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Keywords:

  • Actinidia ;
  • CENTRORADIALIS ;
  • flowering;
  • FLOWERING LOCUS D ;
  • FLOWERING LOCUS T ;
  • winter chilling

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • FLOWERING LOCUS T (FT) and CENTRORADIALIS (CEN) homologs have been implicated in regulation of growth, determinacy and flowering.
  • The roles of kiwifruit FT and CEN were explored using a combination of expression analysis, protein interactions, response to temperature in high-chill and low-chill kiwifruit cultivars and ectopic expression in Arabidopsis and Actinidia.
  • The expression and activity of FT was opposite from that of CEN and incorporated an interaction with a FLOWERING LOCUS D (FD)-like bZIP transcription factor. Accumulation of FT transcript was associated with plant maturity and particular stages of leaf, flower and fruit development, but could be detected irrespective of the flowering process and failed to induce precocious flowering in transgenic kiwifruit. Instead, transgenic plants demonstrated reduced growth and survival rate. Accumulation of FT transcript was detected in dormant buds and stem in response to winter chilling. In contrast, FD in buds was reduced by exposure to cold. CEN transcript accumulated in developing latent buds, but declined before the onset of dormancy and delayed flowering when ectopically expressed in kiwifruit.
  • Our results suggest roles for FT, CEN and FD in integration of developmental and environmental cues that affect dormancy, budbreak and flowering in kiwifruit.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plants have evolved mechanisms tointegrate environmental and developmental cues and precisely control the timing of vegetative and reproductive growth. In Arabidopsis thaliana (Arabidopsis), flowering is initiated by multiple pathways (Amasino, 2010) that converge on a small number of integrator genes, including FLOWERING LOCUS T (FT; Kardailsky et al., 1999; Kobayashi et al., 1999), which encodes a major flowering hormone ‘florigen’ (Chailakhyan, 1968; Zeevaart, 2008). The current model has FT protein transported in the phloem from leaves to the shoot apex (Turck et al., 2008), where it interacts with the bZIP transcription factor FLOWERING LOCUS D (FD), to activate floral meristem identity genes APETALA1 (AP1) and LEAFY (LFY; Abe et al., 2005; Wigge et al., 2005) and SUPPRESSOR OF OVER EXPRESSION OF CONSTANS1 (SOC1; Searle et al., 2006). An endoplasmic reticulum membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1), facilitates FT protein transport (Liu et al., 2012) and 14-3-3 proteins act as intracellular receptors for FT (Taoka et al., 2011). FT-like genes are universally conserved in flowering plants and were demonstrated to perform a role of florigen in plants other than Arabidopsis, including tomato (Lifschitz et al., 2006), squash (Lin et al., 2007) and rice (Tamaki et al., 2007; Komiya et al., 2009; Taoka et al., 2011).

The FT protein is a member of a family of phosphatidylethanolamine-binding proteins (PEBP), initially identified in animals as Raf-1 kinase inhibitors (Yeung et al., 1999). In angiosperms, this family consists of three phylogenetically distinct groups: the FT-like proteins, the TERMINAL FLOWER1 (TFL1)-like proteins (Bradley et al., 1997; Ohshima et al., 1997), and the MOTHER OF FT AND TFL1 (MFT)-like proteins (Mimida et al., 2001; Yoo et al., 2004). The MFT-like genes are the likely basal clade found in angiosperms, gymnosperms, lycophytes and bryophytes, and the ancestor of FT/TFL1-like genes that have diverged in seed plants (Hedman et al., 2009; Karlgren et al., 2011). TFL1 is a key repressor of flowering, which maintains the inflorescence meristem by preventing expression of AP1 and LFY (Ratcliffe et al., 1998, 1999). Further duplication events gave rise to multiple genes within these groups. For example, Arabidopsis has two FT-like genes, FT and TWIN SISTER OF FT (TSF; Yamaguchi et al., 2005), while TFL1 is paralogous to ATC, the Arabidopsis thaliana CENTRORADIALIS (CEN) homologue. A divergent external loop confers antagonistic activity on FT and TFL1 and a single amino acid change is sufficient to convert TFL1 to an activator of flowering (Hanzawa et al., 2005; Ahn et al., 2006). Similarly, two FT homologs from sugar beet perform antagonistic functions, one promoting flowering and the other repressing flowering owing to a mutation in the protein external loop (Pin et al., 2010); antagonistic FT-like paralogs were also recently described in tobacco (Harig et al., 2012).

Studies in species with sympodial growth suggested a more general role for FT-like genes in systemic regulation of growth and termination of meristems (Lifschitz & Eshed, 2006; Lifschitz et al., 2006; Shalit et al., 2009), and implicated the tomato FT homolog SINGLE FLOWER TRUSS (SFT) in heterosis for fruit yield (Krieger et al., 2010).

Unlike annual plants, woody perennials undergo successive growing seasons with vegetative growth before transition to reproductive maturity, followed by cycles of coordinated vegetative and reproductive growth. In temperate regions, these cycles are interrupted by dormancy periods, which ensure survival in unfavourable conditions. Accumulation of chilling during dormancy is often necessary to ensure budbreak and flowering in spring. Studies on FT- and TFL1-like genes in Populus spp. began to shed light on possible mechanisms underlying the regulation of vegetative to reproductive transition, growth and dormancy cycles and the coexistence of vegetative and floral meristems on the same shoot (Böhlenius et al., 2006; Hsu et al., 2006, 2011; Mohamed et al., 2010; Rinne et al., 2011). Two FT genes, FT1 and FT2, have functionally diverged to regulate reproductive onset and vegetative growth, respectively (Hsu et al., 2011). CEN/TFL1 genes were implicated in regulation of maturity, axillary meristem identity and release from dormancy (Mohamed et al., 2010). Similarly, apple has two FT-like genes with differential expression patterns and potentially distinct roles (Kotoda et al., 2010) capable of interacting with transcription factors implicated in cell growth and leaf and fruit development (Mimida et al., 2011). Ectopic expression of apple FT and downregulation of TFL1 both accelerated flowering (Kotoda et al., 2006, 2010). Precocious flowering was also observed upon ectopic expression of a citrus FT homolog in trifoliate orange (Endo et al., 2005). In grape, based on expression irrespective of flowering, VvFT might have a role other than flowering control (Sreekantan & Thomas, 2006; Carmona et al., 2007). From the knowledge gathered to date, it seems that the FT/TFL1 module provides potential to fine-tune developmental regulatory mechanisms and opportunities for improvement of agriculturally important traits (Jung & Müller, 2009; Zhang et al., 2010; Yeoh et al., 2011; Iwata et al., 2012).

