PL190228B1 - Gene constructs of plant origin and their application - Google Patents

Abstract

1. A method of modifying plant flowering. It is characterised in that it comprises plant transformation with a construct, containing a complete or partial DNA sequence coding ATH1 product under control of a promoter operating in plants.

Description

The invention relates to a method of modifying flowering in plants comprising transforming plants with a construct containing a complete or partial DNA sequence encoding an ATH1 product under the control of a plant-operative promoter. Plants are different from animals. The body of a mature plant is formed post-embryo by the continuous activity of the apical shoot and root meristems. The shoot apical meristem is formed during plant embryogenesis and together with the cotyledons, the hypocotyl, the embryonic root and the root meristem it outlines the basic plan of plant structure.

The root apical meristem arises as a cluster of about one hundred cells and is the source of the entire above-ground part of the plant ⁷ . During the vegetative phase of plant development, this meristem gives rise to leaves (rosette), stem and resting lateral meristems. This is followed by the formation of secondary inflorescences, stem leaves and final flower meristems upon induction of flowering. Flowering involves a complex of the interactions of gene products that regulate the change in shoot meristem identity. Factors determining the expression levels of these genes are genotype and environmental stimuli such as photoperiod, temperature and light quality. It is still largely unknown how this change is achieved under the influence of stimuli.

One of the most important events in a plant's life cycle is the decision to enter the reproductive phase. A wide range of environmental and endogenous signals controls the transition from the vegetative to the reproductive phase. Important signals are the length of the day, temperature (vernalization), availability of water and nutrients, and a range of phytohormones, in particular gibberellins (GA). These signals induce a change in the identity of the vegetative apical meristem, known as the flowering transition, and this transition is the formation of the flower meristem. While the leaf buds are the product of the vegetative apical meristem, the flower meristem produces seedlings that differentiate into secondary inflorescences during early generative growth and into flowers later at this stage. In plant breeding research, the control of these processes is a major concern for many crops. This is of particular importance for rosette plants such as lettuce, spinach and sugar beet, which experience a rapid elongation (firing) of the stem after flowering, rendering the crop unusable.

The transition from vegetative to reproductive growth is a critical developmental stage, and since it is the first stage of sexual reproduction, it is of great importance in agriculture, horticulture and plant breeding. Farmers may want to accelerate or delay flowering time, or prevent it altogether: for example to prevent "popping" in eg lettuce or sugar beet. Better understanding of the molecular biology of flowering

190,228 plants will enable it to be controlled or influenced in various ways, providing important practical benefits to agriculture.

In PCT publication WO96 / 14414, the use of the Constans (CO) gene to modify flowering mechanisms is disclosed.

The present invention proposes a method of influencing the transition of plants from vegetative to reproductive growth by providing transformed plants in which the transition is delayed or accelerated by the expression of specific transgenes influencing the process. Such genes may be constitutively expressed or only expressed in response to external stimuli, for example environmental or chemical.

ATH1 is the Arabidopsis thaliana homeobox gene. This gene is described by Quaedvlieg et al. In Plant Cell 7, 117-129, 1995 (incorporated herein by reference): its sequence is given in Fig. 1 of this publication. It was isolated from a collection of light-induced transcription factors. It is expressed in young seedlings and flowers. ATH1 mRNA levels in etiolated seedlings are very light dependent (phytochrome) and also show light adaptation.

We have now established that the ATH1 protein product is involved in the developmental change from vegetative to generative growth. As a result of the ATH1 :: GUS research and preliminary 35S :: ATH1 research, we concluded that ATH1 was involved in the transformation of the vegetative apical meristem into a flower meristem. Specifically, ATH1 acts as an antigiberelin, by repressing GA synthesis or a possible GA response pathway: illustrated in Example 6.

Our research on the ATH1 :: GUS constructs showed that in young, light-growing seedlings, ATH1 is expressed in all three layers of the apical shoot meristem and leaf buds. In young, still developing leaves, ATH1 is expressed in the vascular tissue. This expression disappears in developed leaves. Expression of ATH1 in the meristem is largely restricted to the vegetative phase of development. As soon as Arabidopsis begins to bloom (change from vegetative to generative development) and the shoot apical meristem becomes the flower meristem, ATH1 expression in the meristem decreases. During the flowering phase, ATH1 is produced at a low level in the developing vascular tissue of the stem. Later in plant development, when flowers appear, ATH1 is expressed in various parts of the young plant (flower bed, calyx sepals, or stamen vascular tissue). Our hypothesis that ATH1 is involved in the control of phase transition from vegetative to generative growth is further supported by the flowering time phenotypes overexpressing ATH1 in sense and antisense forms. Ectopic plants expressing the antisense ATH1 show an early flowering phenotype; in contrast, most plants transferring constructs to overexpress sense ATH1 are late flowering. A small proportion of plants carrying the constructs for overexpression is, due to ATH1 reduction by co-suppression, early flowering, as in the case of overexpression of antisense ATH1, and the phenotype of these plants resembles the terminal flower mutant (Shannon and MeeksWagner, 1991) and phenotypes when overexpressing LEAFY (Weigel Nilsson, 1995), APETALA 1 (Mandel and Yanowski, 1995) and CONSTANS (Putteril et al., 1995). Based on these results, in conjunction with data for ATH1 :: GUS, we concluded that ATH1 is involved in controlling the phase of transition from vegetative to generative growth in Arabidopsis thaliana and is likely to be a flowering time regulating gene.

Consequently, the transition can be promoted by inhibiting expression of the ATH1 gene, or delayed or prevented by promoting such expression.

The invention relates to a method of modifying the flowering of plants which comprises transforming the plants with a construct comprising a complete or partial DNA sequence encoding an ATH1 product under the control of a plant-operative promoter.

Preferably, the flowering process in plants is promoted by transforming the plants with a construct that inhibits ATH1 protein production, more preferably the construct is adapted to express antisense RNA into RNA produced by the ATH1 gene.

In a further preferred embodiment of the invention, the flowering process in plants is delayed by transforming the plants with a construct that promotes the production of recombinant ATH1 protein.

