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First published online 30 June 2004
doi: 10.1242/dev.01231

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CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis

Hailong An^1,*, Clotilde Roussot^1,*, Paula Suárez-López^1,, Laurent Corbesier¹, Coral Vincent¹, Manuel Piñeiro^1,, Shelley Hepworth¹, Aidyn Mouradov¹, Samuel Justin², Colin Turnbull² and George Coupland^1,

¹ Max Planck Institute for Plant Breeding, Carl von Linne Weg 10, D-50829 Cologne, Germany
² Department of Agricultural Sciences, Imperial College London, Wye Campus, Wye, Kent TN25 5AH, UK

View larger version (105K): [in a new window]   Fig. 1. CO expression pattern in CO::GUS transgenic plants. Histochemical localisation of GUS activity in CO::GUS Landsberg erecta (Ler) plants grown in 16-hour LDs. (A) 12-day-old seedling grown on MS medium. (B) Shoot apex section of an 11-day-old seedling grown on soil. (C) Transverse section of the inflorescence stem of a 38-day-old plant grown on soil. (D) Mature leaf of a 30-day-old seedling grown on soil. (E) Transverse section of an adult leaf. P, phloem; pX, protoxylem; X, xylem. Scale bars: 50 µm.

View larger version (40K): [in a new window]   Fig. 2. CO somatic sectors created by Cre/lox-mediated excision of 35S::GUS from the CO intron. (A) Excision of 35S::GUS from CO restores early flowering on co-2 mutants. (Left) Wild-type plant. (Middle) Plant homozygous for co-2 and CO(35S::GUS) exposed to heat shock flowers late. (Right) Plant homozygous for co-2, HS::CRE and CO(35S::GUS) exposed to heat shock flowers early. This correlates with excision of the 35S::GUS gene as detected by PCR (data not shown). (B) Comparison of flowering times of progenitor co-2 CO(35S::GUS) lines with those of wild-type and co-2 mutant plants. The transgenic progenitor lines do not carry HS::CRE and flower at the same time as co-2 mutants. Heat shock does not affect their flowering time. In the absence of exposure to heat shock, plants homozygous for co-2 CO (35S::GUS) HS::CRE, such as line 42(8)9, also flower at the same time as co-2 mutants. (C) GUS staining illustrates patterns of GUS-negative sectors obtained in plants homozygous for co-2 HS::CRE and heterozygous for CO(35S::GUS) heat shocked 7 days after pollination. Two cotyledons and five first leaves are stained from 18 different plants exposed to heat shock. Different sector patterns indicate excision of the 35S::GUS marker at different times during shoot development. (D) Effects on flowering time of heat shocking co-2 CO(35S::GUS) HS:CRE plants 7 days after pollination. The flowering time of heat-shocked plants is intermediate between that of wild type and co-2 mutants. Flowering time is measured as leaf number. Rosette and cauline leaf number is shown separately. (E) Flowering times of the progeny of two co-2 CO(35S::GUS) HS:CRE plants that were heat shocked either as imbibed seeds (Plant 1) or 10 days after germination (Plant 2). The progeny were harvested from individual inflorescences, and scored as early (similar to wild type) or late (similar to the co-2 mutant). Plants (n=25-40) were scored from each inflorescence, and the proportion of plants scored as early flowering from each inflorescence is shown. Branch 1 of Plant 1, and the main shoot and branch 2 of Plant 2, showed no early flowering plants.

