Expression of Flower Promoting Genes in Sweet Orange during Floral Inductive Water Deficits

in Journal of the American Society for Horticultural Science

Cool ambient temperatures (5 to 20 °C) and water deficit are the only factors known to induce flowering in sweet orange (Citrus sinensis). Whereas the effects of cool ambient temperatures on flowering have been described extensively, reports on the mechanisms underlying floral induction by water deficit in sweet orange (and other tropical and subtropical species) are scarce. We report changes in the accumulation of transcripts of four flower-promoting genes, CsFT, CsSL1, CsAP1, and CsLFY, in sweet orange trees in response to water deficit or a combination of water deficit and cool temperatures under controlled conditions. Exposure to water deficit increased the accumulation of CsFT transcripts, whereas transcripts of CsSL1, CsAP1, and CsLFY were reduced. However, when water deficit was interrupted by irrigation, accumulation of CsFT transcripts returned rapidly to pre-treatment levels and accumulation of CsSL1, CsAP1, and CsLFY increased. The accumulation of CsFT transcripts in trees during the combined water deficit and cool temperatures treatment was higher than in trees exposed to either factor separately, and accumulation of CsAP1 and CsLFY transcripts after the combined treatment was also higher. These results suggest that water deficit induces flowering through the upregulation of CsFT and that CsFT is the leaf integrator of flower-inducing signals generated by the exposure to water deficit and cool temperatures in sweet orange.

Abstract

Cool ambient temperatures (5 to 20 °C) and water deficit are the only factors known to induce flowering in sweet orange (Citrus sinensis). Whereas the effects of cool ambient temperatures on flowering have been described extensively, reports on the mechanisms underlying floral induction by water deficit in sweet orange (and other tropical and subtropical species) are scarce. We report changes in the accumulation of transcripts of four flower-promoting genes, CsFT, CsSL1, CsAP1, and CsLFY, in sweet orange trees in response to water deficit or a combination of water deficit and cool temperatures under controlled conditions. Exposure to water deficit increased the accumulation of CsFT transcripts, whereas transcripts of CsSL1, CsAP1, and CsLFY were reduced. However, when water deficit was interrupted by irrigation, accumulation of CsFT transcripts returned rapidly to pre-treatment levels and accumulation of CsSL1, CsAP1, and CsLFY increased. The accumulation of CsFT transcripts in trees during the combined water deficit and cool temperatures treatment was higher than in trees exposed to either factor separately, and accumulation of CsAP1 and CsLFY transcripts after the combined treatment was also higher. These results suggest that water deficit induces flowering through the upregulation of CsFT and that CsFT is the leaf integrator of flower-inducing signals generated by the exposure to water deficit and cool temperatures in sweet orange.

Under natural conditions, citrus (Citrus sp.) trees are induced to flower by seasonal exposure to either cool ambient temperatures (5 to 20 °C) or water deficit (Cassin et al., 1969; Moss, 1969). In subtropical regions where citrus are grown, cool temperatures during the winter induce flowering the next spring, whereas in tropical regions, flowering is induced during the dry season with flowering after the first effective rains of the rainy season (Cassin et al., 1969). For both stimuli (cool temperatures and water deficit), the intensity of the induced flowering depends on both the time of exposure to the inductive stimuli and the intensity at which the stimuli was present (Cassin et al., 1969; Southwick and Davenport, 1986). For cool temperatures, the maximum flowering intensity in sweet orange has been reported to be obtained after 8 weeks of exposure to temperatures between 10 and 15 °C (Moss, 1969). For water deficit, no comparable studies have been reported in sweet orange, but exposure to moderate or severe water deficit for 5 weeks in ‘Tahiti’ lime (Citrus latifolia) produced maximum flowering intensity compared with shorter exposures and milder deficit (Southwick and Davenport, 1986). Under field conditions in a humid subtropical climate, exposure to 60 to 75 d of drought has been related to optimum flowering (Albrigo et al., 2006).

