidopsis is known as abscisic acid-insensitive3 (ABI3) or a homologue thereof.
The invention provides a method wherein said RING finger or RING-H2 protein or functional fragment thereof is at least functionally equivalent or homologous to a plant RING finger or RING-H2 protein as shown in FIGS. 1A and 1B. A functional fragment of a RING finger protein contains for example the RING finger (C3HC4) motif:

    
    
        Cx(2)Cx(9,39)Cx(1,3)Hx(2,3)Cx(2)Cx(4,48)Cx(2)C (SEQ ID. No: 16)

and a functional fragment of a RING-H2 proteins contains for example the RING-H2 (C3H2C3) motif:

        Cx(2)Cx(9,39)Cx(1,3)Hx(2,3)Cx(2)Cx(4,48)Cx(2)C (SEQ. ID. NO: 17)

or a motif functionally equivalent thereto. The above used coding is according to the IUPAC-rules: C stands for cysteine, H stands for histidine and x codes for any amino acid. The numbers between the brackets show the number of repetitions, so for example, x(9,39) means a stretch of 9 to 39 amino acids residues, where each residue can be any amino acid. In TGRING1 this motif comprises amino acids 248-288 of SEQ. ID. NO: 15.

    
    


The invention also provides an isolated and/or recombinant nucleic acid encoding a plant RING finger or RING-H2 protein or functional fragment thereof comprising an amino acid sequence that is preferably at least 50%-60% homologous, more preferred 70% homologous and even more preferred 95% homologous to the sequence in FIG. 3, and retains a similar function [i.e. is “essentially equivalent” to the sequence in FIG. 3]. “Essentially equivalent” as used herein means a functional equivalent, e.g. deliberate amino acid substitution may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, and/or the amphipathetic nature of the residues as long as the biological activity of the polypeptide is retained. Homology is generally over the full-length of the relevant sequence shown herein. As is well-understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for “conservative variations”, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Deliberate amino acid substitution maybe made on the basis of similarity in polarity, charge, solubility, hydrophobicity, and/or the amphipathetic nature of the residues as long as the biological activity of the polypeptide is retained. In a preferred embodiment, all percentage homologies referred to herein refer to percentage sequence identity, e.g. percent (%) amino acid sequence identity with respect to a particular reference sequence can be the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Amino acid similarity or identity can be determined by genetic programs known in the art.
The invention provides a method wherein said RING finger or RING-H2 protein act as a ubiquitin-protein ligase (E3) or subunit of such an enzyme. Modification with chains of ubiquitin (Ub) constitutes a primary mechanism by which proteins are targeted for proteasomal degradation. Protein ubiquitination is accomplished through a complex process involving ubiquitin-activating enzymes (E1s), ubiquitin-conjugatingenzymes (E2s) and, in some cases, specificity-conferring ubiquitinligases (E3). The E1 enzyme acts first and passes Ub to an E2 enzyme ready for targeting to a protein substrate (target protein). RING finger or RING-H2 protein, which may act as nucleic acid or protein binding domains may also act as ubiquitin protein ligase (E3) or subunits of such enzymes. Ubiquitin ligases E3 are loosely defined as proteins or protein complexes are responsible for substrate recognition and promoting polyubiquitin ligation to the substrate marking it for degradation by the 26S proteosome. E3 enzymes assemble multiubiquitin chains on a variety of regulatory proteins and thus targets them for proteolysis by the 26S proteosome.
The invention provides a method according to the invention wherein said RING finger or RING-H2 protein may interact at least with a target protein and/or a ubiquitin conjugating enzyme (E2) allowing transfer of ubiquitin residues from said ubiquitin conjugating enzyme to said target protein. E3s may interact with E2s to form thioester linkages with ubiquitin before modifying target proteins. Some E3s do not act as ubiquitin carriers that form thioester intermediates, but instead act as bridges between E2s and target proteins that provide a favorable environment for the transfer of ubiquitin. The RING finger or RING-H2 protein may provide a site of interaction with E2 that allows for the direct transfer of Ub from E2 to target lysines. This reaction results in the formation of an isopeptide bond between the C terminus of ubiquitin and the epsilon amino group of a lysine residue in the protein substrate. Lysine residues within the attached ubiquitin residues can serve as acceptor sites, resulting in the assembly of a multiubiquitin chain, which can to function as a recognition signal for the 26S proteasome.
The RING finger or RING-H2 protein may have other roles in ubiquitination reactions than providing binding sites for E2s. RING finger or RING-H2 protein can instead function as allosteric activators of E2s. The invention further provides a method according to the invention wherein said RING finger or RING-H2 protein act as an allosteric activator of a ubiquitin conjugating enzyme (E2). It is understood that said RING finger or RING-H2 protein acts in pathways other than ubiquitin or in ubiquitin-like pathways, to modulate a developmental transition of a plant.
In particular, the invention provides such a nucleic acid derived from a bulb. A bulb as used herein is a modified underground stem which has one or more buds enclosed in fleshy modified leaves or scales which supply energy to the bud(s) when they start to grow, for example onions and tulips have bulbs. Also, the invention provides a vector comprising a nucleic acid according to the invention, and host cell comprising a nucleic acid or vector according to the invention. An example of such a vector is the plant transformation vector pBIN19, which is often used to deliver a piece of DNA, for example a piece of DNA encoding the TGRING1 protein or functional fragment thereof, to the genome of a plant. The procedure or method for preparing a transformant can be performed according to the conventional technique used in the fields of molecular biology, biotechnology and genetic engineering.
