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  • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
  • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
  • A01H5/10—Seeds
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• Nucleic acid sequences and peptides/proteins of the FT family providing flower- repressing properties in tobacco and transgenic plants transformed therewith
• the present invention relates to novel nucleic acid sequences, amino acid, peptide and protein sequences derived thereof, and plants and their progeny transformed therewith.
• the nucleic acid sequences confer a delay or inhibition of flowering.
• the invention relates to methods for engineering non-flowering plants in order to ensure containment of transgenic plants, especially for such, that can propagate vegetatively.
• the transition from vegetative to reproductive growth is an important feature of the life cycle of plants. Accurate timing of the initiation of flowering is essential for plants to ensure the reproductive success. In agriculture and forestry, this transition is also very important because it significantly influences yield and biomass.
• the development of flowers is an obstacle in respect to the aim of producing a high amount of biomass, since development of the flowers in plants is often accompanied by termination of the vegetative growth and senescence. Therefore, a modulation of the time of flowering, specifically a delay thereof, should result in an increase of biomass because the plant is enabled to convert its full energy into vegetative growth and senescence of the plant material is inhibited or at least deferred.
• PEBP phosphatidyl ethanolamine-binding protein
• SAM shoot apical meristem
• FT has a species spanning universal role in promoting flowering, such as in dicotyledonous species like Arabidopsis, tomato, poplar, apple, cucurbits, sugar beet (Pin et al., 2010), and many others.
• FT expression is activated in the phloem companion cells of the leaves by the B-box zinc finger transcription factor CONSTANS (CO) under inductive long-day conditions (LD) due to the fact that the CO protein is only stabilized in the light.
• CONSTANS B-box zinc finger transcription factor CONSTANS
• LD inductive long-day conditions
• FT protein enters the sieve elements of the sieve tubes and is transported via the mass flow to the SAM, where it interacts with the bZIP transcription factor FD; both together activate the downstream targets of floral development such as the second floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1 ) and the floral meristem identity gene APETALA1 (AP1 ).
• SOC1 floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1
• APETALA1 APETALA1
• TSF TWIN SISTER OF FT
• TSF is a direct regulatory target of CO and acts as a floral promoter, thus operating redundantly to FT.
• SD non-inductive short day conditions
• TFL1 is a floral repressor and responsible for inflorescence architecture, thus being functionally antagonistic to its relative FT.
• substitution of a few defined amino acids can convert TFL1 into a flowering inducer and FT into a flowering repressor, respectively (Hanzawa Y., Money T., and Bradley D. (2005).
• a single amino acid converts a repressor to an activator of flowering.
• TFL and FT Both proteins, TFL and FT were reported to interact with the same co-factor FD in the shoot apex, that way regulating transcriptional repression or activation of target genes:
• the TFL/FD complex represses the transcription of floral meristem identity genes, which are activated by the FT/FD complex.
• MFT MOTHER OF FT AND TFL1
• BFT BROTHER OF FT AND TFL1
• FT and TFL 1 are regarded as belonging to the same family and have 57% identity in their amino acid sequence, they function as antagonists in the development of flowering and, moreover, each represent a phylogenetically different subfamily of the PEBP family.
• RNA interference RNA interference
• RNAi RNA interference
• flowering of the plant is only delayed, but never inhibited.
• protein FLC of Arabidopsis which can be used as a repressor of flower development, in order to defer the time of flowering, see WO 2000/050615.
• Transgenic plants expressing an inhibitor of flowering which is under the control of a conditional or latent promoter will former or later develop flowers - either undesired, or for obtaining progeny. Thus, sooner or later such plants will necessarily be the source for the production of pollen grains. It is evident that control of such pollen is difficult, in case the plants belong to commercially interesting crops, grown outside from greenhouses. Therefore, outcrossing with wild relatives and with corresponding crop plants is still possible and spreading of transgenes into nature cannot be circumvented.
• nucleic acid coding for an amino acid sequence capable of suppressing or delaying the development of blossoms or flowers which preferably belongs to the FT clade by most of its amino acids and motive, but which in any case exhibits a partial sequence which deviates from all sequences and motive of FT proteins known in the art, imparting new and unique features to the plants transformed with constructs including said nucleic acid.