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The use of sequences to inhibit or promote gene expression is well understood. The complete gene sequence, under the control of a promoter that works efficiently in the plant, will generally overexpress the gene product, leading to an enhancement of the effect of the protein thus produced. Sometimes the level of a gene product is lowered: this is called "co-suppression". Downgrading of the gene product is also generally achieved by using dominant negative mutations or by reversing the orientation of the gene sequence relative to the promoter such that an "antisense" messenger RNA is produced.

The DNA construct used in the method of the invention may be an "antisense" construct producing "antisense" RNA or a "sense" construct (encoding at least a portion of a functional protein) producing "sense" RNA. An "antisense" RNA is an RNA sequence that is complementary to a base sequence in its corresponding mRNA: complementary in the sense that each base (or most bases) in an antisense sequence (read in the 3 'to 5 direction) has the ability to pair with corresponding bases (G of C, A of U) in the mRNA sequence read in the 3 'to 5' direction. Such antisense RNA can be produced in a cell by transforming with an appropriate gene construct organized to produce a transcript, at least a portion of the sequence of which is complementary to at least a portion of the coding strand of the corresponding gene (or a DNA sequence substantially homology thereto). A "sense RNA" is an RNA sequence that is substantially homologous to at least a portion of the corresponding DNA construct in the correct orientation so as to produce a transcript with a sequence identical to at least a portion of the coding strand of the corresponding gene (or a DNA sequence substantially homonymous thereto). Appropriate sense constructs can be used to inhibit gene expression (as described in International Patent Application WO01 / 08299) or a sense construct encoding and expressing a functional protein can be transformed into a plant to overproduce the protein.

The DNA constructs used in the method of the invention may contain a base sequence of at least 10 bases (more preferably at least 35 bases) for transcription into RNA.

Theoretically, there is no upper limit to a base sequence - it can be as long as the corresponding mRNA produced by the cell, but for convenience it will generally be sufficient to use sequences between 100 and 1000 bases in length. The preparation of such constructs is described in more detail below.

As the source of the DNA base sequence for transcription, a suitable cDNA or genomic DNA or synthetic polynucleotide may be used. The isolation of appropriate ATH1 sequences from Arabidopsis is described in Quaedvlieg et al., Supra; similar methods can be used to isolate ATH1 sequences from other plants. They may have a greater or lesser degree of homology with the Arabidopsis ATH1 sequences. In this way, sequences encoding a complete or substantially complete protein can be obtained. DNA sequences of appropriate length can be cut for use via restriction enzymes. When genomic DNA is used as the source of a partial base sequence for transcription, it is possible to use both intron and exon regions or a combination of both. In order to obtain a construct suitable for modifying ATH1 expression in plant cells, a cDNA sequence as found in a cDNA for a protein or a gene sequence as found in a plant chromosome may be used. Recombinant DNA constructs can be made using standard techniques. For example, a DNA sequence for transcription can be generated by treating a vector containing such sequence with a restriction enzyme to excise the appropriate fragment. The DNA sequence for transcription can also be generated by the attachment and ligation of synthetic oligonucleotides, or by polymerase chain reaction (PCR) to generate appropriate restriction sites at each end. The DNA sequence is then cloned into the vector downstream of the promoter and upstream of the terminator. If antisense DNA is required, cloning is performed so that the cut sequence is reversed from its orientation in the strand from which it was cut.

In an RNA-expressing construct, the antisense strand that was previously the template strand becomes the coding strand, and vice versa. The construct will therefore encode an RNA by sequence

190,228 bases, which is complementary to some or all of the mRNA sequence for a protein. Thus, the two RNA strands are complementary not only in their base sequence but also in orientation (5 'to 3').

In a construct expressing the sense RNA, the template, and the coding strands retain the destiny and orientation of the original plant gene. Sense RNA expressing constructs encode an RNA with a base sequence that is homologous to some or all of the mRNA sequence. In constructs that express a functional protein, the entire coding region of a gene is linked to transcriptional control sequences capable of being expressed in plants.

For example, the constructs used in the method of the invention may be prepared as follows. An appropriate vector containing the desired base sequence for transcription (such as the pATH1 cDNA clone) is treated with a restriction enzyme to excise this sequence. The resulting DNA strand is cloned (if necessary in the reverse orientation) into a second vector containing the desired promoter sequence and the desired terminator sequence. Suitable promoters include the 35S promoter of the cauliflower mosaic virus and the promoter of the tomato polygalacturonase gene (Bird et al., 1988, Plant Molecular Biology. 11: 651-662) and other growth-regulated plant promoters. Suitable terminator sequences include one from the Agrobacterium tumefaciens nopaline synthase gene (3 'nose).

In the DNA construct used in the method of the invention, the transcription initiation region may be derived from any plant-functional promoter. The transcription initiation region may be positioned so as to obtain transcription of a DNA sequence encoding an mRNA that is complementary to a sufficiently large sequence of bases in the mRNA encoding the ATH1 protein (making the DNA construct a full or partial antisense construct).

The transcription initiation region (or promoter) functioning in plants can be a constitutive promoter (such as the 35S cauliflower mosaic virus promoter) or a promoter inducible or regulated in development as required. For example, it may be desirable to modify the activity of the protein at certain stages of the plant's development. The use of a constitutive promoter will influence protein levels and functions in all parts of the plant, while the use of a tissue-specific promoter will allow more selective control of gene expression and related functions. Thus, antisense or sense RNA is only produced in an organ where its action is required.

The DNA constructs used in the method of the invention can be inserted into plants to regulate the expression of the ATH1 gene leading to modification of plant characteristics (in particular flowering). Depending on the nature of the construct, the production of the ATH1 gene product may be increased or decreased either over time or at a particular stage in the life of the plant. In general, as would be expected, protein production is only increased by constructs that express RNA homologous to substantially complete endogenous mRNA for the protein. Full-length sense constructs may also inhibit protein expression. Constructs containing an incomplete DNA sequence that is shorter than the sequence corresponding to a complete gene generally inhibit gene expression and protein production, whether or not they are structured for sense or antisense RNA expression.