Preferably, such a host cell comprises a plant cell, allowing the generation of a plant or part thereof comprising a nucleic acid according to the invention. ‘Plant cell’, as used herein, amongst others comprises seeds, suspension cultures, embryos, meristematic regions, callous tissues, protoplasts, leaves, roots, shoots, bulbs, gametophytes, sporophytes, pollen and microspores. The target plant may be selected from any monocotyledonous or dicotyledonous plant species, such as for example ornamental plants, vegetables, arable crops etc. ‘Dicotyledons’ (and all scientific equivalents referring to the same group of plants) form one of the two divisions of the flowering plants or angiospermae in which the embryo has two or more free or fused cotyledons. ‘Monocotyledons’ (and all scientific equivalents referring to the same group of plants) form one of the two divisions of the flowering plants or angiospermae in which the embryo has one cotyledon. ‘Angiospermae’ or flowering plants are seed plants characterized by flowers as specialized organs of plant reproduction and by carpels covering the ovaries. Also included are gymnospermae. Gymnospermae are seed plants characterized by strobili as specialized organs for plant reproduction and by naked sporophylls bearing the male or female reproductive organs, for example woody plants.
‘Ornamental’ plants are plants that are primarily in cultivation for their habitus, special shape, (flower, foliage or otherwise) colour or other characteristics which contribute to human well being indoor as cut flowers or pot plants or outdoors in the man made landscape, for example bulbous plant species like Tulipa, Freesia, Narcissus, Hyacinthus etc. ‘Vegetables’ are plants that are purposely selected or bred for human consumption of foliage, tubers, stems, fruits, flowers of parts of them and that may need an intensive cultivation regime. ‘Arable crops’ are generally purposely bred or selected for human objectivity's (ranging from direct or indirect consumption, feed or industrial applications such as fibers) for example soybean, sunflower, corn, peanut, maize, wheat, cotton, safflower and rapeseed.
In particular, the invention provides use of a nucleic acid or a vector according to the invention in a method for modulating a plant. Preferably, said method comprises modulating a developmental transition of said plant.
Preferably, said modulation is achieved by providing said plant with a nucleic acid encoding a RING finger or RING-H2 protein or functional fragment thereof. The invention is for example considered useful to replace forcing of plants with the purpose to accelerate a transition, for example to obtain early flowering bulbs for sale, but can equally well be applied to delay transition in plants where that may be required.
In a particular embodiment, the invention provides a method, for example useful in in-vitro propagation of bulbs, such as tulip bulbs, comprising transient expression of said nucleic acid encoding a RING finger of RING-H2 protein. Other examples of transitions which can be influenced or modulated (accelerated or delayed) with the described invention comprise germination, stratification, bolting, the onset of flowering, fruiting, flowering timing, low temperature induced stem elongation, dormancy breaking or bulb induction, whereby said nucleic acid can also be transiently expressed. The invention additionally provides a plant (if desired only transiently) provided with a nucleic acid encoding a RING finger or RING-H2 protein or functional fragment thereof.
The invention also provides a method for a determining a stage in a or leading up to a developmental transition of a plant comprising determining expression of a RING finger or RING-H2 gene product (mRNA or protein or functional fragment thereof in said plant or parts thereof, and/or comprising determining ubiquination and/or proteolysis of a target protein which interacts or is affiliated with said RING finger or RING-H2 gene product (mRNA or protein or functional fragment thereof). Such a method is useful in a method for selecting a plant for being at a transition phase of its development comprising determining expression of a RING finger or RING-H2 gene product functional fragment thereof in said plant or parts thereof, for example to determine whether said plant is in or close to a distinct developmental phase such as for example germination, stratification, bolting, the onset of flowering or fruiting.
The invention further provides a method for selecting a plant for being at a transition phase of its development comprising determining expression of a RING finger or RING-H2 gene product or functional fragment thereof and/or comprising determining ubiquination and/or proteolysis of a target protein which interacts or is affiliated with said RING finger or RING-H2 gene product (mRNA or protein or functional fragment thereof. Plants with the desired characteristics are now easily selectable and herein also provided.
The invention also provides a method for obtaining a plant or progeny thereof having a desired quality trait related to developmental transitions comprising determining expression of a RING finger or RING-H2 gene product or functional fragment thereof in said plant or parts thereof, and/or comprising determining ubiquination and/or proteolysis of a target protein which interacts or is affiliated with said RING finger or RING-H2 gene product (mRNA or protein or functional fragment thereof.
It is for example now possible to more easily select or breed plants with a desired developmental transition pattern. For example to obtain tulips or other plants with a reduced cold requirement for flowering, or a plant of which the germination of the seeds is either delayed or accelerated, it is now possible to check the progeny of different crosses for expression levels of a RING finger or RING-H2 mRNA or protein or functional fragment thereof regulating said quality (cold requirement or time of seed germination).