• said nucleic acid can be used for the generation of transgenic plants, preferably crops, which are either delayed in flowering or, even more preferred, are non- flowering and remain non-flowering over more than one vegetation period.
• the invention focuses thereby on the usage of plants, which can be multiplied vegetatively, e.g. tobacco or potato.
• Nt FT1 - 4 The inventors of the present invention were able to identify four FT homologs in tobacco, designated as Nt FT1 - 4 which phylogenetically belong to the FT subfamily, but have antagonistic functions in flower development. The function of these proteins was examined by overexpression. Surprisingly, it could be shown that plants overexpressing Nt FTs exhibit complete different phenotypes, ranging from very early flowering shoots in tissue culture (Nt FT4) to nonflowering, 9 month old and more than 5 m high giants (Nt FT1 -3). Usually, a tobacco plant of the variety SR1 flowers around 6 to 8 weeks after germination and will reach a height of about 1 to 1.5 m at that time.
• Figure 10 shows a protein alignment of exemplarily chosen members of the plant PEBP family. Columns of complete identical amino acids in all sequences in the alignment are designated with * ; conserved substitutions are designated with :; semiconserved substitutes are designated with .. Asterisks mark amino acids essential for At FT vs. At TFL1 function (Ahn et al., 2006, see sbove) letters in italic mark amino acids mediating Bv FT1 vs. Bv FT2 function (Pin et al., 2010, see above). There is one non-conserved region where AtFT, NtFT4, as well as the flower- promoting protein from sugar beet, BvFT2, show the identical "YAPGW" motive.
• the present invention provides a new class of proteins and nucleic acids coding for said proteins wherein the proteins (1 ) have repressing properties, and (2) include the motive "NAPDIIDS” in place of the "YAPGW” motive of all flowering promoting proteins. Furthermore, this motive also differs from the already characterized “NAPQQ” motive of the flower repressing BvFT1 in the identical protein region. Preferably, (3) the proteins belong to the FT clade.
• the term "belonging to the FT clade” shall preferably have the meaning that the proteins can be classified phylogenetically into the FT clade and/or share at least 50%, preferably 80% and most preferably 100% amino acids which are identically conserved in each of AtFT, AtTFL.1 , BvFT1 and BvFT2 (see amino acid columns marked with * in Fig. 10), and/or share at least 70%, preferably 80% and more preferably 90% of the amino acid sequence chain within any of the proteins AtFT, AtTFL.1 , BvFT1 and BvFT2.
• the inventors found the motive "APDIIDS", and even more the motive "NAPDIIDS” contributing to flower repressing properties of the said and related peptides and proteins, and therefore found for the first time that the potato gene StSP5G (from Solanum tuberosum) which is reported until now to be a potential inhibitor for the tuber development in potatoes and the tomato gene SISP5G (from Solanum lycopersicum) as well as proteins derived thereof, play an important role in the repression of flowering of potatoes and tomatoes and of transgenic plants transformed therewith.
• StSP5G from Solanum tuberosum
• SISP5G from Solanum lycopersicum
• the invention further provides nucleic acids and proteins or peptides which can be expressed by said nucleic acids, wherein the sequence of the nucleic acids is partly or fully that of one of SEQ.ID No. 1 , 2, 3, and 4 ( Figures 1 to 4), or wherein the sequence of the proteins or peptides is partly or fully that of one of SEQ.ID No. 5, 6, 7, and 8 ( Figures 5 to 8), preferably as indicated in the dependent claims.
• any of the nucleic acid sequences of the invention can be under the control of a promoter.
• the promoter can be a cell specific, temporally induced promoter, originally present in tobacco plants, preferably a promoter naturally controlling the genes FT1 to FT4, thereby inducing expression of FTs in the phloem companion cells.
• the promoter can be a tobacco-derived tissue specific or cell specific, over the course of time constitutive active promoter like the FD promoter (wherein FD is a co-factor of FT) which is preferentially expressed in the SAM, the tissue of floral induction.
• the promoter can be derived from another plant, e.g. the cell specific, temporally induced promoter of the Arabidopsis FT, or that of the sucrose transporter AtSUC, both active in the phloem companion cells of source leaves.
• the promoter could further be a tissue specific or cell specific, over the course of time constitutive active promoter like that of the Arabidopsis FD, driving expression in the SAM.