The DNA construct used in the method of the invention is transformed into a target plant cell. The target plant cell can be part of a whole plant, or it can be an isolated cell or part of a tissue that can be regenerated into a whole plant. The target plant cell can be selected from any monocotyledonous or dicotyledonous plant species. Plants can be derived from a transformed plant cell by regenerating transformants and by producing successive generations of transformant progeny.

The constructs used in the method of the invention can be used to transform any plant using an appropriate transformation technique to produce plants of the invention. Both monocot and dicot cells can be transformed by a number of methods known in the art. In many cases, such plant cells (especially when they are dicotyledonous plant cells) can be grown to regenerate a whole plant, which then reproduces into successive generations of genetically modified plants. You can apply any

190 228 a suitable method of plant transformation. For example, dicotyledons such as tomato and melon can be transformed using Ti Agrobacterium plasmid technology, such as described by Bevan (1984, Nucleic Acid Research, 12: 8711-8721) or Fillatti et al. (Biotechnology, June 1987, 5: 726-730). The plants transformed in this way can be propagated sexually either by cell or tissue culture. Monocotyledonous plants can be transformed using a gene gun. Other ways to transform plants include microinjection and electroporation.

Examples of genetically modified plants according to the invention include cereals, for example rice and corn, wheat, barley, oats and rye. Other important seed products are oilseed rape (canola), sugar beet, sunflower seeds, soybeans and sorghum. Most crops are grown annually from seed, and the production of any kind of seed depends on the plant's ability to flower, pollinate, and produce seeds. Controlling the flowering time is important in horticulture. Horticultural plants whose flowering can be controlled include lettuce, chicory, Brassica vegetables including cabbage, broccoli, cauliflower; and cloves and geraniums.

The main property of the modified plants according to the invention is early or late flowering. Genotypes in which ATH1 protein production is inhibited generally bloom early; genotypes in which it is stimulated bloom late. Other effects on the phenotype can also be observed, e.g. dwarfism in e.g. tobacco.

Controlling flowering time can be useful for several reasons. For example, flowering can be controlled to deliver flowers or fruit at a time most suitable for sale. In producing hybrids, the flowering of the male and female parents can be coordinated. This is most conveniently done by using inducible gene promoters responsive to external stimuli, for example by the use of chemical compounds. An example of such a promoter is the isoform II gene promoter

Glutathione S-transferase, activated by the use of a known herbicide safener (WO 93/01294 for ICI).

Launch control can be economically important for many crop species. For example, in the case of sugar beet, it will be of great use to produce varieties that have a reduced tendency to fire when exposed to cold. Processing plants will be able to extend their lifetime over time, with significant savings in overall costs. Fire-resistant varieties could be sown very early in the season (February) or even the previous year in autumn (assuming winter frosts are not a problem). In addition, varieties in which firing is increased can be bred faster: crossbreeding can be done annually instead of biennially as before.

An early-flowering sunflower could extend its geographic range. Could be bred further north (north of Paris) and possible in drier regions such as parts of Spain, avoiding drought periods in late summer.

In the case of vegetables, firing can be controlled for, for example, lettuce and chicory. This would make it easier to grow the crop over the summer. Existing varieties tend to launch rapidly in summer conditions. For grasses, reducing (or not) firing is beneficial for forage types (improves feed quality) and recreational types (better lawn quality).

Sometimes it is beneficial to control expression of the transgene over time so as to stop it after flowering begins or to inhibit naturally occurring genes until flowering begins. This can be done by using the ATH1 promoter to control the expression of the transgene or the transcription of DNA homologous to a natural gene. Thus, a further separate part of the invention is to provide a DNA construct containing the ATH1 promoter linked to heterologous DNA in such a way as to cause its transcription in plant cells; and plant cells transformed with such dNa constructs.

ATH1 is expressed in the vegetative apical meristem and a decrease in this expression correlates with the transition to flowering. Forced constitutive expression of ATH1 results in a dramatic repression of the flowering transition in both Arabidopsis and tobacco: thus, in Arabidopsis, firing is delayed.

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Conversely, repression of ATH1 results in an early flowering phenotype. Our results suggest that ATH1 extends its function to modulate GA biosynthesis or response. We expect the ATH1 gene to form the basis of a particularly useful fire-control system.

Day length and transition to flowering

The transition to flowering has been particularly well studied in Arabidopsis thaliana. This species became a model system for studying the transition to flowering: at the genetic level by isolating flowering mutants, at the molecular level by cloning genes whose products are involved in controlling the transition to flowering. Arabidopsis is a typical rosette plant in which the vegetative leaves are tightly packed due to the reduced lengthening of the internodes. After flowering, the newly formed internodes rapidly lengthen ("shooting"). In most Arabidopsis ecotypes, day length is the most important factor in determining the flowering transition. Arabidopsis is an optional Long Day (LD) plant, which means that the transition to flowering is accelerated over long days (16 hour light / 8 hour dark cycle), but this is not an absolute requirement. Under long day (LD) conditions, the transition to flowering begins rapidly with only a few rosette leaves (~ 7 leaves on day 16-20 for the Col-0 ecotype). During short day (SD) breeding, e.g. 8 h. lights / l 6 hours darkness, it takes much longer to flower (~ 60 days) and full leaf rosettes are formed with more than ~ 30 leaves per rosette (Col-0 ecotype).

Gibberellic acid (GA) and the transition to flowering

It has long been known that treatment with gibberellins promotes the transition to flowering in many plant species. Most of the species in which GA treatment can induce flowering are long day or cold-requiring plants, and many will normally grow rosettes under non-induction conditions. Furthermore, several experiments suggest that endogenous GA levels are involved in controlling the transition to flowering: conditions that induce transition to flowering may have an effect by elevating endogenous GA levels possibly at or near the apical meristem.