The invention is further explained in the detailed description without limiting the invention thereto.
DETAILED DESCRIPTION
Plant Material and RNA Isolation
Tulip bulbs were harvested in the summer (July) and stored at 20° C. until at least two weeks after stage G (September). All storage treatments were performed in dark ventilated rooms. Bulbs were then either transferred to 5° C. for low temperature treatment, or to 17° C.
For the differential display analysis, after transfer to 5° C. or 17° C., samples were taken after 4, 8 and 12 weeks. The same was done in another year for the Northern blot analysis of differential expression during temperature treatment. For the Northern blot analysis of the different organs after planting, bulbs were transferred to 17° C. in September until the end of December and then transferred to 5° C. for 12 weeks. After planting and growing at 23° C. (12 hr light/12 hr dark), organ samples were taken at the indicated days after planting. All samples were frozen in liquid nitrogen and stored at −80° C. or freeze dried. RNA was isolated using CTAB as described [3]. Northern blots were performed using formaldehyde/agarose gels as described [1]. Hybridization was performed at 68° C. using 50% formamide buffer, and an α32P-CTP labeled RNA probe.
Cloning Procedure
Differential display PCR (DDRT-PCR) was performed as described [2]. The differential band was excised, and reamplified using the same conditions as during the DDRT-PCR, except that dNTP concentration was 100 μM. The PCR product was cloned. All cloning of PCR products was in the pGEM-T vector (Promega, Madison USA). After sequencing, two nested 5′ RACE (Rapid Amplification of cDNA Ends) reactions were performed using the Marathon kit (ClonTech, Palo Alto USA) with fragment specific primers. The resulting fragment was cloned and sequenced. The total fragment was now reamplified and cloned using new primers from the 5′-end (5′-GTCGTCGGCTTCCCCTCCGCCAAG-3′ SEQ. ID. NO: 1) and 3′-end (5′-TTTAACCACAATAGCTCATTGCAAGGCTTC-3′ SEQ. ID. NO: 2) and proofreading enzyme (KlenTaq, ClonTech). Two clones were sequenced on both strands and found to be identical. Requested EST clones were also sequenced on both strands. Sequencing was performed on an automated sequencer (ALFexpress, Amersham Pharmacia Biotech, Uppsala, Sweden), using kits recommended by manufacturer.
Sequence Analysis
Sequences were analyzed using DNASIS (Hitachi Software, Olivet, France) and GeneRunner (Hastings Software Inc, Hastings on Hudson, USA) software packages. Database searches were performed using the NCBI Blast server Multiple alignments were performed at EBI ClustalW server, using standard settings. Alignments were shaded using Boxshade 3.2 (ISREC, Epalinges s/Lausanne, Switzerland).
Results
Cloning and Sequence of TGRING-H2 and Similar EST Sequences
Using differential display a screening was performed for mRNA's expressed at a higher level in the bottom internode of dry stored tulip bulbs at 5° C. compared to those stored at 17° C. A 218 bp fragment was found to be differential and was cloned. Because DDRT-PCR fragments often derive from the non-coding 3′-end, a 5′-RACE reaction was performed. The resulting fragment was sequenced and new 5′- and 3′-primers were designed to reamplify the full-length cDNA using proofreading enzyme.
The resulting 1222 bp clone contains an open reading frame of 327 amino acids with a predicted molecular mass of 36.7 kD. The cDNA is either full length or close to full length because there is an in frame stop codon preceding the predicted start codon. The transcript size was also confirmed on a Northern blot (not shown). The encoded acidic protein (isoelectric point 5.02) has as most significant feature a C-terminal RING-H2 domain, a variant RING-finger domain. It is predicted to be targeted to the cytoplasm. The protein will be further referred to as TGRING1.
Database searches revealed two partially sequenced EST-sequences and one published Arabidopsis sequence, CIP8 [COP-1 Interacting Protein 8; [13]] with significant homology beyond the RING-H2 domain. The EST-clones, rice EST C10402 and arabidopsis EST TAI386, were requested and full length sequenced. Recently, the sequence of the Arabidopsis EST TAI386 sequence has been published as AIP2, an ABI3 interacting protein [8], and will be further referred to as such. The rice EST will be further referred to as OSRING1.
A multiple alignment shows that the two EST sequences are highly homologous to the tulip sequence over the whole length of the protein. OSRING1 shows 58% identity and 72% similarity over 300 amino acids, and AIP2 51% and 64% respectively over 301 amino acids. The homology of CIP8 to TGRING1 is highest in the C-terminal part, but extends beyond the RING-H2 domain (FIG. 1A). Further database research revealed that, although many sequences from every eukaryotic phylum contain the RING-H2 domain, some contain a RING-H2 domain with a conserved motif consisting of 25-40 amino acids directly N-terminal to the RING-H2 domain. These extended RING-H2 domains are found in sequences from plant, animal and viral origin (FIG. 1B).
TGRING1 Expression
The expression level of TGRING1 in bottom internodes is higher during the low temperature treatment compared to the control treatment (FIG. 2A). It is higher in the earlier part of the treatment than in later stages of the treatment. TGRING1 expression was examined in different tissues after planting of the bulbs. Expression in the internodes is examined at the moment when they just start to elongate rapidly and expression in the flower when it shows the first colouring. Roots and leaves show no rapid growth phase, so expression is examined at an arbitrary moment (FIG. 2B). The highest expression is detected in flower tissue and the lowest in the leaves.
We showed that one of the underlying mechanisms of low temperature induced stem elongation in tulips is a gradual change in the sensitivity for auxin in the bottom internodes over the course a low temperature treatment of a few months. This was shown by an increase in auxin induced internodal elongation and auxin induced gene expression of primary auxin response genes, after longer periods of low temperature treatment. By using differential display we have isolated a cDNA clone that is expressed at higher levels during the low temperature treatment, as compared to a control treatment. It codes for a protein with a C-terminal RING-H2 domain.