In arabidopsis (Arabidopsis thaliana), several mechanisms for the regulation of seasonal flowering have been identified during the last 15 years (see Amasino, 2010, for a review). In these proposed mechanisms, the genes Flowering locus T (FT), Apetala1 (AP1), Leafy (LFY), and Supressor of overexpression of Constans 1 (SOC1) play a pivotal role either integrating signals from flowering-promoting or flowering-inhibiting stimuli [FT or SOC1 (Abe et al., 2005; Corbesier et al., 2007; Lee and Lee, 2010; Samach et al., 2000)] or initiating the morphogenesis of floral organs in the shoot meristem [AP1 and LFY (Mandel and Yanofsky, 1995; Weigel and Nilsson, 1995)]. In citrus, putative orthologs of these genes have been isolated and characterized (Nishikawa et al., 2007; Pillitteri et al., 2004b; Tan and Swain, 2007). Exposure to cool floral-inductive temperatures increased the expression (transcript accumulation) of CsFT (Nishikawa et al., 2007), which is consistent with the function of FT in arabidopsis as the ultimate flowering-promoting signal (Abe et al., 2005). In contrast, expression of CsAP1 and CsLFY increased only toward the end of a floral-inductive temperature treatment (Pillitteri et al., 2004a), which is consistent with their function in arabidopsis as floral meristem identity genes (Mandel and Yanofsky, 1995; Weigel and Nilsson, 1995) and with earlier reports of the microscopical differentiation of floral meristems in citrus only after floral induction is over (Abbot, 1935). Although the patterns of expression of CsSL1 (the ortholog of arabidopsis SOC1) have not been reported in response to floral inductive stimuli, the conservation of its function in citrus as a floral promoter is supported by the early flowering phenotype of arabidopsis SOC1 mutants constitutively expressing CsSL1 (Tan and Swain, 2007). In arabidopsis, SOC1 integrates signals from the photoperiod, vernalization, gibberellin, and autonomous floral regulatory pathways (Lee and Lee, 2010).

Although CsFT, CsAP1, CsLFY, and CsSL1 are involved in the regulation of flowering in citrus, the specific mechanisms regulating the expression of these genes must have evolved in citrus to respond to stimuli different to those inducing flowering in arabidopsis based on differences in their flowering response. In arabidopsis, plants are induced to flower by the transition from short to long photoperiods and by treatment with gibberellins under non-inductive short days (Blázquez et al., 1998); citrus flowering, however, can be considered mostly photoperiod insensitive (Moss, 1969), and gibberellins inhibit rather than promote flowering (Monselise et al., 1964). Furthermore, whereas in citrus, temperatures between 5 and 20 °C induce flowering (Moss, 1969), in arabidopsis, flowering could be induced by higher (greater than 23 °C) ambient temperatures (Balasubramanian et al., 2006; Blázquez et al., 2003) or enabled by low vernalizing (less than 5 °C) temperatures (see Kim et al., 2009, for a review). In addition, no effects of water deficit as a floral-inductive factor have been reported for arabidopsis, whereas in citrus (and other subtropical and tropical species), water deficit is a key floral inductive factor (Albrigo and Galen-Saúco, 2004).

The objective of this work was to determine the expression patterns of CsFT, CsAP1, CsLFY, and CsSL1 under floral-inductive water deficit and a combination of cool floral-inductive temperatures and water deficit in sweet orange trees; these two conditions are typical of the floral-inductive periods in humid subtropical and tropical climates where citrus is grown.

Materials and Methods

Plant material and experimental conditions.

The experiments were conducted using two- to three-year-old potted (12-L pots) ‘Washington Navel’ sweet orange rooted cuttings (≈1 m tall) with fully matured foliage (no tender shoots). Before the start of the experiments, trees were kept in a greenhouse with a minimum temperature of 23 °C (maximum temperature was 31 °C) and watered every 2 d to saturation. While in this greenhouse, trees were exposed to natural variations in photoperiod (12.5/11.5 to 11/13 h, day/night) and photosynthetic photon flux (PPF) between 1210 and 1340 μmol·m−2·s−1. Tree water status was monitored before and throughout the experiments by measuring the midday stem water potential (ψs) using the pressure chamber method (Scholander et al., 1965) on leaves covered in an aluminized bag for at least 1 h before measurement (McCutchan and Shackel, 1992). The midday stem water potential of the trees before the beginning of the experiment was –1.1 ± 0.10 MPa.