The aim of this study was to extend the analysis of FT/TFL1-like genes in woody perennial vines. Kiwifruit (Actinidia spp.) are perennial vines with horticultural importance and features of development specific to woody perennials, including a juvenile period before establishment of flowering competence (Ferguson, 1990), growth spread over two seasons (Brundell, 1975a,b; Walton et al., 1997) and a period of low temperatures (winter chilling) required to resume bud growth (Brundell, 1976). The most important commercial kiwifruit cultivars belong to Actinidia chinensis and Actinidia deliciosa, two closely related species with differences in winter-chilling requirements (Wall et al., 2008). It is unclear if photoperiod plays any role in kiwifruit flowering (Snelgar et al., 2007) and there is discrepancy in reports on the timing of floral commitment in latent shoot buds (Fabbri et al., 1992; Snelgar & Manson, 1992; Snowball, 1996; Walton et al., 1997, 2001). These buds are established in the first growing season and contain undifferentiated, dome-shaped axillary meristems with a potential to differentiate into flowers after the winter dormancy period (Walton et al., 1997). The timing of establishment of dome-shaped meristems (Walton et al., 1997), combined with expression of a LEAFY homolog (Walton et al., 2001) and low levels of accumulation of flower-specific MADS-box genes PISTILLATA and AGAMOUS (Varkonyi-Gasic et al., 2011), support the view that floral commitment occurs in the spring of the first year, followed by differentiation in the spring of the second year.

In this study we characterize a kiwifruit FT and a CEN gene by expression analysis and ectopic transgenic analysis. We demonstrate interaction with a FD-like bZIP transcription factor, which is functionally conserved in Arabidopsis and is downregulated in dormant buds in response to cold treatment when FT is induced in response to winter chilling. This work highlights the conservation and divergence in strategies perennial plants employ to regulate their flowering, growth and dormancy cycles and adds to better understanding of the range of FT/TFL1-like gene action in woody perennials.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials

Kiwifruit plant material was collected from two female cultivars, ‘Hayward’ (A. deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) and ‘Hort16A’ (A. chinensis Planch.). Plants were grown in the orchard under natural conditions and according to standard practice. Tissue was collected from at least three individual plants and whenever possible, sampling was performed at midday to avoid potential variations.

Sampling for gene expression analysis of root, stem, leaf, flower, fruit and bud tissue, axillary buds in field-grown plants during the season and early stages of shoot development was described previously (Walton et al., 2001; Varkonyi-Gasic et al., 2011; Wu et al., 2012). Fruit sampling was performed as per Richardson et al. (2011). Additional samples were collected from ‘Hort16A’ and ‘Hayward’ plants grown in the Plant & Food Research orchard near Te Puke, New Zealand. ‘Hort16A’ flower and latent bud samples were collected in spring 2010 and leaf and flower samples were collected during the spring and summer season of 2006–2007.

To test gene expression upon exposure to cold, ‘Hort16A’ and ‘Hayward’ woody shoots (canes) were collected in autumn 2007 and subjected to a range of treatments. In the first experiment, the canes were defoliated and exposed to 4°C and 0.5°C for up to 14 d in continuous dark before bud dissection. A minimum of five canes were used per each treatment and each cane contained a minimum of eight buds. A subset of canes was used to monitor budbreak. In the second experiment, the lower sides of ‘Hort16A’ canes containing leaves were immersed in water and exposed to room temperature or 4°C in long day (16 h light), short day (8 h light) and continuous light (24 h light) conditions for 7 d before bud dissection. In an additional experiment, ‘Hort16A’ and ‘Hayward’ canes were collected in autumn 2008 and exposed to 0.5°C for up to 35 d, before tissue sampling. A subset of canes was removed after 7 d and tissues enriched in periderm, phloem and xylem were obtained by peeling off the stem layers. A subset of canes was removed after 21 d and maintained at room temperature for 7 d before phloem tissue sampling. A minimum of five canes were used per treatment and each cane contained a minimum of eight buds. Additional biological replicate samples were collected in spring 2012 from ‘Hort16A’ plants grown in the Plant & Food Research glasshouse in Auckland, New Zealand. The plants were maintained at ambient temperature or exposed to 6°C for 8 wk. In addition, canes were excised and exposed to 4°C and 0.5°C for 1 wk and 2 wk. Three canes from separate plants were used per each treatment and a single bud or stem internode sample from each cane was collected.

Budbreak assay on excised canes

Measurement of winter chilling requirement and subsequent budbreak can be performed on excised canes (Snelgar et al., 1997; Snowball, 1997). Actinidia chinensis ‘Hort16A’ and A. deliciosa ‘Hayward’ canes exposed to 4°C and 0.5°C for up 14 d were dissected into cuttings. Ten cuttings per treatment were immersed in water and exposed to room temperature and continuous light to monitor the time of visible budbreak. The number of days required for all ‘Hort16A’ cuttings to break buds was recorded as 100% budbreak; the number of days required for budbreak in at least one cutting was also recorded; the difference represents the measure of synchronization of budbreak. Budbreak on ‘Hayward’ cuttings was less efficient and some failed to break buds, particularly if exposed to cold for a short period of time. For that reason, 50% budbreak was measured.

