ANR gene expression in white clover Graphical abstract Page |1 ANR gene expression in white clover Page |2 Research Article Spatio-Temporal Profile of the Anthocyanidin Reductase Gene Expression in White Clover Flowers Shamila W. Abeynayake1,2,3, Stephen N. Panter1, Nadia Efremova4, Aidyn Mouradov and German C. Spangenberg1,2 *, 1,5 Department of Environment and Primary Industries, Biosciences Research Division, AgriBio, La Trobe University, Bundoora, Victoria 3083, Australia, 1 La Trobe University, Bundoora, Victoria, 3083, Australia, 2 Department of Agroecology, Aarhus University, Forsøgsvej 1, 4200, Slagelse, Denmark 3 Phytowelt GreenTechnologies GmbH, Stöckheimer Weg 1, D-50829 Köln, Germany 4 School of Applied Sciences, RMIT University, Bundoora, Victoria 3083, Australia 5 *Corresponding author: RMIT University, School of Applied Sciences, Bundoora, Victoria 3083, Australia; Tel: 062 3 99257144; Fax: 062 399257100 aidyn.mouradov@rmit.edu.au Received; Accepted Abstract Proanthocyanidins (PAs) are polymers of flavan 3-ol subunits that are naturally produced by some plants and ameliorate pasture bloat in livestock. Genes encoding proteins involved in the biosynthesis, intracellular transport and compartmentalisation of PAs and their precursors are tightly regulated by transcription factors. In this study, the expression pattern of an anthocyanidin reductase (ANR) gene was shown to correlate with the accumulation pattern of PAs and/or their monomers in white clover (Trifolium repens L.). The sub-cellular localisation patterns of an ANR-GFP fusion protein and proanthocyanidins and/or monomers are consistent with biosynthesis of flavan 3-ols in the cytoplasm prior to PA accumulation in the vacuole of cells in flowers. Characterisation of the white clover ANR gene provides a valuable resource for further work aiming to improve bloat safety in white clover by elevating PA levels in leaves. Keywords: condensed tannin, BANYULS, transgenic, in-situ hybridisation Abbreviations: ANR, anthocyanidin reductase, ER, endoplasmic reticulum, GFP, green fluorescent protein, LAR, leucoanthocyanidin reductase, PA, proanthocyanidins ANR gene expression in white clover Page |3 1. Introduction Flavonoid compounds, including anthocyanins, flavonols and proanthocyanidins, are a large group of secondary metabolites from plants with a wide range of biological roles (reviewed by Lepiniec et al. 2006). Proanthocyanidins, or condensed tannins, are bioactive polymers of flavan 3-ols. Of significant agricultural interest, a threshold level of proanthocyanidins (24% of dry weight) in livestock feed binds to dietary proteins in the rumens of sheep and cattle, slowing the degradation of protein by gut microbes and the formation of protein foams that can trap digestive gases (Aerts et al. 1999). This process ameliorates pasture bloat in sheep and cattle and also reduces the loss of dietary amino acids via conversion to ammonia in sheep. The biosynthesis and regulation of proanthocyanidins has been substantiated by studies of transparent testa (tt) mutants in Arabidopsis that lack oxidised proanthocyanidins and have seeds that are relatively light in colour (Shirley et al. 1995; Lepiniec et al. 2006). The proanthocyanidin-specific branch of the flavonoid pathway starts with the conversion of 2,3-flavan 3,4-diols either to anthocyanidins and 2,3-cis-flavan 3-ols by anthocyanidin synthase (ANS, E.C. 1.14.11.19) and anthocyanidin reductase (ANR, syn BANYULS, E.C. 1.3.1.77) or to 2,3-trans-flavan 3-ols by leucoanthocyanidin reductase (LAR, E.C. 1.17.1.3) (Abrahams et al. 2003; Tanner et al. 2003; Xie et al. 2003). Anthocyanidins are the branch point between the biosynthetic pathways leading to production of proanthocyanidins and anthocyanins, which are generated by O-glycosylation, O-acylation and O-methylation of anthocyanidins (for a detailed review, see Lepiniec et al. 2006). An established model for proanthocyanidin biosynthesis, shows that flavan 3-ols are produced within a complex of flavonoid-pathway enzymes anchored to the cytoplasmic side of the endoplasmic reticulum and transported to the vacuole by a mechanism involving several proteins with transport functions or by transport vesicles (for review, see Zhao et al. 2010). Components of the transport machinery for compartmentalisation of flavan 3-ols include: a glycosyltransferase, a glutathione S-transferase that binds and transports glycosylated