Although this is a feature shared by a lot of proteins, both in arabidopsis and other species, only two arabidopsis sequences showed homology over the whole length of TGRING1. One of these, AIP2, has a sufficiently high homology to be a true homologue. This is also true for rice OSRING1, which has slightly higher identity and similarity because it is of monocotyledonous origin, as TGRING1. The Arabidopsis CIP8 is a more distantly related protein. The extended RING-H2 domain shared by these four proteins is conserved beyond the plant kingdom, which indicates a special function for this domain.
The Arabidopsis proteins AIP2 and CIP8 are isolated as proteins interacting with developmental regulators. CIP8 interacts with COP1 through specific interaction of the RING-H2 domain of CIP8 and the RING-finger domain of COP1 [13]. The interaction of AIP2 with ABI3 is mediated by the C-terminal half of AIP2, containing the RING-H2 domain [8]. But surprisingly, it interacts with the B2 and B3 domains of ABI3, which do not contain a RING-finger domain. So either the C-terminal part of AIP2 that does not contain the RING-H2 domain interacts with ABI3, or the RING-H2 domains can interact with different domains with no apparent sequence homology, despite the fact that the RING-H2 domains are highly similar in sequence (43% identity and 71% similarity between the RING-H2 domains of AIP2 and CIP8).
Low temperature induced stem elongation can be viewed as a form of dormancy breaking. Stratification, vernalisation and dormancy breaking in tree buds are similar processes driven by low temperature. Interesting now is that the TGRING1 arabidopsis homologue AIP2 is an ABI3 interacting factor. The Arabidopsis abscisic acid-insensitive3 (abi3) mutant was originally isolated for its ability to germinate in the presence of inhibiting concentrations of abscisic acid (ABA; [7]) and has a role in embryo maturation and dormancy maintenance [9]. Recent publications have shown that ABI3 also has a role in vegetative quiescence processes. The abi3-4 mutant shows a rapid induction of flowering and ABI3 is expressed in the apex of dark grown seedlings [8;11;12]. Another recent publication proposes a function for the RING finger domain in targeted ubiquitin-mediated proteolysis. RING finger proteins act therein as E3 ubiquitin protein ligases [6].
Taken all these things together we show here that the low temperature induced TGRING1 expression has a role in targeting the tulip homologue of ABI3 for proteolysis, and thereby removing the state of dormancy, allowing the plant to grow under favourable conditions. Analysis of AIP2 in Arabidopsis during processes as stratification and vernalization and its influence on ABI3 protein levels are ways to further show this phenomenon, considering that the fact remains that two totally different approaches to analyze similar phenomena as seed dormancy and low temperature induced flowering in tulip result in the cloning of homologous proteins having the above described RING-H2 motif.
The expression of AIP2 during cold stratification.
Purpose of the Experiment
Determine the expression pattern of the arabidopsis homologue AIP2 of the cloned tulip TGRING1 during seed stratification by low temperature.
Experimental Setup
Dry Arabidopsis thaliana seeds of the Cape Verdian ecotype (Cvi, planting date 21 june, harvested november 1, start experiment november 11, all in 2001) were sown on wet filter paper and either put in the dark at 20° C. or at 4° C. for 1, 3, 5 or 7 days. Per treatment, a part of the seeds was after treatment transferred to 20° C. in the light (16 hrs light/8 hrs dark) to determine germination percentage; the rest of the seeds were frozen in liquid nitrogen and stored at −80° C. for RNA isolation. Germination percentage was determined 7 days after seeds were placed at 20° in the light. RNA extraction and Northernblotting was performed as described for the experiments on tulip.
Results
FIGS. 4a and b show the expression level of AIP2 after the indicated amount of days of treatment. The AIP2 probe hybridized with a single band of approximately 1200 bp. The expression level is strongly increased 1 day after incubation at 4° C., both compared to dry seeds (twelvefold higher) and seeds incubated at 20° C. (fivefold higher). The expression stays higher during the low temperature treatment, compared to the 20° C. treatment.
FIG. 3c shows the germination percentage of the seeds after the different treatments. The seeds were not very dormant, yet the low temperature treatment increased the germination percentage, compared to seeds kept at 20° C. Germination was 100% after three or more days at 4° C. The results show that the arabidopsis AIP2 mRNA is strongly upregulated during cold stratification. This shows that the AIP2 protein is not only conserved in sequence but also in function, moreover it is even functionally conserved in different developmental processes requiring low temperature induction, namely low temperature induced stem elongation in tulip and seed stratification in arabidopsis. The increased expression during cold stratification correlates with an increased germination percentage after the treatment. Because AIP2 interacts with ABI3 and has a RING-H2 domain it is likely that the dormancy breaking occurs by proteolytic targeting of the ABI3 protein via ubiquitination, thus removing the state of dormancy. AIP2 would then act as a monomeric E3 ubiquitin ligase. By analogy the tulip TGRING1 protein would have the same function in the process of low temperature induction of stem elongation, which can be seen as a form of dormancy breaking. This experiment shows that AIP2 and homologous proteins are clear markers for dormancy breaking. It also shows the involvement of a RING domain protein in dormancy breaking in another process as low temperature induced stem elongation in tulips, namely cold seed stratification.