Arabidopsis mutants deficient in GA biosynthesis (GA series) or insensitive to this hormone (GAI series) show a late-flowering phenotype under non-induction conditions, and the severely disturbed GAI-3 mutant is also late-flowering under induction conditions (Wilson et al., 1992) ).

Contribution of GA in controlling the transition to flowering through ATH1

We tested whether exogenous GA could overcome the inhibitory effect of constitutive ATH1 expression in tobacco. Apparently, GA spraying had the ability to "rescue" the early flowering phenotype in tobacco plants constitutively expressing ATH1. The contribution of GA was also demonstrated by the phenotype of reduced internode elongation of tobacco plants constitutively expressing ATH1. These findings suggest that ATH1 acts as a repressor of Ga biosynthesis or, alternatively, of a GA response. The dominant effect of ATH1 overexpression on the flowering transition, in combination with the reversal of this effect by endogenously added GA, suggests many applications in a variety of crops. This is of particular interest because deregulation of ATH1 expression does not lead to pleiotropic phenotypes and the reversal of the phenotype is complete. Complete recovery means that there will be no problem with breeding or duplicating ATH1 transformants and therefore maintaining transgenic lines, which can be a serious problem for flowering mutants, is simple. By using the GA transformation, plants can be reverted to wild-type development at any time, plants bloom normally and seed is formed normally.

It is also possible to inhibit ATH1 overexpression in genetically modified plants obtained by the process of the invention by treating the plants with gibberellin, for example gibberellin A3 or A4 / A7.

Harvest index increase

When plants are grown close together, there is shadow avoidance syndrome, in which plants respond to far-red radiation reflected from their neighbors. The most visible

This results in a rapid and dramatic increase in leaf stem and petiole growth rate with leaf growth, production of storage organs and reproductive development. It is known that by overexpressing phyA genes in tobacco, the shadow avoidance response can be overcome, leading to an increased harvest index (Robson et al., 1996). The yield index is expressed as leaf biomass in proportion to total biomass. Overexpression of ATH1 in tobacco causes a reduction in stem growth, while growth and leaf number remain unchanged or even reduced compared to the wild type. The ATH1 overexpression and phyA overexpression phenotypes are similar, suggesting the possibility of using ATH1 overexpression to increase the crop index for crops.

The invention will be further described with reference to the following examples and experiments, which illustrate certain aspects of the present invention; and with reference to drawings, where:

Figure 1 shows the DNA sequence for the ATH1 cDNA;

Figure 2 is a diagram of the pWP90 plasmid;

Figure 3 is a diagram of the plasmid pMOG23;

Figure 4 is a diagram of the plasmid pVDH275;

Figure 5 is a bar graph showing dwarfism caused by constitutive ATH1 expression in tobacco plants at 90 days of age;

Figure 6 is a graph showing the effect of gibberellins on the height of ATH1 overexpressing tobacco plants;

Figure 7 is a bar graph showing flowering time (as number of rosette leaves formed) for plants with decreased and increased ATH1 expression compared to wild-type Arabidopsis (ecotype C24).

General methods

Plant material and growth conditions

The wild-type Arabidopsis thaliana genotypes used were Columbia and C24. GenATH1 is located on chromosome 4 between markers mi431 (96.9 cM) and 06455 (97.9 cM). Arabidopsis thaliana Columbia was used in plant transformation experiments using a vacuum infiltration protocol, while Arabidopsis thaliana C24 was used in plant transformation experiments using a root transformation protocol.

Plants were grown in a growth room under fluorescent light with a 16 hour light period followed by an 8 hour dark period at a constant temperature of 22 ° C.

To measure flowering time, seeds were soaked and placed at 4 ° C for 4 days to break the dormant period and sown into soil. The germinating seedlings were usually covered for the first 1-2 weeks to prevent drying out.

Transformation of Arabidopsis plants

Binary constructs containing the ATH1-GUS chimeric genes and the 35S-antisense ATH1 genes were transformed into Arabidopsis thaliana ecotype C24 using the Agrobacterium tumefaciens mediated root transformation method of Valvekens et al. (1988). Transformants were selected on medium containing 50 mg / L kanamycin.

The binary constructs containing the chimeric 35S-ATH1 genes were transformed into the Columbia ecotype Arabidopsis thaliana using a vacuum infiltration protocol (Bent et al. (1994); Betchold et al. (1993)) with some modifications. Plants were grown separately in 5.5 cm dishes. Plants were transformed after the appearance of the first pods on the secondary flower shoots.

900 ml of Agrobacterium tumefaciens cultures containing the appropriate construct were grown overnight before the day of infiltration, cells were harvested by centrifugation and resuspended in an equal volume of infiltration medium containing 2% instead of 5% sucrose. Plants were infiltrated by immersing whole rosettes and flower shoots at 100 mm Hg for 10 minutes.

Transformant seeds were selected on medium containing 50 mg / L kanamycin.

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Example 1 ATH1 Expression Analysis Total RNA Isolation

Total RNA from plants was isolated according to De Vries et al. (1988) with some minor modifications: (1) plant tissue was ground in liquid nitrogen in the presence of half the volume of phenol / extraction buffer mixture and heated at 65 ° C in a water bath and (2) ) RNA was ethanol / sodium acetate precipitated before and after LiCl precipitation.

RNAase protection analysis

The HindIII-XhoI fragment from phagemid ATH1 was cloned into pBlu-scriptSK (-) (Stratagene) and digested with HindIII to generate the template for T7 RNA polymerase. The ATH1 RNA probe protected a 140 nucleotide fragment. The RNA probe was synthesized using RNAT7 polymerase (Pharmacia) and the buffer described by the manufacturer, with the difference that 160 pCi [32p] mu (gQQ cj / mujjnoi) was used. RNAse protection was performed using 10 pg of total RNA and 10 pg of tRNA according to the protocol described by Sambrook et al. (1989). The digested mixture contained 600 U / ml RNase T1 (Gibco BRL) and 20 pg / ml RNase A (Boehringer). RNA: RNA hybrids were analyzed by sequencing gels electrophoresis (6% polyacrylamide / 7M urea) and visualized by autoradiography.