flavan 3-ols across the cytoplasm and a multidrug and toxic compound extrusion (MATE) transporter coupled to a H+/ATP-ase on the tonoplast to provide active transport of glycosylated flavan 3-ols into the vacuole. The mechanism for polymerisation of flavan 3-ols into proanthocyanidins is not well understood, but may involve either laccase activity or spontaneous condensation within the vacuole (Lepiniec et al. 2006). The production of proanthocyanidins is spatially restricted and developmentally controlled at the transcriptional level in many plants, including Arabidopsis, barrel medic, grapevine and white clover (Lepiniec et al. 2006; Bogs et al. 2005; Pang et al. 2007; Abeynayake et al. 2012). Proanthocyanidin biosynthesis and the subcellular transport of flavan 3-ols in Arabidopsis have been shown to be regulated by six genes, TT1, TT2, TT8, TT16 and TTG1. The proteins encoded by three of these genes, TT2 (R2R3-MYB), TT8 (bHLH) and TTG1 (WD40) function as a ternary transcriptional complex regulating the expression of BANYULS and the tissue- and cell-specific accumulation of proanthocyanidins (reviewed by Lepiniec et al. 2006). TT2 is expressed only in the innermost cell layer of the inner seed integument and regulates the expression of DFR, ANS and BANYULS. TT8 also regulates expression of DFR and BANYULS in siliques, and TTG1 controls BANYULS ANR gene expression in white clover Page |4 expression, mainly through its effect on the function of TT8. MADS box (TT16), zinc finger (TT1) and WRKY (TTG2) classes of transcription factors control the expression of BANYULS indirectly by activating early steps in flavonoid biosynthesis or by regulating the development of proanthocyanidin-producing cells (reviewed by Lepiniec et al. 2006). Down-regulation of an ANR gene isolated from white clover that is normally expressed during early flower development was shown to increase anthocyanin accumulation, but reduce proanthocyanidin levels in flowers of transgenic white clover plants (Abeynayake et al. 2012). The current study aimed to characterise this gene further by identification of putative cis regulatory elements within its promoter and by in situ hybridization to visualize the pattern of ANR transcript accumulation in floral tissues. Furthermore, production of the TrANR protein was visualised in specific cells of floral tissues of transgenic white clover plants expressing a translational fusion between the TrANR protein and the green fluorescent protein of Aequorea victoria under the control of the TrANR promoter. Materials and Methods Plant growth conditions White clover plants were vernalised in a controlled growth room for 6 weeks at 5 oC with an -2 -1 8 h photoperiod and a light intensity of 41+/s at canopy height. Flowering was then induced in a controlled growth cabinet (Enconair) by growing plants for 4 weeks at -2 -1 22oC with a 16 hour photoperiod and a light intensity of 240+/-30 s at canopy height. Characterisation of the white clover ANR gene A genomic clone containing TrANR was identified in a custom white clover BAC library by a PCR-based strategy, using the primers 5`-CACTGCAAAACCACCCACTT-3` and 5`TGCTTGAAACTGAACCCTTCTT-3`. Sequence was obtained from the promoter region of TrANR using BAC DNA extracted with the Large Construct Kit (QIAGEN) and a primer walking strategy involving Sanger sequencing. Sequence of the TrANR gene was also obtained using a GS-20 ‘454’ pyrosequencing strategy according to the manufacturer’s recommendations and Newbler sequence assembly software, version 1.1.02.15 (Roche). The genomic sequence of the white clover (Trifolium repens L.) ANR gene (TrANR) has been deposited in Genbank (accession number GU300807). Preparation of recombinant plasmids for plant transformation A 1066 bp PCR fragment containing the coding region of TrANR minus the stop codon was amplified from a previously-characterised cDNA clone (Sawbridge et al. 2003; Abeynayake et al. 2012) using the primers 5`-attB1-GCACTAGTGTGTATAAGTTTCTTGG-3` and 5`-attB2ATTCTTCAGTGCCCCCTTAGTCTTA-3`. GATEWAY® cloning was used to insert this PCR product upstream of and in-frame with the gfp coding region and under the control of an enhanced CaMV 35S promoter and the CaMV 35S terminator in a plant transformation ANR gene expression in white clover Page |5 vector. generating p35S:TrANR-GFP (Figure 1). The CaMV 35S promoter in this vector was replaced by a 1310 bp promoter fragment flanked by AscI