FIGURE LEGENDS
FIG. 1A Multiple alignment of TGRING1 and homologous proteins from Arabidopsis and rice. Residues are shaded black when more than 50% of the residues is identical and gray when more than 50% of the residues is similar. Fully conserved residues are indicated by an asterisk, conserved substitutions by a colon, and semi conserved substitutions by a dot. The RING-H2 domain is underlined, and the N-terminal extension of the RING-H2 domain is underlined by a dashed line.
FIG. 1B Multiple alignment of the extended RING-H2 domains of proteins from animal, plant and viral origin. Shading is as indicated for A. Species are indicated as follows: Mm is Mus Musculus (mouse), Rr is Rattus rattus (rat), At is Arabidopsis thaliana, Os is Oryza sativa (rice), Hs is Homo sapiens (human), Tg is Tulipa gesneriana and Hh is human Herpesvirus. Database accession numbers are: PRAJA1 NP—032879, NDAP1 BAA06979, RHC2a AAC69860, os329 BAA88184, hs311 BAA91254 and ICPO P28284.
FIG. 2A Expression of TGRING-1 in the bottom internode of tulip during low temperature treatment (5° C.) compared to a control treatment (17° C.).
FIG. 2B Expression of TGRING-1 in different tissues after planting. As a reference, the expression level in the bottom internode at day 0 after planting is shown. This expression level is the same as that at week 12 of the 5° C. treatment in FIG. 2A (different exposure times are used for FIG. 2A and FIG. 2B). The other internode samples were taken at the moment that they started to grow rapidly: leaves and roots show no exponential growth phase and samples were taken at an arbitrary moment; flower tissue was sampled when colouring started. 25 micrograms of total RNA was used per lane. Equal loading was checked by ethidium bromide staining (not shown).
FIG. 3
Nucleic acid of TGRING1 (SEQ. ID. NO: 14)
The predicted 327 amino acid (SEQ. ID. NO: 15)open reading frame is coded for by bases 104-1182 and is indicated above the nucleic acid sequence. The bases coding for the RING-H2 domain are underlined.
FIG. 4A
Expression of AIP2 during treatment of imbibed seeds at either 4° C. or 20° C. An RNA probe was used complementary to bp 665-1120 of the AIP2 mRNA sequence (GenBank accesion ATH251087). The band corresponds to a size of approximately 1200 bp. 8 μg of total RNA was loaded per lane; the bottom panel shows a methylene blue staining of the blot to confirm equal loading. ds indicates dry seeds.
FIG. 4B
Quantification of the intensities of the bands in FIG. 4a. 
FIG. 4C
Percentage germination of the seeds after the indicated treatments.