Generation of ATH1-GUS chimeric constructs

A Spel-NcoI fragment containing approximately 1300 nucleotides from the ATH1 promoter sequence was isolated. After the NcoI site ends with Klenow polymerase, this fragment was inserted into single Smal / Xbal sites in the binary vector pBilO1.1, which contained the GUS gene (Jefferson et al., 1987), creating a translational fusion between the ATH1 promoter and the GUS gene. The protein encoded by this chimeric gene consists of 42 amino acids of ATH1 linked to the GUS protein. The binary construct was named tH1.4. tH1.4 was transformed into Agrobacterium tumefaciens LBA4404 competent cells (Gelvin and Schilperoort, 1988). Arabidopsis lines (C24 ecotype) were transformed as described below.

In situ localization of GUS activity in Arabidopsis thaliana lines transgenic for ATH1 -GUS

Seedlings and plant tissues were harvested and stained for 1 to 16 hours at 37 ° C in a solution containing 0.5 mg / ml X-Gluc (Biosynth AG) dissolved in N-dimethylformamide, 0.1% Triton Χ-100, 0.5 mM K4Fe (CN) 6-H ₂ O, 0.5 mM K ₃ Fe (CN) ₆ and 50 mM phosphate buffer, pH 7.2.

After X-Gluc staining, plant tissues were fixed overnight in a solution containing 1% glutaraldehyde and 4% formaldehyde in 50 mM sodium phosphate, pH 7.2. Then the seedlings were dehydrated in the following stages: in 10%, 30%, 50%, 70%, 90% and 2 x 100% ethanol. Large plant tissues were pre-embedded in 1% agarose (Sigma). Infiltration and embedding in Technovite 7100 (Kulzer, Hereaus) were performed according to the manufacturer's instructions. 4 μ sections were prepared using a Reichert-Jung 1140 rotary device with a disposable Adams steel knife. Sections were stained with 0.1% ruthenium red (Sigma) in distilled water for 2 minutes at room temperature and photographed in a Zeiss Axioskop using Kodak Professional Ektar 25 film.

The seedlings were fixed and dehydrated as above. Technovit 7100 was infiltrated for 1 day. The seedlings were then transferred to a construction made of celluloid film (Amvois), double-sided adhesive tape, foil, double-sided tape. In the last three layers, a central region was cut out to accommodate seedlings. The seedlings were then placed in Technovit 7100 solution and the central region was covered with another film. After curing overnight at room temperature, a plastic plate containing seedlings was obtained. In order to obtain sections of plate-embedded seedlings, the celluloid film material was removed and the plate was cut along the longitudinal section of the respective seedling regions. Sectioning, staining, and photographing were performed as described above.

Localization of ATH1 expression

ATH1 gene expression was analyzed by RNAse protection analysis (Quaevlieg et al., 1995). High levels of ΑΤΉ1 mRNA were detected during early seedling development (days 2-6) and in the flowers of mature Arabidopsis plants. Location of the chambers 10

The expression of the ATH1 gene was determined by introducing the ATH1-GUS tH1.4 chimeric construct into Arabidopsis thaliana. Various tissues were stained with X-gluc, and whole plant slices and tissue sections were made to visualize GUS activity (see below).

ATH1 expression during vegetative development

The growth tip of a 5-day-old light-grown seedling is flat and consists of a double-layered tunic surrounding the underlying body. At this stage, the meristem has the first leaf buds initiated (Mischke and Brown, 1965).

In plants transformed with tH1.4, high levels of GUS activity were present in the shoot tips. Shoot apex sections showed that high GUS activity is evident in all three layers of the shoot apical meristem and extends through the sub-apical region, extending further where the vascular bundle of the hypocotyl branches into the cotyledons. High GUS activities were also present in the initial pair of leaves.

ATH1 expression during the transition to flowering and inflorescence development.

Initially, during the flowering phase, the apical shoot meristem develops into a stem, stem leaves and secondary inflorescences. As the inflorescence develops, the flower meristem produces flower buds. In plants transformed with tH1.4, GUS activity decreased in the flower meristem during the transition phase. There was no detectable GUS activity in the meristem. Low levels of GUS activity were present in the rib zone. Later, when the flowers appeared, GUS activity was present in various parts of the young flowers (flower bottom, calyx sepal or stamen vascular tissue).

Example 2

Fusion construction of the ATH1 open reading frame promoters The ATH1 cDNA was cloned into the unique EcoRI / XhoI restriction sites into the well known and commercially available pBluescript SK (-) vector (Stratagene).

2.1. Fusion of the 35S promoter with CaMV to the ATH1 open reading frame

A BamHI / SnaBI fragment containing 1573 nucleotides of the ACTH1 cDNA sequence (the BamHI site was created by PCR mutagenesis, 35 nucleotides after the translation start site) was isolated and inserted into single BamHI / SmaI cloning sites in the pWP90 vector, which contains the 35S CaMV double promoter and the NOS terminator (see Fig. 2), resulting in a transcriptional fusion between the dual 35S CaMV promoter and cDNAATH1. This construct, named cH1.24, was then cut with the restriction enzymes SstI / EcoRV, and the resulting SstI / EcoRV insert was then inserted into single BamHI / SmaI restriction sites in the binary vector pMOG23 (see Figure 3). The binary construct was named tH1.2. tH1.2 was transformed into Agrobacterium tumefaciens pGV2260 competent cells (Caplan et al., 1985). Arabidopsis lines (Col-0 ecotype) were transformed by vacuum infiltration as described below.

2.2 Constructing a fusion of the 35S promoter from CaMV with the antisense frame ATH1

An EcoRI / SnaBI fragment containing approximately 1830 nucleotides of the ACTH1 cDNA sequence was isolated and inserted into single Smal / EcoRI cloning sites in the pWP90 vector (see Figure 2), resulting in a transcriptional fusion between the 35S CaMY double promoter and the ATH1 antisense frame. The resulting construct was named cH1.22. The EcoRV / SstI insert from cH1.22 was then cloned into single Smal / SacI restriction sites in the binary vector pMOG23 (MOGEN) (see Fig. 3). This binary construct, named tH 1.1, was transformed into competent Agrobacterium tumefaciens LBA4404 cells. Arabidopsis lines (C24 ecotype) were transformed as described below.