and XhoI sites that had been amplified by PCR from a BAC genomic clone of the white clover ANR gene using the primers 5`TTCGTGGCGCGCCTCCATTAGATTAGTACAATGACG-3` and 5`CGGCTCGAGTTTCACTAAGAAACTTATACACAC-3` to make pTrANR:TrANR-GFP. All PCR products and the ligation sites in p35S:TrANR-GFP and pTrANR:TrANR-GFP, were verified by Sanger sequencing (Figure 1). . Generation of transgenic white clover plants Transgenic white clover plants (Trifolium repens L. cv Mink) containing pTrANR:TrANR-GFP were generated by Agrobacterium-mediated transformation using cotyledonary explants and selection with 50 mg/L kanamycin sulphate (Ding et al. 2003). Transgenic white clover plants containing a GFP gene, encoding GFP with or without the C-terminal HDEL signal for retention in the ER, under the control of the CaMV 35S promoter were provided by Emma Ludlow (Ludlow, 2006 - unpublished data). Screening of putative transgenic white clover plants DNA was extracted from leaf tissue of putative transgenic plants using the Wizard DNA purification kit (Promega) and plants were screened by real-time PCR for the presence of the npt2 selectable marker gene using the primers 5`-GGCTATGACTGGGCACAACA-3` and 5`-ACCGGACAGGTCGGTCTTG-3` (Dorak et al. 2006). PCR reactions were set up using a SYBR Green PCR Master Mix (Applied Biosystems), according to the manufacturer’s thermal cycler (Stratagene) with: 10 mins at 95oC; 40 cycles of 30 s at 95oC, 30 s at 60oC and 30 s at 72oC; 1 min at 95oC, 30 s at 55oC and 30 s at 95oC. In-situ hybridisation Preparation of tissues and in situ hybridisation were performed as previously described, in order to visualise the pattern of TrANR transcript accumulation within floral tissues of white clover plants (Efremova et al. 2004). Images were taken under bright–field illumination using a Zeiss Axiophot microscope equipped with plan-NEOFLURAL objectives, a JVC KY-F70 digital camera and Discus software. Confocal and epifluorescence microscopy To examine subcellular localisation of GFP, tissues of transgenic and wild type white clover plants were hand-sectioned using a scalpel blade and mounted on glass slides in 10% v/v glycerol and were then examined using a Leica TCS SP2 confocal microscope. For comparison, Arabidopsis leaves bombarded with the p35S:TrANR-GFP were also examined after incubation for 24 h on solid MS media (Mathur et al. 2003). ANR gene expression in white clover Page |6 Results Sequence characterisation of the TrANR promoter region In order to identify potential regulatory sequences, the sequence of the putative promoter region of TrANR obtained from a BAC genomic clone was compared to a public database of cis-acting promoter elements (http://www.dna.affrc.go.jp/PLACE/signalscan.html). Two regions identified contained a combination of potential bHLH and MYB-binding sites within 1 kb of the TrANR start codon (Figure 2). The first region, between 340 and 379 bp upstream of the start codon, contains potential bHLH- and MYB-binding sites located 28 bp apart. The second region, between 109 and 164 bp upstream of the start codon, contains these two sites spaced 44 bp apart. Another potential MYB-binding sequence, similar to the highaffinity P-binding site of maize, was found 250 bp upstream of the start codon of TrANR, between the two MYB/bHLH-binding regions (Grotewold et al. 1994). Other potential ciselements within the TrANR promoter region included a putative LFY-binding site (CCAATGT) and a number of putative CArG boxes that are targets for MADS-box transcription factors. TrANR transcripts are present in epidermal cells of immature floral organs in white clover Immature, 50% open, and mature inflorescences from white clover plants were divided in half transversely, allowing flower development to be represented by six stages, the youngest being the upper half of immature inflorescences (stage 1) and the most developed being the lower half of mature inflorescences (stage 6). In situ hybridisation was used to identify cell types of immature white clover flowers in which the TrANR gene is expressed (Figure 3). The TrANR transcript was not detected in the least developed flowers within immature inflorescences (stage 1, data not shown). No signal was detected in the negative control treatment when a sense strand-specific probe was used with stage 2 flowers (Figure 3A). When stage 2 flowers were hybridised to an antisense probe, accumulation of TrANR transcripts was