Phylogeny

Predicted protein sequence alignment was performed using Vector NTI version 9.0.0 (Invitrogen) clustal w (opening 15, extension penalty 0.3). Phylogenetic analyses were conducted using mega version 3.1 (Kumar et al., 2008) using a minimum evolution phylogeny test and 1000 bootstrap replicates.

Gene isolation and vector construction

The AcFT coding sequence was amplified from A. chinensis bud cDNA, cloned into pGEM-T Easy (Promega, Madison, WI, USA) and subcloned as a SpeI–XhoI fragment into corresponding sites of pSAK778 binary vector (Drummond et al., 2009). A clone containing the full-length cDNA of AdCEN was obtained from Plant & Food Research Actinidia expressed sequences tag (EST) library (Crowhurst et al., 2008) and cloned as a SpeI–XhoI fragment into corresponding sites of pSAK778 binary vector. The AcFD coding sequence was amplified from A. chinensis bud cDNA, introduced into pDONR221 (Invitrogen) and transferred to pHEX2 (Hellens et al., 2005). Construction of these plant transformation vectors placed each cDNA between the CaMV 35S promoter and the ocs 3′ transcriptional terminator. The pSAK778 35S–AcFT construct was digested with SacI–SpeI to remove the 35S promoter fragment. Subsequently, the SUC2 promoter amplified from pAF12 (Stadler et al., 2005) was cloned as a SacI–SpeI fragment. The resulting plasmids were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. The primer sequences used for gene amplification are presented in the Supporting Information, Table S1.

Plant transformation and growth of transgenic plants

Agrobacterium tumefaciens-mediated Arabidopsis transformation was performed as described previously (Clough & Bent, 1998; Martinez-Trujillo et al., 2004). Seeds of transgenic plants were selected on half-strength Murashige and Skoog (MS) medium supplemented with kanamycin and placed in a growth room under a long-day (21°C, 16/8 h light/dark) or short-day regime (21°C, 8/16 h light/dark). Actinidia eriantha transformation was performed according to Wang et al. (2006). Transgenic and control lines were established and maintained in a glasshouse under ambient light and temperature conditions for 18 months, exposed to 8 wk of chilling at 6°C and transferred to the glasshouse in spring.

RNA extraction and expression studies

Total RNA was isolated from Arabidopsis seedlings using the Trizol reagent (Invitrogen) and from kiwifruit tissue, as described by Chang et al. (1993). Reverse transcription (RT) was performed using RNA treated with DNase I (Invitrogen), an oligo(dT) primer and the superscript III reverse transcriptase (Invitrogen). Quantifications using real-time PCR were performed with the FastStart DNA Master SYBR Green I mix (Roche Diagnostics) using the LightCycler 1.5 instrument and the LightCycler Software version 4 (Roche Diagnostics). Oligonucleotide primers (Table S1) were designed to produce amplification products of 100–150 nucleotides. The specificity of primer pairs was confirmed by melting curve analysis of PCR products and agarose gel electrophoresis followed by sequence analysis. Products were quantified against the standard curve using dilutions of a sample with the highest expression and the expression was normalized to kiwifruit ACTIN (GenBank accession number FG403300), chosen as a reference gene based on low variability of expression, good stability index values and similar expression patterns to those detected with additional housekeeping genes (Wu et al., 2012). Arabidopsis ACT2 (At3 g18780) was used as a reference for transgenic Arabidopsis studies. Error bars shown in the quantitative PCR data represent the means ± standard error (SE) of three replicate PCR reactions.

Yeast two-hybrid assays

Full-length coding sequences of Arabidopsis and Actinidia genes were amplified using a two-step Gateway PCR with gene-specific and adapter oligonucleotides (Table S1), cloned into pDONR221 and subcloned into pDEST32 (pBDGAL4, bait) and pDEST22 (pADGAL4, prey; Invitrogen). Bait and prey constructs were transformed into yeast strains PJ69-4α and PJ69-4a, respectively (James et al., 1996), for selection on minimal media lacking Leu (bait) or Trp (prey), followed by mating on YPAD plates and selection on minimal media lacking both Leu and Trp. The screening was performed on media lacking Trp, Leu and His and supplemented with 0, 1, 5, 25 or 100 mM 3-amino-1,2,4-triazole. Plates were incubated for 4 d at 20°C and scored for growth. The screening was performed in duplicate.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Identification of FT- and TFL1-like genes from kiwifruit

We mined the Actinidia EST database (Crowhurst et al., 2008) for sequences with homology to Arabidopsis FT and TFL1. An A. chinensis fruit cDNA and an A. deliciosa shoot bud cDNA were identified and designated AcFT and AdCEN (Table S2). Further amplification and sequencing were deployed to obtain the full-length A. chinensis and A. deliciosa FT and CEN cDNAs and the A. chinensis genomic sequences. Near-identical FT and identical CEN cDNA sequences were obtained from both Actinidia species. They will be referred to as kiwifruit FT and kiwifruit CEN (GenBank accession numbers JX417423 and JX417424). High similarity and conservation of sequence in amino acid positions critical for function (Fig. 1a) combined with the similar genomic structure to Arabidopsis FT- and TFL1-like genes (Fig. 1b) and phylogenetic analysis (Fig. 1c) confirmed that the genes identified belong to appropriate PEBP gene families.

image

Figure 1. Kiwifruit FT and CEN genes. (a) Amino acid alignment of Arabidopsis FT, TSF, TFL1 and ATC proteins and predicted kiwifruit FT and CEN proteins. Amino acid positions critical for function are indicated with arrows. The two-letter prefix denotes the species where the sequence was identified initially; Ac, Actinidia chinensis; Ad, Actinidia deliciosa. (b) Kiwifruit FT and CEN gene structure compared with Arabidopsis PEBP family. (c) Phylogenetic tree of kiwifruit FT and CEN (arrows) and FT- and TFL1-like predicted proteins from other plant species, constructed using a minimum evolution phylogeny test and 1000 bootstrap replicates.