Two experiments were conducted at separate times: the first experiment tested the effect of floral-inductive water deficit only on the expression of CsFT, CsAP1, CsLFY, and CsSL1; whereas the second experiment tested the effect of floral-inductive water deficit and floral-inductive cool temperatures on the expression of the same genes. Both experiments were conducted in temperature-controlled growth rooms illuminated with fluorescent lights (800 μmol·m−2·s−1 PPF at the canopy level) with a 11/13-h (day/night) photoperiod. In the first experiment, trees were exposed to floral-inductive water deficit at a constant non-floral-inductive temperature (23 ± 1 °C) for 60 d, whereas in the second experiment, trees were exposed to floral-inductive water deficit at a constant floral-inductive temperature (12 ± 1 °C) for 40 d. After each treatment was over, trees were transferred back to the greenhouse and irrigation was restored to promote bud sprouting.

Water deficit was imposed by withholding irrigation until ψs reached –2.0 ± 0.12 MPa (moderate water deficit) and then kept at this level by irrigating the trees with a volume of water that matched the daily weight loss of the trees. This level of water deficit (≈–2 MPa) was reached between Days 15 and 20 after the beginning of the experiment. The cool temperature treatment was applied by setting the temperature of the growth room at 12 °C at the beginning of the experiment.

Sample collection, conservation, and RNA extraction.

Samples for gene expression analysis were collected at 10-d intervals starting on the day when the treatments were applied. CsFT expression was measured in leaves, whereas CsAP1, CsLFY, and CsSL1 were measured in axillary buds; this choice of tissues was based on the most likely site of functional expression of these genes inferred from works in arabidopsis (Corbesier et al., 2007; Wigge et al., 2005) and citrus (Furr et al., 1947; García-Luís and Kandušer, 1995; García-Luis et al., 1989). Leaf and bud samples were randomly collected from the three most apical nodes in mature six- to seven-node-long shoots because flowering is most likely to occur at these positions (Sauer, 1954; Valiente and Albrigo, 2004). At each time, six shoots were sampled on each tree; thus, leaf and bud samples consisted of a pool of six leaves or buds per tree. All samples were collected between 1445 and 1515 hr local standard time (Lake Alfred, FL), immediately frozen in liquid nitrogen, and stored at –80 °C until used.

Total RNA was extracted from the collected samples using a phenol–chloroform precipitation method (Chomczynski and Sacchi, 1987) and purified using silica membranes (Epoch Biolabs, Sugar Land, TX) with on-column DNase digestion (Qiagen, Germantown, MD). RNA quality was evaluated by spectrophotometry and denaturing gel electrophoresis and quantified by spectrophotometry (Sambrook et al., 1989).

Quantitative reverse transcription–polymerase chain reaction.