2.3. Fusion of the heat shock promoter to the ATH1 open reading frame

By PCR mutagenesis, an additional BamHI site was created in pTT19, a vector containing the promoter, leader and 77 nucleotides of the coding sequence of the Hspl8.2 heat shock gene from Arabidopsis thaliana (Takahashi and Komeda, 1989). An additional BamHI site is located in the untranslated leader at nucleotide -710 from the start of translation of Hsp18.2.

By restriction with BamHI, the 5 'untranslated leader and 77 nucleotides of the Hsp18.2 coding sequence were removed. The remaining construct was named leaderless pTT19.

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The HindIII / BamHI fragment of this leaderless pTT19, containing only the Hsp18.2 promoter sequence, was fused to the BamHI / EcoRI fragment containing the entire ATH1 cDNA sequence, resulting in a transcriptional fusion of the Hsp.18.2 promoter with the untranslated leader and the coding sequence. The BamHI and EcoRI sites were created by PCR mutagenesis, resulting in a BamHI restriction site at the beginning of the ATH1 cDNA sequence and an EcoRI restriction site immediately after the TAA stop codon. The resulting HindIII / EcoRI fragment was inserted into the single HindIII / EcoRI restriction sites of the pWP90 vector (see Figure 2) and this new construct was then partially digested with HindIII and EcoRV restriction enzymes.

The largest HindIII / EcoRV fragment was then inserted into the binary vector pBIN 19 cut with HindIII / SmaI (Frisch et al., 1995). This construct was named HspH1.

A transcriptional fusion was also performed between the Hsp18.2 promoter and the ATH1 coding sequence without a leader sequence. An additional BamHI site in cDNAATH1 was created by PCR mutagenesis immediately prior to the start of translation. Digestion of this BamHI site in conjunction with digestion of a single Xhol site in cDNAATHI resulted in a fragment of approximately 680 nucleotides in length containing the ATHI coding sequence. This 680 nucleotide fragment was replaced with an approximately 980 nucleotide fragment that is generated after digestion of HspHl with BamHI / XhoI restriction enzymes. The result is HspHIB, a transcriptional fusion between the leaderless ATH1 coding sequence and the HspI8.2 promoter.

Both HspH1 and HspH1B were transformed into Agrobacterium tumefaciens LBA4404 competent cells. Arabidopsis lines (C24 ecotype) were transformed as described below.

2.4 Fusion of the pea plastocyanin promoter to the ATH1 open reading frame

A transcriptional fusion between the pea plastocyanin promoter and the ATH1 coding sequence can be made by inserting the ATHI coding sequence into the single BamHI and SaII restriction sites of pVDH275 (Pwee and Gray, 1993; Last and Gray, 1989) (see also Figure 4). In the ATH1 coding sequence, an additional SalI site (immediately before the start of ATG in ATH1) and BamHI (immediately after the TAA stop signal in ATH1) can be created by PCR metagenesis. The resulting construct in which the ATH1 coding sequence is inserted between the pea plastocyanin promoter and the nos terminator can be transformed into Agrobacterium tumefaciens cells, followed by plant transformation.

Introduction of additional ATH1 copies into Arabidopsis

Additional copies of ATH1 can be introduced into Arabidopsis plants by transforming their additional ATH1 loci containing the ATH1 promoter and the ATH1 coding sequence. This can be done by fusing the SnaBI / NcoI fragment of the cDNAATH1 of about 1000 nucleotides with the restriction fragment of the SstI / EcoRI cDNAATHI of about 250 nucleotides to pBI101.1 containing the Agrobacterium nose terminator (Jefferson et al., 1987). The resulting fragment can be fused to an approximately 3.5 kb NcoI restriction fragment from the genomic ATH1 clone (Quaedvlieg et al., 1995). The NcoI / EcoRI fragment thus generated, containing the ATHI promoter, ATH1 coding sequence and the nos terminator, can be inserted into the NcoI / EcoRI cut pMTL23 cloning vector (Chambers et al., 1988). The StuI / EcoRI restriction fragment of the resulting construct can then be inserted into the binary vector pMOG23, transformed into Agrobacterium cells, and then transformed into the plants.

Example 3

Influence on flowering traits by using the 35S CaMV / ATH1 promoter gene fusion. Flowering time measurement

Flowering time was measured by counting the number of leaves, including cotyledons, in the rosette at the time the flower bud emerged. A close correlation between leaf number and flowering time has previously been shown (Koorneef et al., 1991; Bagnall (1993)).

ATH1 overexpression leads to flowering delay

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To further understand the role of ATH1 in plant development, the full-length ATH1 cDNA sequence was ligated to the constitutive tobacco mosaic virus 35S promoter and the thus generated 35S: ATH1 chimeric gene was transformed into Arabidopsis Col-0 ecotype using a vacuum infiltration method. Six independent primary transformants were thus obtained.

All these transgenic lines were self-crossed. From each independent transgenic line, 40 distinct seeds were germinated in the soil and checked for altered phenotypes compared to wild-type plants. Four of the six lines showed an altered flowering time phenotype. In three of these lines all plants were delayed in flowering (approximately 14 rosette leaves to flowering compared to 10 rosette leaves in wild-type Col-0 plants). In the remaining lines, about 85% of the plants showed the same delayed-flowering phenotype, while 15% of the plants showed an early-flowering phenotype (after about 7 rosette leaves) as shown due to the lack of ATH1 RNA. These early flowering plants also showed a terminal flower phenotype, often with incomplete flowers and mutated flower organs.

Example 4

Early flowering by antisense ATH1 expression.

Like ectopic ATH1 overexpression, inhibition of ATH1 gene function can also be used to influence flowering time. The inhibition of gene function is mediated by constitutive over-expression of antisense ATH1.