• promoters or promoters available from other sources can be used as well, for example the spatial and temporal strong constitutive active, viral Cauliflower Mosaic Virus (CaMV) 35S promoter.
• CaMV viral Cauliflower Mosaic Virus
• the promoter may be constitutive, but this feature is not a necessary one.
• nucleic acids and peptides/proteins of the present invention are preferably used for an enhancement of biomass per plant/per time unit, via a modulation (deferment) of the time of flowering, or complete suppression thereof.
• Nt FT1 - 4 the nucleic acid sequence of which is indicated in SEQ ID Nos.
• ATC A. thaliana Centroradialis
• BFT A. thaliana Brother of FT and TFL1
• FT A. thaliana Flowering Locus T
• MFT A. thaliana Mother of FT and TFL1
• Nt CET1 , 2, 4 N. tabacum Centroradialis-like genes from tobacco
• Nt FT1 -4 N. tabacum Flowering Locus T
• TFL1 A. thaliana Terminal Flower 1
• TSF A. thaliana Twin Sister of Flowering Locus T.
• Nt FTs have a similar genomic structure among themselves and to FT genes from other species (exemplarily compared to At FT) with four exons interrupted by three introns. While the length of the exons is highly conserved the length of the introns differs among the Nt FTs.
• Nt FT1 - 4 To validate the phylogenetic classification of Nt FT1 - 4, an amino acid sequence alignment of those putative tobacco FTs with the flower-promoting Arabidopsis FT as well as with the flower- inhibiting Arabidopsis TFL1 and its tobacco homologs CET1 , CET2 and CET4 was created (Figure 10).
• the potential tobacco FTs show a relative high overall sequence identity from -70% (Nt FT3 with Nt FT4) to -89% (Nt FT1 with Nt FT3) to each other and -62% (Nt FT2) to -73% (Nt FT4) with At FT. In contrast, they show less sequence identity to tobacco CETs (-52%) and to the Arabidopsis TFL1 (-52%).
• Nt FT1 - 4 was ectopically overexpressed under the control of the strong and constitutive cauliflower mosaic virus 35S promoter (35S:Nt FT) in tobacco. After agrobacteria mediated transformation up to 7 independent transgenic lines for each construct were regenerated.
• FIGS 11A and 11 B The photographs (A) and (B) show shoots of plants wherein the coding region of Nt FT4 was cloned downstream of the constitutive promoter of the cauliflower mosaic virus (35S) and introduced into tobacco by transformation. Only shoots with flower-like structures could be regenerated, while shoots arrested in development and did not form roots, thereby abolishing the regeneration of mature plants. Therefore, they could not regenerate to mature plants.
• the phenotype was nearly identical to that caused by the overexpression of the Arabidopsis FT (35S:At FT), which served as a control in this experiment ( Figures 11C and 11 D).
• transformants of the constructs 35S:Nt FT1, 35S:Nt FT2 and 35S:Nt FT3 developed almost normal shoots in tissue culture. Plantlets of all three constructs with different expression levels were propagated by cuttings (in order to get two clones of each line with identical expression levels) and cultured in tissue culture until plantlets developed roots. Afterwards, transgenic clones of each line were transferred to phytotrons with one clone cultivated under LD (long day) and the other under SD (short day) conditions and flowering time was measured. Under these conditions, co-cultivated wild-type control plants started to produce flowers after four (LD) and five (SD) weeks indicating that flowering was delayed under SD.
• LD long day
• SD short day
• Nt FT1, Nt FT2 or Nt F73-transgenic plants developed differentially and exhibited mild, moderate and severe phenotypes with respect to flowering time and growths under both cultivation conditions. This could be observed as shown in Figures 12 A to 12 F: Representative transgenic tobacco lines overexpressing Nt FT1 (A, D), Nt FT2 (B, E) or Nt FT3 (C, F) were grown under long- (A to C) or short-day (D to F) conditions.
• transgenic lines with a mild phenotype started flowering only a few days later than WT plants, in phenotypic moderate lines flowering was retarded for approximately one week. Solely for 35S:Nt FT3 construct no moderate phenotype could be observed.
• all mildly affected plants displayed a phenotype comparable to WT plants and flowering time was only slightly delayed ( ⁇ 3d) whereas moderately affected plants developed first flowers 1 to 1 .5 weeks later and showed a slightly reduced internode length.