Five hundred nanograms of total RNA were used for cDNA synthesis in a 20-μL reaction with oligo dT primers (SuperScriptIII®; Invitrogen, Carlsbad, CA). One microliter of the synthesized cDNA was used for two-step (95 °C denaturation and 60 °C for 1 min annealing and extension) quantitative polymerase chain reaction (qPCR) in a 20-μL reaction using SYBR green detection (SYBR® Premix ExTaq™II; Takara, Mountain View, CA) on an Applied Biosystems 7500 FAST real-time PCR system (Life Technologies, Carlsbad, CA). Primers for qPCR were: 5′-CGGCGGAAGGACTATGAC-3′ and 5′-TGTGAGAAAGCCAGAGAGGAA-3′ (CsFT), 5′-CAGCCAGAGAATCTAACAAACG-3′ and 5′-TCAGTTTTGTGGTGGTATTGCC-3′ (CsSL1), 5′-CCCTGGAGTGCAACAACCT-3′ and 5′-CTGATGTGTTTGAGAGCGGT-3′ (CsAP1), and 5′-TCTTGATCCAGGTCCAGAACATC-3′ and 5′-TAGTCACCTTGGTTGGGCATT-3′ (CsLFY). CsGAPDH was used as reference gene (5′-GGAAGGTCAAGATCGCAATCAA-3′ and 5′-CGTCCCTCTGCAAGATGACTCT-3′). All qPCR assays were validated for specific amplification and optimized for amplification efficiencies between 1.88 and 2.05 with a linear dynamic range of six log10 cycles before the experiments. The sequence of the primers to amplify CsLFY was obtained from Nishikawa et al. (2009), whereas all other primer sequences were designed in-house. CsFT primers on this study were designed based on the sequence of CiFT3 reported by Nishikawa et al. (2009). The CiFT3 sequence was selected over CiFT2 or CiFT because, reportedly, CiFT3 correlated better to floral-inductive treatments (Nishikawa et al., 2009). Relative gene expression was calculated as a fold change ratio using Pfaffl’s method (Pfaffl, 2001) with sliding-window efficiencies calculated for each reaction using the sliwin function in the qpcR R package (Ritz and Spiess, 2008).

Flowering data.

The number of inflorescences and new vegetative shoots was counted on the flush induced by the experimental treatments. Counts were made on all six- to seven-node-long shoots formed during the previous year present in each tree. The type of inflorescence (leaf-abundant, leaf-deficient, leafless, and single flowers) formed was also registered. Flowering data are presented as the average number of inflorescences per shoot (six to seven nodes long) per tree.

Statistical analysis.

Both experiments were conducted separately using a completely randomized design with four single-tree replicates per treatment. Levels of gene expression were analyzed with analysis of variance using a repeated measurements model. Flowering data were analyzed using t tests. Mean fold change of transcript levels was transformed to a logarithmic scale (log2) for statistical analysis, but the untransformed data are presented in the graphs. Statistically significant differences were declared at P ≤ 0.05. All statistical analyses were executed in R (R Development Core Team, 2011).

Results

Flowering response induced by water deficit treatment at floral-inductive and non-inductive temperatures.

In both experiments, trees that had been exposed to water deficit produced more inflorescences than well-irrigated trees (Tables 1 and 2). At 23 °C, very few inflorescences (0.2 per shoot) were induced in irrigated trees, whereas trees that were exposed to water deficit produced an average of 2.0 inflorescences per shoot (Table 1). Most of the inflorescences formed in trees under water deficit were of the leaf-abundant type (more leaves than flowers). In addition to inflorescences, trees that had been exposed to water deficit also produced an average of 1.1 vegetative shoots compared with no new vegetative buds formed in irrigated trees. The newly formed vegetative shoots sprouted at more basal positions along the shoot, whereas inflorescences were formed at more apical positions (data not shown). These data indicate that the water deficit treatment applied was effective at inducing flowering in the experimental trees.

Table 1.

Effect of water deficit applied for 60 d at 23 °C on bud sprouting and flowering of two- to three-year-old sweet orange cuttings.z

Table 1.

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Table 2.

Effect of water deficit applied for 40 d at 12 °C on bud sprouting and flowering of two- to three-year-old sweet orange cuttings.z

Table 2.

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At 12 °C, new inflorescences were induced in irrigated trees, but trees that had been exposed to water deficit produced almost twice as many inflorescences per shoot (Table 2). Leaf-abundant inflorescences were the most common type of inflorescence formed. Trees under water deficit produced significantly (P ≤ 0.05) more leaf-abundant inflorescences and single flowers than irrigated trees, whereas no differences between both treatments were detected for leaf-deficient, leafless inflorescences and new vegetative shoots. These data indicate that the combined effect of water deficit and cool temperature on floral induction was greater than the effect of cool temperature alone.

Effect of water deficit on the accumulation of CsFT, CsSL1, CsAP1, and CsLFY transcripts at non-inductive temperatures (23 °C).