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Further interrogation identified additional TFL1-like and MFT-like genes (Table S2), but only a single FT gene was present. Amplification with degenerate oligonucleotide primers and rapid amplification of 3′ ends (3′RACE) in vegetative and reproductive tissues, combined with amplification of the genomic region and promoter sequence (data not shown), all suggested the presence of a single FT gene.

Kiwifruit FT and CEN regulate flowering time in transgenic Arabidopsis

To establish if kiwifruit FT and CEN perform a role in flowering, their cDNAs driven by the cauliflower mosaic virus (CaMV) 35S promoter were ectopically expressed in Arabidopsis Col-0 plants. FT promoted floral transition both in inductive long-day and non-inductive short-day conditions (Fig. 2a,b). Ectopic expression of CEN delayed bolting in inductive conditions and affected flower development (Fig. 2c).

image

Figure 2. Conservation of kiwifruit FT and CEN function in Arabidopsis. (a) Flowering time was recorded as number of leaves produced by the primary inflorescence stem after bolting. Progeny of three 35S-AcFT transgenic lines were chosen for analysis. Error bars represent standard deviation for eight individual plants. LD, long day; SD, short day. (b) Example of early flowering in short-day conditions and expression analysis of 35S-AcFT primary transgenic plants. (c) Flowering was repressed in 35S-AdCEN transgenic plants. Four primary transgenic lines were chosen for analysis, including three with severe delay in inflorescence stem development and one with a weak phenotype. Line #1 failed to produce flowers and abnormal flowers were produced on lines #2 and #3. (d) Expression of kiwifruit FT under the constitutive 35S promoter or vascular SUC2 promoter could substitute for the lack of endogenous FT in the ft-l mutant. Flowering time was recorded as number of leaves produced by the primary inflorescence stem after bolting. Progeny of one ft-1 35S-AcFT and three ft-1 SUC2-AcFT transgenic lines were chosen for analysis. Error bars represent standard deviation for eight individual plants. (e) Phenotypes and expression analysis of one ft-1 35S-AcFT and three ft-1 SUC2-AcFT primary transgenic plants. Transgene expression was normalized against ACT2; error bars represent SE for three replicate reactions (b, c, e).

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To establish if kiwifruit FT is functionally conserved and active in the vascular tissue, a complementation study of the late-flowering Arabidopsis ft-1 mutant was performed. Kiwifruit FT was able to substitute for the lack of endogenous FT in Arabidopsis when expressed under the control of both the constitutive 35S promoter and the vascular-specific SUCROSE TRANSPORTER 2 (SUC2) promoter (Fig. 2d,e).

Kiwifruit FT and CEN interact with a kiwifruit FD-like protein

FT interacts with transcription factors, including the bZIP transcription factor FD in the shoot apical meristem, which is essential to initiate floral meristem development (Abe et al., 2005; Wigge et al., 2005). Interrogation of the Actinidia EST database identified a sequence in shoot buds encoding a predicted protein with homology to Arabidopsis FD and FD PARALOG (FDP), with the conserved bZIP domain and a C-terminal SAP (Ser-Ala-Pro) motif (Fig. 3a). Ectopic expression driven by the 35S promoter was sufficient to restore the delayed flowering of the fd-1 Arabidopsis mutant (Fig. 3b) and the gene was designated kiwifruit FD (GenBank accession number JX417425). Yeast two-hybrid assays were used to test the interactions between kiwifruit FT, CEN and FD. Arabidopsis FT, TSF, TFL1, FD and FDP with well-documented interaction patterns were used as controls (Abe et al., 2005; Wigge et al., 2005; Jang et al., 2009; Hanano & Goto, 2011). It was found that FD, FDP and kiwifruit FD were capable of homodimerization and heterodimerization and interacted with FT, TSF and kiwifruit FT. Kiwifruit CEN interacted weakly with FD and FDP and a moderate interaction was demonstrated with kiwifruit FD (Table 1).

Table 1. Kiwifruit and Arabidopsis protein interactions detected by yeast two-hybrid assays
pBD (bait)pAD (prey)mM 3AT
pDEST22FTTSF TFL1 FD FDP AcFT AdCEN AcFD
  1. ++, very strong interaction; +, strong interaction; +/−, weak interaction; −, no interaction. Kiwifruit FD bait (in grey) was excluded from analysis because of strong auto-activation. 3AT = 3-amino-1,2,4-triazole.

pDEST320
FT+++0
TSF+++0
TFL10
FD+/−+/−+/−+/−+/−+5
FDP+/−+/−+/−+/−+25
AcFT++++++1
AdCEN+/−+0
AcFD++++++++++++100
image

Figure 3. Conservation of kiwifruit FD. (a) Amino acid alignment of Arabidopsis FD, FDP and a predicted kiwifruit FD protein. The bZIP domain and the SAP motif implicated in the interaction with FT are underlined. (b) Expression of kiwifruit FD under the constitutive CaMV 35S promoter could substitute for the lack of endogenous FD in the fd-l mutant. Three primary transgenic lines were chosen for analysis. The expression was normalized against ACT2. Error bars represent SE for three replicate reactions.