spatially restricted to epidermal cells of petals and carpels (Figure 3B-E) and to a lesser extent, to stamens (Figure 3E). Detection of TrANR transcripts in more mature flowers at stages 3-6 was masked by progressive accumulation of a brown metabolite, potentially representing oxidised proanthocyanidins and flavan 3-ols within TrANRexpressing cells (Figure 3F-I). Confocal imagines revealed small vesicle-like structures of between 0.1 and 3 μm in size in the cytoplasm of epidermal cells of fresh petals from immature flowers (Figure 3J-K). The TrANR protein is produced in epidermal cells of immature petals, carpels and stamens PCR analysis showed that five out of the eight white clover lines that were generated as a result of Agrobacterium-mediated transformation with pTrANR:TrANR-GFP contained the construct. GFP fluorescence was detected in immature flowers of one of these five transgenic lines and images of this line were taken using epifluorescence and confocal scanning laser microscopy (Figure 4). GFP fluorescence was detected only in epidermal cells of petals, carpels and stamens (Figure 4). GFP fluorescence was initially seen in epidermal ANR gene expression in white clover Page |7 cells located on the abaxial side of petals and progressed to epidermal cells on the adaxial side during flower development (Figure 4B). A GFP signal was seen in epidermal cells located on both the abaxial and adaxial sides of carpels (Figure 4B-C) and in immature embryos (Figure 4C-D). A mosaic pattern of GFP fluorescence that was similar to the pattern of proanthocyanidin accumulation detected by DMACA staining was seen in the epidermal layer of carpels and stamens (Figure 4E-H). Background autofluorescence made it very difficult to detect GFP fluorescence in trichomes (data not shown). A reticulate pattern of GFP-fluorescence was seen in optical sections from confocal images of epidermal cells in floral organs from plants containing pTrANR:TrANR-GFP (Figure 5A-C). The fluorescence pattern seen in transgenic white clover lines ectopically expressing a C-terminal fusion between GFP and the HDEL signal for retention of proteins in endoplasmic reticulum membranes (Figure 5D) was similar. When expressed under the control of a CaMV 35S promoter, GFP fluorescence was not seen in the ER of epidermal cells of Arabidopsis leaves after particle bombardment (Figure 5E) and was seen in the nucleus and cytoplasm but not in the ER of cells in transgenic white clover plants (Figure 5F). Discussion Down-regulation of the white clover ANR gene, similar to ANR genes in Arabidopsis and Medicago truncatula, results in the diversion of intermediates from proanthocyanidin to anthocyanin production in white clover flowers (Abeynayake et al. 2012). This study showed that the presence of TrANR transcripts is consistent with the accumulation of proanthocyanidins and/or their monomers in epidermal cells of petals, stamens, carpels, as well as in trichomes of sepals in developing white clover flowers. Hence, TrANR is considered a marker of proanthocyanidin production in white clover, as well as being a target for engineering bloat safety in this plant (Aerts et al. 1999; Abeynayake et al. 2012). As in Arabidopsis, proanthocyanidin biosynthesis in white clover is developmentallyregulated, spatially restricted and correlates well with the presence of ANR transcripts. In Arabidopsis, PA production is restricted to seed coats and appears to be triggered by fertilisation of flowers. However, PA biosynthesis and TrANR expression are high in immature flowers of white clover plants and PAs can be detected in both flowers and seed coats (Figure 3, Abeynayake et al. 2012). Functional analysis of the TrANR promoter, which contains a similar range of gene regulator binding sites as observed in the promoter of BANYULS (Debeaujon et al. 2003) showed that gene expression of TrANR is coordinated spatiotemporally with proanthocyanidin accumulation in white clover flowers (Figures 2, 3 and 4). The presence of a conserved arrangement of MYB- and bHLH-binding motifs in the TrANR promoter (Figure 2) suggests that this gene, like BANYULS, may be regulated by a transcription factor complex (Baudry et al. 2004). Recently, Medicago truncatula and Trifolium arvense genes encoding TT2-like MYB factors have been identified that upregulate proanthocyanidin production when overexpressed in leaves of transgenic legumes that otherwise contain only