The full-length antisense ATH1 cDNA was ligated to the constitutive 35S promoter of the cauliflower mosaic virus and the chimeric 35S.'antisense ATH1 gene thus generated was transformed into Arabidopsis ecotype C24 using the Valvekens root transformation protocol. Twenty-two independent transforms were obtained and all were self-crossed. From each line, 10 separate seeds were germinated in the soil and checked for altered phenotypes compared to wild-type C24 plants. In five of these lines, the plants showed an early-flowering phenotype: flowering commenced after the formation of six to ten rosette leaves compared to approximately twenty leaves in wild-type plants.

Example 5

Alteration of flowering time in Nicotiana tabacum by overexpression of ATH1

As in Arabidopsis, ectopic overexpression of ATH1 cDNA (driven by the 35S promoter of cauliflower mosaic virus) in tobacco (Nicotiana tabacum cv. Samsun) also leads to a delay in flowering time compared to wild type tobacco plants. In 35S..ATH1 tobacco plants, flowering time was delayed by weeks or months. These plants were also stunted. This dwarf trait, like the flowering phenotype, is clearly correlated with the level of transgene expression. In the most severe cases, the plants did not bloom at all and only reached a fifth of their normal height, while in less severe cases the plants delayed flowering only a week or two and reached about four-fifths of their normal height. Leaf number and shape were normal in all transformed plants.

Examples 6-8

The following examples illustrate the effect of GA on transgenic plants according to the invention. As noted above, ATH1 overexpression effectively inhibits shooting (flower induction). We hypothesized that ATH1 might be a repressor of GA synthesis or the GA response pathway (we believe the former). The following examples illustrate and support this hypothesis.

General methods

Tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were transformed using the leaf disc procedure (Horsch et al., 1985). Transgenic plants were selected on MS medium (Murashige and Skoog, 1962) containing 300 mg / ml kanamycin and 2% sucrose. After transfer to soil, the plants were grown in a greenhouse at 22 ° C under light conditions of 16 hours of daylight, when supplemented with artificial light. The effect of gibberellins (GA) was tested by applying 100 mM GA3 to the leaves (spraying)

190 228 in a solution containing 100 ml / l of Triton Χ-100. Control plants were sprayed with a solution containing only Triton Χ-100. Spraying started 60 days after sowing, when the wild-type plants were about 2.5 cm tall, and continued at intervals of 3 to 4 days. Plant height was measured every 3 to 4 days and continued until flowering occurred as determined by the appearance of flower buds.

Example 6

Constitutive overexpression of sense ATH1 leads to delayed flowering

6.1 ATH1 overexpression in tobacco

In order to constitutively overexpress the ATH1 gene in transgenic plants, its coding region was placed under the control of the 35S CaMV promoter and the resulting construct was transformed into tobacco (Nicotiana tabacum cv. Samsun NN). Forty independent kanamycin-resistant plants were obtained, of which only five showed detectable expression of the transgene. ATH1 mRNA levels varied from high in HME # 4, # 10, # 30 plants to medium / low in H10E # 35 and # 37 plants. Depending on the expression level of ATH1, flowering in these plants was delayed from weeks to months compared to wild-type plants which bloom after 3-5 months depending on the season. In the most severe case (H10E # 4), the plants never bloomed until maturation (> 15 months after planting). H10E # 10 and # 35 plants, which showed high ATH1 expression, bloomed after 15 months, while medium / low expression plants, H10E # 35 and # 37 did not bloom until 6 months. Like the altered flowering phenotype, plants overexpressing ATH1 showed reduced stem growth, resulting in plant stunting. There is a clear correlation between the degree of dwarfism of growth and the level of transgene expression (see Fig. 5). In the most severe case, the plants only reached a fifth of their normal height. Leaf number varied from twice as high as wild-type to normal in all transgenes.

6.2 ATH1 overexpression can be reversed by GA3

The ATH1 overexpression phenotype can be reversed to the wild-type phenotype by using GA3. The foliar application of GA3 to the tobacco plants of Example 6.1 (spraying with 100 mM GA3 at three to four days intervals) resulted in a complete restoration of the wild-type stem length (Figure 6). This was also true for the late flowering phenotype (data not shown).

Example 7

ATH1 overexpression in Arabidopsis

To obtain further data on the role of ATH1 in plant development, full-length ATH1 cDNA was fused to the constitutive 35S promoter of the cauliflower mosaic virus and the chimeric 35S: ATH1 gene was transformed into Arabidopsis ecotype Col-0 using a vacuum infiltration method. Six independent primary transformants were thus obtained and all transgenic lines were self-crossed. From each independent transgenic line, 40 distinct seeds were germinated in the soil and checked for altered phenotypes compared to wild-type plants. Four of the six lines showed an altered flowering time phenotype. The seeds from these plants did not germinate well, and if this did, the plants stopped at the seedling stage. Both effects could be reversed by transferring the plants to a culture medium containing 10-5 M GA3 and culturing them in this medium for three days. After recovery and transfer to soil, plants developed normally except for the late flowering phenotype. Under short-day conditions, transgenic plants produce significantly more rosette leaves (vegetative amounts) than wild-type plants (about 40 rosette leaves and 100 days after germination and the plants are still not flowering, compared to about 30 rosette leaves before flowering for wild-type plants) ). Under LD conditions, most of these plants experience a partial reversal of generative to vegetative growth, evidenced by the formation of overhead rosettes (vegetative leaves) on the inflorescence stem. Plants (C24 ecotype) containing an extra copy of the ATH1 cDNA under the control of the Hspl8.2 heat shock gene promoter (HspHiB plants) also showed a late-flowering phenotype. Even without heat shock (it is known that this promoter also has basic activity without induction) plants containing this construct bloom much later under LD conditions than plants of the type

190,228 wild-type (30.5 rosette leaves produced in wild-type versus 61 rosette leaves produced in HspH1B plants - see Fig. 7).