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Kiwifruit FT and CEN are differentially expressed

To gain further insight into the role of kiwifruit FT and CEN, their expression in kiwifruit vegetative and reproductive organs and at various stages of bud development were interrogated by reverse transcription quantitative PCR (RT-qPCR; Fig. 4). The FT transcript accumulated in the leaf, flower and fruit. It was detectable in the stem, but could not be detected in the root or terminal vegetative buds. The overall levels of accumulation were very low (Fig. 4a). The kiwifruit CEN transcript was detected in vegetative terminal buds and in the roots and accumulation was higher than that of FT (Fig. 4b). Both transcripts were barely detectable in the axillary bud sample collected before dormancy.

image

Figure 4. Opposite patterns of kiwifruit FT and CEN expression. (a,b) Relative expression of kiwifruit FT and CEN in Actinidia chinensis ‘Hort16A’ (open rectangles) and Actinidia deliciosa ‘Hayward’ (closed rectangles); R, root; S, stem; L, leaf; F, flower; Fr, fruit; TB, terminal buds; AB, axillary buds. The expression of each gene was normalized against ACTIN (ACT). (c) Relative expression of kiwifruit CEN, FT, CDKB1, SEP4 and PI in A. deliciosa ‘Hayward’ axillary buds during the growth and dormancy cycle. The shading indicates the period where there was no visible growth. The schematic below indicates the position of collected buds pre-dormancy and during dormancy; sampling post-dormancy represents the new developing shoot. F, floral buds; L, latent buds. The expression of each gene was normalized against ACTIN and expressed as fold upregulation to one of the time-points. Error bars represent SE for three replicate reactions. (d,e) Actinidia chinensis ‘Hort16A’ early shoot and flower development from dormant bud (stage 1) to visible floral bud (stage 5). The stages were described previously (Polito & Grant, 1984; Varkonyi-Gasic et al., 2011). (f,g) Relative expression of kiwifruit FT and CEN during Achinensis ‘Hort16A’ shoot bud dormancy (stage 1) and early shoot and flower development (stages 2–5) (h,i) Relative expression of kiwifruit FT and CEN in basal (floral) and distal (latent) buds of Achinensis ‘Hort16A’ elongating shoots. The expression of each gene was normalized against ACTIN. Error bars represent SE for three replicate reactions.

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Kiwifruit axillary buds represent sites of vegetative, inflorescence and floral initiation (Fig. S1) and were further analysed in samples collected at regular intervals over the period of 1 yr. Growth and floral differentiation was monitored through expression of the cell cycle gene CDKB1 (Walton et al., 2009), the floral meristem and floral organ identity gene SEPALLATA4 (SEP4) and the floral organ identity gene PISTILLATA (PI; Varkonyi-Gasic et al., 2011). The accumulation of CEN transcript was highest in latent buds during summer, but declined in autumn before the establishment of dormancy. Accumulation of FT transcript was first detected in dormant buds in winter and later after resumption of growth and flower differentiation. The initial accumulation of FT transcript preceded accumulation of transcripts associated with growth, floral meristem and floral organ development (Fig. 4c) and occurred after a period with daily minimal temperatures close to or below freezing (Fig. S2).

Next, a more detailed analysis in dormant buds and during early stages of shoot emergence was performed, using excised woody shoots (canes) exposed to cold. In this manner, the fluctuations in field conditions were minimized, budbreak and shoot growth were synchronized and the sampling was done at shorter intervals coinciding with well-defined stages (Fig. 4d,e) described previously (Polito & Grant, 1984; Varkonyi-Gasic et al., 2011). Both transcripts were detected in shoots emerging from axillary buds post dormancy, with opposing expression patterns, significant accumulation of FT transcript in dormant buds and low relative expression of CEN (Fig. 4f,g). With further growth, expression of CEN was confined to latent buds and FT transcript accumulated in developing floral buds (Fig. 4h,i).

Expression of FT is highly dynamic, developmentally regulated and not clearly associated with flowering

Differential rates of accumulation of FT transcript in axillary buds suggested a highly dynamic expression correlating with developmental and seasonal changes. To establish if the expression of FT in other organs also varied with developmental stages and seasons, leaf, flower and fruit samples were collected and analysed by RT-qPCR. Sampling was performed at midday and from fully exposed leaves to minimize potential variation (Fig. S3). The FT transcript was barely detectable in seedling leaves, but accumulated in large leaves collected from the basal end of the shoot of mature plants in spring, when flowering occurred. Accumulation decreased significantly in basal leaves of mature plants in summer, during fruit development (Fig. 5a).

image

Figure 5. Seasonal and developmental regulation of FT expression. (a). Relative expression of kiwifruit FT in Achinensis ‘Hort16A’ basal leaves of seedlings and mature plants in spring (November) and summer (January). (b) Relative expression of kiwifruit FT in basal and distal leaves of an Achinensis ‘Hort16A’ growing shoot. A cell-cycle gene CDKB1 was used to monitor cell divisions and the size of basal and distal leaves is indicated below. (c) Relative expression of kiwifruit FT in basal and distal leaves of Achinensis ‘Hort16A’ and Actinidia deliciosa ‘Hayward’ floral (F) and vegetative (V) shoots. (d) Relative expression of kiwifruit FT during Achinensis ‘Hort16A’ flower development. The sampling was performed in the spring months and corresponds to flower development stages 8, 9 and 10 (Brundell, 1975a). (e) Relative expression of kiwifruit FT during Achinensis ‘Hort16A’ fruit development as described by Richardson et al. (2011); DAA, days after anthesis. The expression was normalized against ACTIN. Error bars represent SE for three replicate reactions. (f) Schematic summarizing accumulation of FT transcript (blue colouring) during plant maturation and organ development. Accumulation of the transcript is confined to large basal leaves, flowers (small ovals) and fruit (larger circles).

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A separate experiment focused on distinguishing FT expression levels in basal and distal leaves, which subtend developing flowers and latent buds, respectively. Leaf samples were collected at regular intervals during the spring months from mature, flowering plants. No or very low accumulation was detected in small basal leaves collected in September, during early stages of shoot emergence and floral bud development. The accumulation of FT transcript was detected in larger basal leaves in October, coinciding with visible flower development, while the distal leaves showed no, or very low accumulation. The accumulation in basal leaves declined in November, at the time of anthesis, and no clear correlation was identified between the size of the leaf or the growth rate measured by accumulation of CDKB1 (Fig. 5b). Accumulation of FT transcript was detected in large basal leaves of flower-bearing shoots, as well as in shoots without flowers collected in late spring (Fig. 5c).

An increase in the accumulation of FT transcript was also detected during flower and fruit development, followed by decline at anthesis and with fruit ripening (Fig. 5d,e). The dynamic nature of this accumulation of FT transcript during development is summarized in a schematic shown in Fig. 5f.