trace levels of proanthocyanidins (Hancock et al. 2012; Verdier et al. 2012). In both cases, enhanced proanthocyanidin production in the transgenic plants was correlated with ectopic expression of ANR and other genes encoding enzymes required for proanthocyanidin biosynthesis. Identification of targets for these MYB factors in promoters ANR gene expression in white clover Page |8 of structural genes, including ANR, and characterisation of the mechanism underlying asymmetrical pattern of TrANR expression in epidermal layers of floral organs (Figure 3) would improve our understanding of proanthocyanidin pathway regulation in white clover. The characterised TrANR promoter region lacks motifs that would be expected to direct asymmetrical gene expression (eg: Watanabe and Okada, 2003), but trans-activation is still a possibility. A widely accepted model for compartmentalisation of flavonoids involves the organisation of the flavonoid enzymes in metabolons that are anchored to the cytoplasmic face of the ER membrane (Hrazdina and Wagner, 1985). Confocal imaging of transgenic white clover flowers expressing a TrANR-GFP fusion construct clearly showed localisation of the fusion protein within the ER in epidermal cells of petals (Figure 5). Unlike most of the known ER-targeted proteins, TrANR is predicted to be a soluble protein without known signal peptides for transport into any cellular compartment, including the ER. Also, transient expression of MtANR-GFP (Pang et al. 2007) and TrANR-GFP fusion proteins (Figure 5E) in epidermal cells of Arabidopsis leaves did not result in GFP fluorescence in the ER and this was consistent with the lack of functional proanthocyanidin biosynthesis machinery. This suggests that interaction between TrANR and membrane-bound enzymes of the flavonoid pathway,, such as C4H, is required for localisation of TrANR to the ER (Hrazdina and Wagner 1985) . In this study, spherical vesicle-like structures were seen in the cytoplasm of proanthocyanidin-accumulating epidermal cells of petals within immature white clover flowers (Figure 3J-K). Although biochemical confirmation is needed, the structures might represent transport vesicles facilitating the movement of flavan 3-ols or proanthocyanidins from the cytoplasm to the vacuole. Some studies have provided evidence for vesiclemediated transport of proanthocyanidins from the endoplasmic reticulum to the vacuole in plants (eg: Parham and Kaustinen 1977; Zobel, 1986). ANR gene expression is thus a useful marker for the presence of proanthocyanidin biosynthetic machinery in both Arabidopsis and white clover plants. Isolation of specific cell types that express TrANR and accumulate proanthocyanidins from transgenic plants with the pTrANR:TrANR-GFP construct should allow co-ordinately regulated genes involved in proanthocyanidin biosynthesis to be identified. This finding should facilitate metabolic engineering to reconstitute the proanthocyanidin biosynthesis pathway in white clover leaves for enhanced bloat safety. Acknowledgements The authors thank Matthew Hayes, Noel Cogan, Daniel Isenegger and Ulrik John for critical comments on the manuscript and are grateful to Rob Glaisher and Catherine Li from La Trobe University for technical help with the confocal and electron microscopy and to Megan McKenzie for GS-20 ‘454’ pyrosequencing of the TrANR BAC clone. The authors would also like to thank Emma Ludlow for providing transgenic white clover plants containing chimeric GFP constructs. This work was supported by the Molecular Plant Breeding Cooperative Research Centre and the Department of Primary Industries, Victoria. ANR gene expression in white clover Page |9 References Abe M., Takahashi T., Komeda Y. (2001), Identification of a cis-regulatory element for L1 layer-specific gene expression, which is targeted by an L1-specific homeodomain protein. Plant J, 26(5): 487-494. Abeynayake S., Panter S., Mouradov A., Spangenberg G. 