• Nt FT1 J
• Nt FT2 K
• Nt FT3 L
• LD long-
• SD short-day
• FIG. 13 Indicate the growth behaviour of phenotypic severe transgenic tobacco lines overexpressing Nt FT1 - 3.
• the photographs (A) to (J) indicate a time series of exemplarily chosen lines overexpressing Nt FT1, Nt FT2 or Nt FT3 grown under long-day conditions. Pictures were taken 8, 1 1 .5 and 29 weeks after transfer (wat) to the phytotron. The wildtype (WT) plant in I and J is 8 weeks old.
• the transgenic lines reached a size of up to 5m, thereby displaying a tremendous increase in biomass: At the end of the experiment, they possessed ⁇ 120 leaves with a maximum size of 65cm in length for mature leaves with an approximately 1 .5 fold size increase as compared to an 8-week-old WT plant ( Figures 13K and L). A similar increase in biomass of about 3.5 fold is also evident for the stem ( Figure 13K). It should be noted that cultivation of the severely overexpressing plants under SD conditions had to be terminated after 6 month when plants were 2m in height and reached the ceiling of the phytotron. Until then, plants developed in the same way as their counterparts grown under LD conditions.
• Figure 14 depicts the growth behavior of phenotypic severe transgenic tobacco lines overexpressing Nt FT1 - 3 under SD conditions.
• Photographs (A) to (J) depict a time series of exemplarily chosen lines overexpressing Nt FT1, Nt FT2 or Nt FT3 grown under SD conditions. Pictures were taken as indicated below each image (wat: weeks after transfer to the phytotron). The wildtype plant in I and J is 8 weeks old.
• the time point of bolting of the severe phenotypic lines correlates with the overexpression levels (Table 3) because plants already bolting at 4 to 6 wat (35S:Nt FT7 L i and 35S:Nt F72 L i) exhibit the lowest expression level within the severe phenotypic plants.
• the spatial and temporal expression profile of the flower-repressing Nt FT1 - 3 and the flower- promoting Nt FT4 was analyzed. For this, total RNA from leaf, apex, stem and root tissue of 4 week-old tobacco plants cultivated under LD and SD conditions was extracted and subjected to qRT-PCR.
• expression levels of the individual Nt FTs are shown in relation to Nt EF1a, which served as the reference gene.
• Nt FT1, Nt FT2 and Nt FT4 were exclusively expressed in leaf tissue under both light conditions, however, the level of transcription for all genes was weak and near the detection limit under LD conditions. This can be seen from
• Figure 15 A and B which indicate that Nt FT1, 2 and 4 are exclusively expressed in leaves under SD (A) as well as under LD (B) conditions, albeit the expression level under LD is near the detection limit. Values have been normalized to the transcript level of the reference gene EF1 a. Although cDNA can be obtained for Nt FT3, the expression level was too low to reliably analyze its spatiotemporal expression by qRT-PCR.
• Nt FT3 1 kb of the Nt FT3 promoter was cloned upstream of the reporter gene GFPER and stably transformed into tobacco plants by transformation.
• the CLSM showed that GFP expression was restricted to the vascular bundle of leaves as shown by a cross section of a leaf petiole in Figure 15 (C). The strongest signal could be observed in the veins of basal leaves, nevertheless expression and therefore fluorescence was weak, indicated by the strong autofluorescence of the xylem due to high laser intensities needed for detection.
• expression of Nt FT3 can be localized to the vascular bundle and more precisely to the companion cells (D, longitudinal section of a petiole).
• the auto-fluorescence of the xylem reflects the low expression level of Nt FT3.
• Arrows in (C) indicate vascular bundles. Arrow heads in (D) mark sieve plates stained with aniline-blue.
• PNI FT3 could be shown at the cellular level to be active in phloem companion cells (CCs), which are typically localized adjacent to sieve elements (SEs), whose sieve plates were stained with the callose-staining dye aniline blue ( Figure 15D).
• CCs phloem companion cells
• SEs sieve elements
• Figure 15D the callose-staining dye aniline blue
• Nt FT1 - 4 total RNA from tobacco seedlings and basal leaves harvested weekly until flowering from tobacco plants cultivated under LD as well as SD conditions was used to estimate expression of Nt FT1 - 4 by qRT-PCR
• the expression levels of Nt FT1 (E), Nt FT2 (F) and Nt FT4 (G) increase gradually during development under SD conditions showing the lowest expression level in seedlings (time point 1 ) and the highest expression level in leaves of flowering plants (time point 6).