Expression of FT in axillary buds and the stem is regulated by temperature

Accumulation of FT transcript in dormant buds in the field (Fig. 4c, Fig. S2) and on defoliated excised canes (Fig. 4f) coincided with exposure to low temperatures. To address if whether this accumulation was directly influenced by chilling, excised canes or whole plants of A. chinensis and A. deliciosa cultivars were exposed to different temperatures and gene expression was measured. These species differ in their chilling requirement in the field (Wall et al., 2008) and the same was demonstrated when defoliated excised canes were kept in controlled environments (Fig. 6a,b). The FT transcript accumulated faster at the lower temperature in both cultivars, but the accumulation was more prominent in A. chinensis ‘Hort16A’ (Fig. 6c), which requires less chilling for budbreak than A. deliciosa ‘Hayward’ (Fig. 6b). No expression of CEN, floral meristem or floral organ identity genes was detectable in axillary buds during exposure to 4°C or 0.5°C for up to 14 d (data not shown), while accumulation of FD transcript declined (Fig. 6d). Additional biological replicate samples confirmed these findings (Fig. S4). There was a decline in the accumulation of both FT and FD transcripts in axillary buds upon cold induction in all light conditions (Fig. S5). Furthermore, accumulation of FT transcript was detected in the stem internode (Figs 6e, S4), particularly in the sample enriched for phloem tissues (Fig. 6f). Exposure of kiwifruit organs other than woody stems to chilling had little effect on the accumulation of FT transcript (data not shown) and no accumulation was detected in the seedling stem (Fig. 6f). The level of accumulation declined upon return into ambient temperature (Fig. 6g).

image

Figure 6. Expression of kiwifruit FT in stem tissues is induced by chilling of excised canes. (a) Visible budbreak on an Actinidia chinensis ‘Hort16A’ cutting. (b) Budbreak assay on excised canes exposed to cold. The number of days at room temperature (RT) required for all Achinensis ‘Hort16A’ and half of Actinidia deliciosa ‘Hayward’ cuttings to break buds was recorded as 100% and 50% budbreak, respectively, and plotted (rectangles); the number of days required for budbreak in at least one cutting was also recorded; the difference (bars within rectangles) is the measure of synchronization of budbreak. ‘Hayward’ canes maintained at RT failed to break buds (N/A). (c) Relative expression of kiwifruit FT in a pooled bud sample dissected from ‘Hort16A’ and ‘Hayward’ canes exposed to 4°C and 0.5°C for up to 14 d. (d) Relative expression of kiwifruit FD in the same sample. (e) Relative expression of kiwifruit FT in stem internodes dissected from ‘Hort16A’ and ‘Hayward’ canes exposed to 0.5°C for up to 35 d. (f) Relative expression of kiwifruit FT in tissues dissected from ‘Hort16A’ canes and seedling stems exposed to 0.5°C for 7 d. (g) Relative expression of kiwifruit FT in the stem phloem tissue dissected from ‘Hort16A’ canes maintained at 0.5°C and room temperature (RT) for an indicated number of days (d). The expression was normalized against ACTIN and expressed as fold upregulation to one of the time-points. Error bars represent SE for three replicate reactions performed on a pooled sample (five canes).

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Similarly, FT transcript accumulated in whole plants exposed to extended cold. After 8 wk at 6°C, the accumulations were similar in both cultivars and accumulation in stem internodes exceeded that detected in the buds (Fig. 7a). Expression of a floral meristem identity gene FRUITFUL (FUL) and detection of a floral organ identity gene PI in some samples was indicative of early stages of bud development (Fig. 7b; Varkonyi-Gasic et al., 2011), and visible budbreak followed upon return into the glasshouse conditions.

image

Figure 7. Expression of kiwifruit FT in stem tissues is induced by chilling of whole plants. The plants were maintained at ambient temperature or exposed to 6°C for 8 wk. (a) Relative expression of kiwifruit FT. (b) Relative expression of kiwifruit FD, FUL and PI after chilling. The expression was normalized against ACTIN. Error bars represent SE for three technical replicate reactions.

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Ectopic expression of kiwifruit FT and CEN in A. eriantha

To establish if FT induces flowering in kiwifruit, A. eriantha was transformed with the same 35S–AcFT construct that was used in Arabidopsis. Actinidia eriantha vines are relatively small, have a low requirement for winter chilling and flower prolifically in glasshouse conditions within 2 yr of transformation (Wang et al., 2006). A 35S–GUS construct was used to transform control plants. In the initial screen, eight 35S–AcFT lines were identified with low to moderate levels of transgene expression, but only five survived for the period of 2 yr, when first flowering was observed on two of the lines. The control plants demonstrated vigorous growth and one of them flowered in the same year, but the plants carrying the 35S–AcFT construct failed to grow vigorously and demonstrated reduced secondary growth, resulting in thin stems, of which a large proportion died (Fig. 8a,b). Only a small number of flowers were produced, some with mild abnormalities in floral patterning (Fig. 8c).

image

Figure 8. Ectopic expression of kiwifruit FT and CEN in Actinidia eriantha. (a) Plants carrying the 35S-AcFT construct failed to grow vigorously and demonstrated reduced secondary growth, resulting in thin stems, of which a large proportion died (arrows). (b) Expression of FT transgene was measured in distal leaves in summer, when expression of endogenous FT is low or undetectable. (c) Elongated and misshapen petals of A. eriantha T1 plant. (d) Kiwifruit CEN transgene expression analysis. (e) Latent bud on A. eriantha 35S-AdCEN T12 and flower buds on a 35S–GUS control (C) plant.