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Xie D., Sharma S.B., Wright E., Wang Z.Y., Dixon R.A. (2006), Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J, 45(6): 895-907. Zhao J., Pang Y., Dixon R.A. (2010), The mysteries of proanthocyanidin transport and polymerization. Plant Physiol, 153(2): 437-443. Zobel A. (1986), Localization of phenolic compounds in tannin-secreting cells from Sambuccus racemosa L. shoots. Ann Bot, 57(6): 801-810. ANR gene expression in white clover P a g e | 12 A XhoI AscI Figures LB RB nptII gfp B CaMV 35S term XhoI nos CaMV 35S prom prom AscI ocs term TrANR LB RB nptII ocs term TrANR nos TrANR prom prom gfp CaMV 35S term Figure 1. Schematic diagram showing the T-DNA regions of transformation vectors for expression of TrANR-GFP translational fusions The p35S:TrANR-GFP (A) and pTrANR:TrANR-GFP (B) transformation constructs were designed to allow the production of a C-terminal translational fusion between the white clover anthocyanidin reductase protein, encoded by TrANR, and green fluorescent protein under the control of an enhanced CaMV 35S promoter and a TrANR promoter, respectively. TrANR prom - Trifolium repens anthocyanidin reductase gene promoter; CaMV 35S prom – enhanced cauliflower mosaic virus 35S promoter; TrANR – Trifolium repens anthocyanidin reductase gene coding region; gfp – green fluorescent protein gene coding region; CaMV 35S term – cauliflower mosaic virus 35S terminator; ocs term – octopine synthase gene terminator; nptII – neomycin phosphotransferase 2 gene coding region; nos prom – nopaline synthase gene promoter; RB – right border of T-DNA region; LB – left border of TDNA region. ANR gene expression in white clover bHLH MYB -379 -340 P a g e | 13 haPBS -250 bHLH -164 MYB -109 TrANR bHLH haPBS -149 -101 AtBAN haPBS: CCTAMYR(G)ASC bHLH: CANNTG MYB: CNGTTR, YAACKG, CCWACC Figure 2. Cis-acting elements in the promoter regions of anthocyanidin reductase genes Positions of selected MYB and bHLH cis-acting elements are shown relative to the start codons of the Trifolium repens and Arabidopsis thaliana anthocyanidin reductase genes (TrANR and BANYULS, respectively). haPBS, high-affinity P-binding site (after Debeaujon et al. 2003). ANR gene expression in white clover P a g e | 14 Figure 3. Cell-specific accumulation of TrANR transcripts in white clover flowers In-situ hybridisation was used to visualise TrANR transcripts in fixed tissue sections from floral organs of white clover cv ‘Mink’. Negative control, involving a sense strand-specific probe and a section through an immature flower (A, stage 2). Transcript accumulation in carpels (B-C) and petals (D-E) of an immature flower (stage 2). Accumulation of a brown metabolite in epidermal cells of a petal (F) and a carpel (G-H) and a multicellular trichome (I) from maturing flowers (stage 5). Vesicles in epidermal cells of petals from fresh flowers collected at stages 3 (J) and 6 (K), viewed by confocal scanning laser microscopy. Crosssections (A,C,E). Longitudinal sections (B,D, F-K). ab, abaxial side; ad, adaxial side; st, stamen; ca, carpel; pe, petals; sp, sepal; tr, trichome. Refer to text and Abeynayake et al. H-K). ANR gene expression in white clover P a g e | 15 Figure 4. Cell-specific production of a TrANR-GFP fusion protein in white clover flowers A fusion construct containing a translational fusion between the white clover anthocyanidin reductase (TrANR) coding region and the gfp coding region, under the control of the TrANR promoter, was expressed in white clover plants. A longitudinal section of petals from a transgenic plant viewed using bright field conditions (A) and with a GFP fluorescence filter set (B). Cross-section of a flower from a transgenic plant viewed with a GFP fluorescence filter set (C). Carpel of a flower from a transgenic plant viewed with a GFP fluorescence filter set (D-E). Carpel from a non-transgenic flower stained for the presence of proanthocyanidins and/or 2,3-flavan 3-ols with DMACA (F). Epidermal cells from a stamen tube of a transgenic plant viewed with a GFP fluorescence filter set (G). Stamen tube of a non-transgenic plant stained with DMACA (H). ab, abaxial; ad, adaxial; e, embryo; ov, ovule; pe, petals; sp, sepal. ANR gene expression in white clover P a g e | 16 Figure 5. Intracellular localisation of a TrANR-GFP fusion protein in white clover flowers and Arabidopsis leaves Visualisation of GFP fluorescence by confocal microscopy (single optical sections): GFP fluorescence in epidermal cells of petals from transgenic white clover plants expressing a TrANR:TrANR-GFP construct (A-C); petal epidermal cells from a transgenic white clover plant expressing a 35S:GFP-HDEL construct (D); a leaf epidermal cell from Arabidopsis thaliana after particle bombardment with a 35S:TrANR-GFP construct (E); GFP fluorescence in epidermal cells of petals from transgenic white clover plants expressing a 35S:GFP construct (F). nc –