• Transcript levels were determined in seedlings (time point 1 ) and basal leaves which were harvested every week until opening of the first flowers (time point 2 - 6).Values have been normalized to the transcript level of the reference gene EF1a.
• Nt FT3 As already noticed by analyzing the spatial expression pattern, the expression level of Nt FT3 under SD and LD as well as expression levels of the remaining Nt FTs under LD were near to the detection limit.
• Nt FT2 and Nt FT4 Similar expression pattern under SD conditions were observed: All genes displayed quite low expression in seedlings, but a successive increase was evident during developmental stages and expression levels of all Nt FTs reached the maximum at the time point of flowering, a fact which appears to be also evident for Nt FT3.
• Nt FT4 seemed to exhibit a generally lower expression level than Nt FT1 and Nt FT2, the increase in Nt FT4 expression (4400 fold) significantly exceeded the increase of Nt FT1 (164fold) and Nt FT2 (936fold) expression at the time point of flowering.
• Figure 15 (H) visualizes the increase of the expression levels. The values of the time points 2 - 6 of each gene were referred to time point 1 (set as 1 for each gene).
• Nt FT4 (encoding for a floral activator) increases to a much higher fold than that of Nt FT1 or Nt FT4 (encoding for floral repressors).
• FT-expression is regulated in a photoperiod dependent manner. Due to the fact that the expression of the tobacco FTs was hardly detectable under LD conditions but increased gradually under SD conditions, it can be concluded that FT expression in tobacco is also photoperiod dependent and that flowering under SD conditions is regulated in an FT-dependent manner.
• the molecular basis of floral induction under LD conditions remains elusive. Due to missing sequence data, it cannot finally be clarified yet if flower induction under LD conditions occurs FT-independent or if further FT orthologs are involved.
• 35S:Nt FT2 was exemplarily overexpressed in the model plant Arabidopsis, a member of the Brassicaceae and a plant that does not possess FTs with repressing function in floral transition.
• 35S:Nt FT2 transgenic Arabidopsis plants were obtained by Agrobacteria-mediated transformation and phenotypically analyzed. It became obvious by analyzing flowering time of the different transformants that the results resemble those obtained from overexpression of 35S:Nt FT1 - 3 in tobacco. Plants with a high expression level of 35S:Nt FT2 exhibit a late flowering phenotype under inductive LD conditions. This is shown in Figures 16A to 16C.
• 35S:Nt FT1 - 3 were overexpressed in the potato variety Solanum tuberosum.
• the transgenic potato plants were obtained by Agrobacteria-mediated transformation and
• floral repression mediated by a repressing Nt FT obviously works species-spanning, and the invention can be used for the transformation of other plants than tobacco as well, e.g. plants of other genera of the Solanaceae family, like the genus solanum (with potato as an example) or even plants of other plant families like the Brassicacea family.
• Nt FT1 - 3 The most noticeable characteristic of Nt FT1 - 3 is, albeit phylogenetic clearly related to the FT- like clade, that all three proteins have flower-repressing function, therefore functionally comparable to TFL1 .
• X-ray analysis of TFL1 and FT from Arabidopsis revealed two typical structural characteristics of these PEBP-family proteins: On the one hand a putative ligand- binding pocket and on the other hand an external loop (Benfield and Brady, 2000; Hanzawa et al, 2005; Ahn et al., 2006). Key amino acids in these structural features have been suggested to be important for FT- versus TFL1 -function in Arabidopsis (Hanzawa et al., 2005; Ahn et al., 2006).
• Tyr85 located at the entrance of the binding pocket, is essential for FT-function, whereas His88 (corresponding position in TFL1 ) mediates TFL1 -function.
• the second crucial amino acid is part of the 14 amino acid comprising external loop encoded by the fourth exon (Segment B), which evolved very rapidly in TFL1 orthologs but is almost invariant in FT orthologs (Ahn et al., 2006).
• Segment B fourth exon
• an Asp144 makes a hydrogen bond with His88
• FT carries a glutamine at the corresponding position (Gln140), which does not interact with the Tyr85.