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By contrast, transformation of the 35SAdCEN construct into A. eriantha resulted in plants with relatively high levels of transgene expression (Fig. 8d) and normal growth. The establishment of dormancy and timing of budbreak were comparable to controls, but four out of five transgenic plants did not produce flowers after 2 yr. At the same time, the controls demonstrated prolific flowering (Fig. 8e).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

A single FT gene is implicated in diverse roles in perennial vines

FT-like proteins were reported to play a universal role in regulation of flowering time, consistent with the role of the flowering hormone, florigen (Turck et al., 2008). However, identification of multiple FT-like genes in a number of annual and perennial species suggested more general roles in development, and the functional diversification of paralogous genes provided a mechanism for diverse functions. For example, only two out of 13 FT-like genes identified in rice (Chardon & Damerval, 2005) were demonstrated to regulate flowering time (Kojima et al., 2002; Komiya et al., 2008); one out of 15 maize FT-like genes was confirmed as primary floral activator (Meng et al., 2011); two out of 10 soybean FT-like genes were implicated in flowering control (Kong et al., 2010); two out of five FT-like genes described in pea and Medicago truncatula were implicated in flowering (Hecht et al., 2011; Laurie et al., 2011); and the potato floral and tuberization transitions were regulated by two different FT-like paralogues (Navarro et al., 2011). In woody perennials, transient activity of two FT paralogs coordinated repeated cycles of reproductive and vegetative growth in mature poplar (Hsu et al., 2011) and expression of only one of two FT genes in apple and one of three FT genes in citrus appeared to be associated with transition to flowering (Nishikawa et al., 2007; Kotoda et al., 2010). However, only one FT gene could be identified in the peach genomic sequence at the Joint Genome Institute Phytozome database (http://www.phytozome.org/peach), suggesting that a single FT gene may be sufficient for normal plant growth and development.

Only one FT sequence was identified in the kiwifruit EST collection and additional analysis of cDNA and genomic DNA all suggested the presence of a single FT gene and multiple TFL1- and MFT-like genes. Similarly, a single FT gene has been reported for the woody perennial vine grapevine, which, together with an MFT and multiple TFL1 genes, represents the Vitis vinifera FT/TFL1 gene family (Carmona et al., 2007; Jaillon et al., 2007; Velasco et al., 2007). Therefore, one FT gene may be sufficient for development of a woody perennial vine and the diversification in function might require additional factors. The expression pattern of kiwifruit FT in a range of kiwifruit tissues was dynamic, age-dependent and correlated with maturity and termination of development, as described or predicted in other plants, including phase change (Hsu et al., 2006), leaf sink-source transition (Shalit et al., 2009), floral patterning (Xi & Yu, 2009), fruit and seed development (Kobayashi et al., 1999; Yamaguchi et al., 2005) and release from winter dormancy (Hsu et al., 2011; Rinne et al., 2011).

Opposite expression patterns of kiwifruit FT and CEN

Both FT/TFL1 antagonism and the role of TFL1-like genes in floral repression have been well-documented (Shannon & Meeks-Wagner, 1991; Ratcliffe et al., 1998). In tomato, the TFL1 homolog SELF-PRUNING antagonizes the effect of the FT homolog SFT to regulate a broad range of developmental events (Shalit et al., 2009). In a perennial relative of Arabidopsis, Arabis alpina, TFL1 regulates vernalization response and maintains the perennial life cycle (Wang et al., 2011). Similarly, TFL1 is a key regulator of perennial growth and seasonality of flowering in strawberry (Iwata et al., 2012; Koskela et al., 2012). In woody perennials, TFL1-like genes have a crucial role in maturity and meristem identity; downregulation of TFL1-like genes accelerated flowering in apple (Kotoda et al., 2006) and Populus, possibly by lowering the amount of FT required for outcompeting the CEN/TFL1 proteins (Mohamed et al., 2010).

Delayed flowering obtained upon constitutive expression of kiwifruit CEN in Arabidopsis and A. eriantha combined with CEN expression profiles, particularly accumulation in latent buds during the time when primordia are established that can give rise to flowers in the following spring, is consistent with the role of TFL1-like genes in maintenance of meristem indeterminacy (Bradley et al., 1997; Ratcliffe et al., 1998; Conti & Bradley, 2007; Mohamed et al., 2010). Kiwifruit CEN may be necessary to prevent flowering in latent buds and ensure that meristems are maintained to be available for flowering in the following year, as described in A. alpina (Wang et al., 2011). Unlike in A. alpina, where the TFL1 homolog increases the duration of required vernalization, kiwifruit CEN is not expressed in dormant buds and is unlikely to have a function during accumulation of winter chilling. The decline in accumulation of CEN transcript before the establishment of dormancy is also different from the hypothesized role of Populus PopCEN1 in measurement of chilling accumulation (Mohamed et al., 2010).

The opposing FT and CEN expression patterns detected in developing shoots further imply that kiwifruit CEN may have a role in development that is opposite to that of FT. Interaction with a common partner, FD, appears to be a conserved mechanism (Pnueli et al., 2001; Abe et al., 2005; Wigge et al., 2005; Danilevskaya et al., 2010; Hanano & Goto, 2011) and is consistent with a role of CEN in the transcriptional repression of FD-dependent events (Hanano & Goto, 2011). Therefore, the FT/CEN balance in developing kiwifruit shoots may have a regulatory role by incorporation of developmental and environmental signals in an FD-mediated manner.

Is kiwifruit FT a florigen?

Conservation of kiwifruit FT function in Arabidopsis, functionality from the vasculature, interaction with both Arabidopsis and kiwifruit FD proteins and expression in mature plants all suggest a conserved, universal role in regulation of flowering time and imply that FT may function as florigen in kiwifruit.