Example 8

Early flowering by antisense ATH1 expression

Like ectopic ATH1 overexpression, disruption of ATH1 gene function may also shed light on ATH1 function in plant development. The disruption of gene function was achieved by constitutive overexpression of antisense ATH1. The full-length antisense ATH1 cDNA was fused to the constitutive 35S promoter of the cauliflower mosaic virus and the chimeric 35S: ATH1 antisense gene was transformed into Arabidopsis ecotype C24 using the Valvekens root transformation. Twenty-two independent transforms were obtained and all were self-crossed. From each line, 10 separate seeds were germinated in the soil and checked for altered phenotypes compared to wild-type C24 plants. In five of these lines, the plants exhibited an early-flowering phenotype: flowering commenced after the formation of six to ten rosette leaves compared to approximately thirty leaves in wild-type plants (see Figure 7).

Example 9

The shadow avoidance answer

When plants are grown close together, shade avoidance syndrome develops, in which plants respond to a lowered red / far red light ratio caused by red light filtering through the leaf canopy. This results in a rapid and dramatic increase in the growth rate of stems and petioles with leaf growth, storage organ production and reproductive development, thus resulting in an increase in the harvest index (where the harvest index is expressed as leaf biomass in proportion to total biomass).

The shade avoidance response is believed to be mainly mediated by phytochrome B and overproduction of phytochrome has been shown to eliminate the shade avoidance response, resulting in an increase in the harvest index of tobacco growing in the fields (Robson et al., 1996).

The absence of phytochrome B also leads to a loss of the shade avoidance response and under induction conditions this leads to a reduction in stem elongation compared to non-induction conditions.

In the laboratory, situations that trigger the shadow avoidance response can be mimicked by adding different far red light flux (Frc) intensities to solid white light (Wc). Under these conditions, wild-type Arabidopsis (C24 wt) plants show the typical shade avoidance response (elongated hypocotyls at Wc + Frc compared to the hypocotyl length at Wc only, while phytochrome B receptor mutants lacking a shadow avoidance response show the opposite response. (decrease in hypocotyl length) Plants with antisense AtHl also show a decrease in hypocotyl length, and in the most severe cases in plants with antisense AtHl (such as AtHl # 3) this decrease is similar to that in seeds of phyB mutants. Thus, it can be concluded that similarly like the lack of active phytochrome B, the loss of Athl results in a loss of the shadow avoidance response.

Shadow avoidance analysis for AtHl antisense plants (such as AtHl), wild-type C24 (wt C24) and photoreceptor mutant (phyB) plants is shown below. The seedlings were grown for 2 days under continuous white light followed by 4 days under the same high light conditions or 4 days under white light supplemented with far red. The length of the hypocotyls was measured after 6 days of growth. The following results were obtained.

C24wt Toilets Wc + Frc = Hypocotyl Length = Hypocotyl Length 5.5 mm 7.5 mm PhyB Toilets = length of the hypocotyl 9.5 mm Wc + Frc = length of the hypocotyl 7.3 mm Antisense Toilets = length of the hypocotyl 9.0 mm Athl # 3 Wc + Frc = length of the hypocotyl 5.0 mm Antisense Toilets = length of the hypocotyl 9.8 mm

190 228

Athl # 7 Wc + Frc = hypocotyl length

Antisense Wc - Hypocotyl Length

Athl # 23 Wc + Frc = hypocotyl length

8.0 m 7.5 mm 7.0 im

Literature cited:

Bagnall, 1993: Ann. Bot. 71-83

Bechtold et al., 1993: C. R. Acad. Sci. Paris, Life Sciences 316: 1194-1199

Bent et al., 1994: Sciece 265: 1856-1960

Caplan et al., 1985: J. Bacteriology 161: 655-664

Chambers et al., 1988: Gene 68: 139-149

Frisch et al., 1995: Plant Mol. Biol. 27: 405-409

Gelvin and Schilperoot, 1988: Plant Molecular Biology Manual:

Dordrecht: Kluwer AcademicPublisher.

Jefferson et al., 1987: EMBO J. 6: 3901-3907 Koomeef et al., 1991: Mol, Gen. Genet. 229: 57-66 Last and Gray, 1989: Plant Mol. Biol. 12: 655-666 Mandel and Yonowski, 1985: Nature 377: 523-524 Putteril et al., 1995: Cell 80: 847-857 Pwee and Gray, 1993: Plant J. 3: 437-449 Quaedvlieg et al., 1995 : The Plant Cell 7: 117-129 Shannon and Meeks-Wagner, 1991: The Plant Cell: 877-892 Takahashi and Komeda, 1989: Mol. Gen. Genet. 219: 365 Takahashi et al., 1992: Plant J. 2: 751-761 Weigel and Nilsson, 1995: Nature 377: 495-500

Horsch, R., Fry, F., Hoffman, N., Eichholtz, D., Rogers, S., and Fraley, R. (1985). A simple and general method for transferring genes into plants. Science 227, 1229-1231. Murshige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15.473-479.

Robson, P.R.H., McCormac, A.C., Irvine, A.S., and Smith, H. (1996). Genetic engineering of harvest index in tobacco through overexpression of a phytochrome gene. Nature Biotechnology 14 (8), 995-998.

Wilson, R.N., Heckman, J.W., and Sommerville, C.R. (1992). Gibberellin is requred for flowering in Arabidopsis thaliana under short days. Plant. Phys. 100, 403-408.

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Publishing Department of the Polish Patent Office. Circulation 50 copies. Price PLN 4.00.

Claims (4)

Patent claims A method of modifying flowering in plants, the method comprising transforming the plants with a construct comprising the complete or partial DNA sequence encoding an ATH1 product under the control of a plant-acting promoter. 2. The method according to p. The method of claim 1, wherein the flowering process in the plants is promoted by transforming the plants with a constituent that inhibits the production of the ATH1 protein. 3. The method according to p. The method of claim 2, wherein the construct is adapted to express the antisense RNA into RNA produced by the ATH1 gene. 4. The method according to p. The method of claim 1, wherein the flowering process in the plants is delayed by transforming the plants with a construct that promotes the production of recombinant ATH1 protein.