View larger version (39K): [in a new window]   Fig. 3. Analysis of photoperiod response by grafting. (A) Transport of phloem-mobile 14C-sucrose across graft union. Tissues were harvested 2 hours after feeding 14C-sucrose (1 µCi, 1.5 nmol) to leaf on graft partner (donor). Graph shows the proportion of the mobile fraction recovered on the other side of the graft union. Defoliation of the receiver shoot was expected to increase the transfer of photosynthate to the receiver shoot, and a significant effect of defoliation is observed (P=0.05 by t-test). (B) Transmission of a photoperiod stimulus across a graft union. Y-grafted Col wild-type plants grown in 8-hour SDs for 70 days were transferred to 16-hour LDs for 7 days. During this time one of the shoots, the SD receptor, was partially defoliated and covered for part of the day so that it was only exposed to SDs. After the 7 days in LDs, the grafted plants were returned to SDs. Flowering was scored 17 days after the start of LD treatment. Disconnected Y-graft plant pairs were treated exactly as grafted except the graft union was severed. Under these conditions, none of the plants exposed only to SDs flowered. (C) Photograph of Y-grafted co-2 mutant and wild-type plants. Developing flower buds on co-2 shoot (right) grafted to Columbia-5 (left) under LD (27 days). (D) Flowering-time of grafted plants. Y-grafts were assembled on 4- to 5-day-old seedlings. The co-2 mutant grafted to the wild-type plants flowered earlier after producing fewer leaves than the co mutant control (P<0.001 for acceleration of flowering in co grafts versus co controls). Plants were held under 16-hour LD (n=9-16). Bars are mean±s.e.

View larger version (85K): [in a new window]   Fig. 4. Misexpression of CO from heterologous promoters. (A) Histochemical localisation of GUS activity in longitudinal sections of promoter::GUS transgenic plants. Shoot apex and cotyledon section of 9-day-old AtSUC2::GUS Ler plant showing phloem-specific expression (pSUC2). Shoot apex and cotyledon section of an 11-day-old F1 plant of CLV1::LhG4xOp::GUS-Op::CO co-2 showing expression in the meristem and phloem (pCLV1). Shoot apex section of an 11-day-old F1 plant of ANT::LhG4xOp::GUS-Op::CO co-2 showing expression in leaf primordia (pANT). Shoot apex section of 9-day-old KNAT1::GUS Ler plant showing expression in meristem (pKNAT). (B) Phenotype of LD-grown co-2 plants carrying transgenic constructs driving CO expression in specific domains. (C) Flowering time of co-2 transgenic plants in which CO is expressed from tissue-specific promoters. Plants were grown either in LDs or in SDs. The minus sign indicates that an experiment was not conducted under SDs. (D) In situ hybridisation of sections of KNAT1::CO co-2 (left) and co-2 mutant (right) plants probed with CO. Arrowheads in D indicate SAM. Scale bars: 100 µm.

View larger version (77K): [in a new window]   Fig. 5. Analysis of CO function in the phloem by in situ hybridisation and confocal microscopy of GFP:CO fusion proteins. (A) RT-PCR analysis of CO and FT mRNA abundance in emerging leaves of Ler, co-2, 35S::CO Ler and SUC2::CO co-2 plants. (B) In situ hybridisation of CO and FT expression in the leaf vasculature of plants grown in LDs (10-hours light/6-hour day extension/8-hours dark). For SUC2::CO co-2 transverse sections are also shown. Scale bar: 25 µm. (C) Confocal images of GFP fluorescence in whole leaf (a,b; using a 5x lens) and leaf epidermis (c; 40x lens) of SUC2::GFP plants; in epidermal cells (d, 40x lens) and vascular tissues (g,h; 63x oil immersion lens) of SUC2::GFP:CO plants; and in vascular tissues of CO::GFP:CO plants (e,f; 63x oil immersion lens). The GFP fluorescence channel is overlaid with red and the transmissible light channels in a,d,e and g. GFP emission fingerprinting is shown in b,f and h. Plants were grown on MS plate in LDs. (D) Confocal image of the apex of a SUC2::GFP:CO plant (using a 10x lens). GFP fluorescence is detected in the vascular tissue (a), but not in the meristem (b).

View larger version (30K): [in a new window]   Fig. 6. Flowering time of co-2 transgenic plants in which FT is expressed in specific tissues, and of SUC2::CO co-2 ft-7 plants. Plants were grown in LDs or in SDs on soil. The minus sign indicates that an experiment was not conducted under SDs.