Transformation of A. eriantha resulted in plants with impaired growth and reduced survival, which is similar to previous reports of growth retardation observed upon overexpression of FT in other species (Lifschitz et al., 2006; Shalit et al., 2009; Kotoda et al., 2010), but none of the surviving plants demonstrated precocious maturity. Therefore, it appears that FT confers termination, but is not sufficient for flower development in kiwifruit, although the small number of transgenic lines may not be sufficient to evaluate the phenotypes fully. The surviving lines had relatively low levels of transgene expression, suggesting that FT might have a detrimental effect on the regeneration or transformation efficiency and survival of transgenic plants. Alternatively, expression of FT might have affected the ability of transgenic plants to recover from exposure to cold, perhaps by slowing general growth and making plants more susceptible to a chilling injury. Attempts to evaluate the function of FT by constitutive expression in transgenic A. chinensis (Wang et al., 2007) failed (data not shown), probably as a result of impaired regeneration competence of the transformed calli upon high levels of expressionof FT, similar to previous reports for apple (Kotoda et al., 2010), and consistent with the role in meristem termination described in tomato (Lifschitz et al., 2006; Shalit et al., 2009). Similarly, transformation of both A. chinensis and A. deliciosa with constitutively expressed Arabidopsis FT was unsuccessful (data not shown).

In addition, accumulation of FT transcript was not clearly associated with the flowering process. Accumulation of this transcript in developing shoots commenced after the initial stages of flower differentiation; the transcript was absent from latent buds at the time when primordia with a potential to develop into flowers in the following season were established (Fig. 4), and it was detected in both floral and vegetative shoots (Fig. 5). It is therefore possible that FT in kiwifruit regulates general growth and termination but may not have a florigenic function, similarly to grapevine, where FT is relatively highly expressed irrespective of flowering (Sreekantan & Thomas, 2006; Carmona et al., 2007).

Conversely, accumulation of FT transcript in basal, source leaves in both flower-bearing shoots and shoots without flowers may provide sufficient FT protein to be translocated into developing latent buds, where floral evocation occurs and dome-shaped meristems, which may give rise to flowers in the following season, are established. Similarly, the accumulation of FT transcript in the stem vasculature during winter chilling could result in accumulation of sufficient FT protein, which could move upon reopening of phloem conduits, as hypothesized for Populus (Rinne et al., 2011), enabling budbreak, shoot elongation and flower differentiation (Fig. 9). The presence of FT could explain why flowers are produced on shoots that develop immediately after winter chilling, during the first flush of spring growth (Grant & Ryugo, 1982), but not on shoots that develop later in spring (Fig. S1). In that case, additional mechanisms may be required to prevent premature bud outgrowth and flower development during chilling, when FT transcript accumulates. Reduced accumulation of FD transcript (Fig. 6) and accumulation of floral repressors including SVP-like genes (Wu et al., 2012) may provide such a mechanism, but further study is required to address these questions and attempt to understand the molecular basis of budbreak and flowering in kiwifruit.

image

Figure 9. Model for FT function in kiwifruit budbreak, flowering and floral commitment in latent buds. Blue colouring denotes accumulation and movement of the FT protein.

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Kiwifruit FT may have a role in dormancy release induced by chilling

Environmental cues are essential in determining growth and flowering time, particularly in seasonally predictable photoperiod and temperature. In trees, timing of dormancy and flowering can be controlled by photoperiod (Böhlenius et al., 2006) and temperature (Hsu et al., 2011), although some species appear to be insensitive to photoperiod. For example, low temperature but not photoperiod regulate both dormancy induction and release in apple, pear (Heide & Prestrud, 2005) and Sorbus species (Heide, 2011). In kiwifruit, dormancy is probably established in response to shortening daylength (Lionakis & Schwabe, 1984), but it is less clear if photoperiod plays any role in flowering (Snelgar et al., 2007). Conversely, temperature has a major effect on kiwifruit budbreak and flowering. Exposure to winter temperatures is essential to enable synchronised budbreak and efficient flowering (Walton et al., 2009).

Exposure of whole plants or excised canes to low temperature also had a major effect on the accumulation of FT transcript in the woody stem. This accumulation was initially faster in A. chinensis ‘Hort16A’ than A. deliciosa ‘Hayward’, correlating with a lower winter chilling requirement and significantly earlier bloom of A. chinensis cultivars than A. deliciosa ‘Hayward’ (Wall et al., 2008). The accumulation of FT transcript in the stem internode was most prominent in the sample enriched with phloem tissues (Fig. 6), which is consistent with a potential mobile signal capacity of FT protein. FT transcript accumulated in the bud itself, although it may reflect accumulation in the stem, because some stem vascular tissue is dissected out with the bud. Experiments in poplar suggested accumulation of FT transcript in the bud leaf primordia (Rinne et al., 2011), and a mechanism where dormancy induces physical isolation of buds by establishment of callose plugs, while chilling reopens signal conduits, thus enabling the movement of FT protein into the meristem. It is possible that a similar mechanism exists in kiwifruit, where both the bud and the stem FT become available for movement (Fig. 9). Accumulation of FT transcript was also demonstrated in citrus stem as a result of floral induction by low temperature (Nishikawa et al., 2007) and this might be a common mechanism used by woody perennial plants.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Rongmei Wu and Peter McAtee for kindly providing kiwifruit cDNA samples, Robyn Lough and Andrew Gleave for cloning and sequencing support, Monica Dragulescu for assistance with transgenic plants, Tim Holmes for photography and Anne Gunson for critically reading the manuscript. The Arabidopsis ft-1 mutant was obtained from the European Arabidopsis Stock Centre, the fd-1 mutant was kindly provided by Robyn Lough and the Arabidopsis SUC2 promoter was kindly provided by Ruth Stadler. The authors declare no conflict of interest. This work was funded by the New Zealand Foundation for Research, Science and Technology grant C10X0816 MeriNET.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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Fig. S1 Schematic diagram of the kiwifruit life cycle.

Fig. S2 Expression of FT in dormant buds.

Fig. S3 Variation in accumulation of FT transcript in Actinidia chinensis ‘Hort16A’ leaves.

Fig. S4 Differential FT and FD accumulation in response to chilling.

Fig. S5 Effect of temperature and photoperiod on accumulation of FT and FD transcripts.

Table S1 Nucleotide sequence of oligonucleotide primers used in this study

Table S2 Actinidia PEBP genes identified in the expressed sequences tag (EST) database