INCORPORATION OF SEQUENCE LISTING
Incorporated herein by reference in its entirety is the Sequence Listing for the application. The Sequence Listing is disclosed on a computer-readable ASCII text file titled, “sequence.txt”, created on Dec. 15, 2008. The sequence.txt file is 25 kb in size.
Literature
REFERENCE LIST


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## Claims

1. An isolated and/or recombinant nucleic acid encoding a plant RING-H2 protein or functional fragment thereof comprising a motif having an amino acid sequence that is at least 95% identical to amino acid residues 248-288 as shown in SEQ. ID. NO. 15, wherein the protein or functional fragment thereof binds to and catalyzes ubiquitination and/or proteolysis of a developmental regulatory protein, wherein said developmental regulatory protein is COP1.

2. The nucleic acid according to claim 1 wherein said plant comprises a bulb.

3. A vector comprising the nucleic acid according to claim 1.

4. An isolated host cell comprising the nucleic acid according to claim 1.

5. A vector comprising the nucleic acid according to claim 2.

6. An isolated host cell comprising the nucleic acid according to claim 2.

7. An isolated host cell comprising the vector according to claim 3.

8. The nucleic acid according to claim 1, wherein said amino acid sequence has one or two amino acid substitutions at a position selected from the group consisting of position 250, 254, 256, 259, and 269 as compared to the amino acid sequence of SEQ ID NO: 15.

9. An isolated and/or recombinant nucleic acid encoding a plant RING-H2 protein or functional fragment thereof, comprising a nucleic acid sequence that is at least 95% identical to SEQ ID NO:14, wherein the protein or functional fragment thereof binds to and catalyzes ubiquitination and/or proteolysis of a developmental regulatory protein, wherein said developmental regulatory protein is COP1 and/or abscisic acid-insensitive3 (ABI3).