• Table 2 shows a partial sequence alignment, illustrating the crucial amino acids of both repressing and activating tobacco FTs described herein, in comparison to FT/TFL1 from Arabidopsis, the flower promoting BvFT2 and to the floral repressor BvFT1 as well as the flower repressors SISP5G from Solanum lycopersicum and StSP5G from Solanum tuberosum..
• Segment B is part of exon four and encodes an external loop which evolved very rapidly in TFL1 -homologs but is almost invariant in FT- homologs. Letters in italic mark the amino acids which are important for the antagonistic function of Bv FT1 and Bv FT2 (Pin et al., 2010).
• Nt FT1 - 3 as well as Bv FT1 contain the two critical amino acid residues (or their conserved substitution), which at the corresponding position are essential for FT- function in Arabidopsis (Tyr-85 and Gln-140). Therefore, these amino acids are not obligatory determining FT-function in tobacco, a fact already described for sugar beet FTs (Pin et. 2010).
• Table 2
• NtFT1 R. LDREWN- APDIIDSRQN . . FHN
• NtFT4 R. .LGRETVY-APG WRQN . . LYN
• Nt FT1 - 3 Another described crucial sequence triad (LYN, located in segment C), which is conserved in FT-homologs and therefore potentially essential for FT-function, is obviously altered in Nt FT1 - 3, however it is present in the floral repressor Bv FT1 (Table 2).
• the latter differs from its flower- inducing ortholog Bv FT2 in three amino acid residues of segment B (italic in Table 2), a substitution of these residues converts the activator into a repressor and vice versa (Pin et al., 2010).
• Nt FT4 So/anaceae-specific activating FT
• the amino acid sequence of the repressing Nt FT1 - 3, the repressing St SP5G and the repressor SI SP5G significantly differs to that of the repressing Bv FT1 , instead exhibiting a conserved insertion of the three amino acids I ID.
• the inventors assumed a species-specific amino acid pattern for repressive versus promotive function of the FTs.
• Example 1 Cloning of the tobacco FT homologs and analysis of their evolutionary relationship
• NucleoSpin® RNA Plant kit Macherey-Nagel
• SuperScriptll Invitrogen
• Nt FT1 - 4 sequence similarity to At FT.
• An alignment of the genomic and cDNA sequences revealed the exon-intron structure of Nt FT1 - 4, which is schematically depicted in Figure 9A.
• all potential Nt FTs have a similar genomic structure among themselves and to FT genes from other species (exemplarily compared to At FT) with four exons interrupted by three introns. While the length of the exons is highly conserved the length of the introns differs among the Nt FTs.
• Nt FT1 - 4 To validate the phylogenetic classification of Nt FT1 - 4, an amino acid sequence alignment of those putative tobacco FTs with the flower-promoting Arabidopsis FT as well as with the flower- inhibiting Arabidopsis TFL1 and its tobacco homologs CET1 , CET2 and CET4 was created using T-Coffee (EMBL-EBI) ( Figure 10).
• the potential tobacco FTs show a relative high overall sequence identity from -70% (Nt FT3 with Nt FT4) to -89% (Nt FT1 with Nt FT3) to each other and -62% (Nt FT2) to -73% (Nt FT4) with At FT.
• Example 2 Characterization of the molecular function of Nt FT1 - 4 by overexpression studies in tobacco
• Nt FT1 - 4 To assess the function of Nt FT1 - 4 in the regulation of flowering time, we next set out to ectopically overexpress the corresponding genes under the control of the strong and constitutive cauliflower mosaic virus 35S promoter (35S:Nt FT) in tobacco. Therefore, the following cloning strategy was performed.
• the pCambia1300 was digested with Nhe I and Afl II and the coding region of the hygromycin was inserted into the Nhe I and Afl II digested pBin19 (Bevan, 1984) resulting in the binary vector pBin19 Hyg.
• the 35S:Nt FT1 - 4 constructs were then excised and inserted into the Hind III digested binary vectors pCambia1300 or pBin19 Hyg resulting in pCambia1300 35S:Nt FT1 and pBin19 Hyg 35S:Nt FT2 - 4.
• the coding region of At FT was amplified from Arabidopsis leaf cDNA, cloned into pCRII ® Topo ® (Invitrogen) and sequenced.
• the coding region of At FT was then amplified by PCR from the cDNA within the vector pCRII ® Topo ® (Invitrogen) using primers containing restriction sites as shown in Table 4 below. PCR products were digested to the corresponding restriction sites and cloned downstream of the constitutive 35S promoter into the pRT104 vector. The 35S:At FT construct was then excised and inserted into the Hind III digested binary vector pCambia1300. All binary vectors were verified by sequencing and subsequently introduced by electroporation into Agrobacterium tumefaciens LBA4404 (Hoekema et al., 1983). For the transformation experiments Nicotiana tabacum cv.
• SR1 plants were grown on MS medium (Murashige and Skoog, 1962) under sterile conditions (LD; 23°C, 100 ⁇ m "2 sec "1 ) and Agrobacterium- mediated transformation was performed as described in Horsch et al. (1986).
• transformants of the constructs 35S:Nt FT1, 35S:Nt FT2 and 35S:Nt FT3 developed almost normal shoots in tissue culture.
• Independent transgenic lines for each construct were regenerated (seven for 35S:Nt FT1, five for 35S:Nt FT2 and three for 35S:Nt FT3) and identical clones of all transgenic and WT tobacco plants were produced by cuttings under sterile conditions, transferred into soil after rooting and grown in phytotrons under long-day (LD;
• Nt FT1 - 4 Each RT sample for Nt FT1 - 4 was assayed in triplicates whereas reference genes, NRT (not reverse transcribed) and NTC (non-template control) controls were assayed in duplicates.
• Transcript levels of the two potential reference genes EF1 a and L25 were examined in each RT sample. Of these genes, EF1 a was found to be the most stably expressed, and this gene was therefore used to normalize transcript levels of Nt FT1 - 4. Relative expression levels were calculated using the REST-MCS software (Pfaffl et al., 2002). Primers used for qRT PCRs are shown in Table 4.
• Nt FT1 - 4 Each RT sample for Nt FT1 - 4 was assayed in triplicates whereas reference genes, NRT (not reverse transcribed) and NTC (non-template control) controls were assayed in duplicates.
• Transcript levels of the two potential reference genes EF1 a and L25 were examined in each RT sample. Of these genes, EF1 a was found to be the most stably expressed, and this gene was therefore used to normalize transcript levels of Nt FT1 - 4. Relative expression levels were calculated using the REST-MCS software (Pfaffl et al., 2002). Primers used for qRT-PCR are shown in Table 4.
• Nt FT1a expression levels of the individual Nt FTs are shown in relation to Nt EF1a, which served as the reference gene.
• Nt FT1, Nt FT2 and Nt FT4 were exclusively expressed in leaf tissue under both light conditions, however, the level of transcription for all genes was weak and near the detection limit under LD conditions ( Figure 15 A and B).
• cDNA can be obtained for Nt FT3
• the expression level was too low to reliably analyze its spatiotemporal expression by qRT-PCR.
• the ER-tagged version of GFP was chosen to prevent diffusion of GFP via the phloem in order to correctly identify GFP expressing cells.
• the cassette consisting of P N t F r 3 :GFP E R and the terminator of the cauliflower mosaic virus was amplified using primers containing Sal I restriction sites. PCR products were digested with Sal I, inserted into the Sal I digested binary vector pBin19 Hyg, verified by sequencing and subsequently introduced by electroporation into Agrobacterium tumefaciens LBA4404 (Hoekema et al., 1983).
• transgenic tobacco lines Five independent transgenic tobacco lines were obtained by Agrobacteria-mediated plant transformation and designated as PNt FT3- GFP er .
• the transgenic plants were transferred into soil after rooting, grown in the green house and four to six week old plants were analyzed by confocal laser scanning microscopy (CLSM) using a Leica TCS SP5 X microscope (Leica Microsystems, Germany) with excitation/emission wavelengths of 488/500-600 nm.
• CLSM confocal laser scanning microscopy
• 35S:Nt FT2 in the model plant Arabidopsis, a member of the Brassicaceae and a plant that does not possess FTs with repressing function in floral transition.
• the 35S:Nt FT2 construct was inserted into the Hind III digested binary vector plabl 2.1 carrying the BASTA resistance gene under the control of mannopine synthase promoter (Post et al., 2012).

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