By the end of the water deficit treatment, accumulation of CsFT transcripts in leaves of trees kept under water deficit had increased ≈22-fold relative to initial levels (Fig. 1). Statistically significant (P ≤ 0.05) differences in the level of accumulation of CsFT were detected by Day 20 after the beginning of the experiment. Over the duration of the experiment, the accumulation of CsFT transcripts increased constantly. However, when water deficit was interrupted by irrigation, transcript accumulation returned to initial and control levels and remained at this level on Day 10 after the transfer. These results indicate that the accumulation of CsFT transcripts was induced by water deficit and was proportional to the duration of the water deficit treatment. In addition, these results show that accumulation of CsFT transcript was only sustained above basal levels for as long as the trees remained under water deficit.

Fig. 1.

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Fig. 1.

Transcript accumulation of CsFT, CsSL1, CsAP1, and CsLFY in two- to three-year-old ‘Washington Navel’ sweet orange cuttings under water deficit at 23 °C. Transcript accumulation was monitored in leaves (CsFT) and buds (CsSL1, CsAP1, and CsLFY) of four-month-old or older shoots using quantitative reverse transcription–polymerase chain reaction. Moderate water deficit (–2 MPa midday stem water potential) was imposed by reducing irrigation frequency and volume. Water deficit was removed on Day 60 (gray line). Symbols represent the means of four single-tree replicates ± se (six shoots/tree). Transcript accumulation is relative to the levels of CsGAPDH and to the level of each gene in “well-irrigated” trees on Day 0.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 2; 10.21273/JASHS.138.2.88

No differences in the level of accumulation of CsSL1 transcripts in buds were detected between trees under water deficit and irrigated trees while trees were exposed to water deficit (Fig. 1). However, after the water deficit was interrupted, levels of CsSL1 transcript accumulation increased 1.5-fold in the buds relative to the levels in the well-irrigated control trees previously exposed to water deficit. These results show that CsSL1 expression was unaffected by water deficit but was promoted after reirrigation.

Accumulation of CsAP1 and CsLFY transcripts in buds was reduced significantly by Day 20 and onward by water deficit with levels of transcripts of these two genes ranging between 0.5- and 0.75-fold relative to the levels in the irrigated controls (Fig. 1). After the interruption of the water deficit treatment, the accumulation of transcripts of both genes increased ≈2-fold and returned to initial and control levels 10 d afterward. These results indicate that water deficit, when present, negatively regulates the accumulation of CsAP1 and CsLFY transcripts and that interruption of the water deficit promoted it.

Effect of water deficit on the accumulation of CsFT, CsSL1, CsAP1, and CsLFY transcripts at floral inductive temperatures (12 °C).

Accumulation of CsFT transcripts in leaves increased steadily once trees were transferred to the growth room at 12 °C (Fig. 2). The increase in the accumulation of CsFT transcripts was greater in trees under water deficit than in irrigated trees. By the end of the treatments, levels of CsFT in trees under water deficit had increased ≈34-fold relative to initial levels, whereas in irrigated trees, the increase was ≈12-fold; 3 d after the trees were transferred back to the greenhouse and irrigation restored in the trees previously under water deficit, the levels of CsFT transcripts had returned to near initial levels. Increasing levels of CsFT transcripts in leaves of well-irrigated trees in response to cool temperatures were in agreement with previous reports in the literature (Nishikawa et al., 2007, 2009). These results, along with the results of the flowering response, suggest that the accumulation of CsFT transcripts was an indicator of the overall level of floral induction perceived by the trees regardless of the stimuli producing it.

Fig. 2.

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Fig. 2.

Transcript accumulation of CsFT, CsSL1, CsAP1, and CsLFY in two- to three-year-old ‘Washington Navel’ sweet orange cuttings under water deficit at 12 °C. Transcript accumulation was monitored in leaves (CsFT) and buds (CsSL1, CsAP1, and CsLFY) of four-month-old or older shoots using quantitative reverse transcription–polymerase chain reaction. Moderate water deficit (–2 MPa midday stem water potential) was imposed by reducing irrigation frequency and volume starting 7 d before transfer from 23 to 12 °C (gray area). Water deficit was removed on Day 40 (darker gray line) and trees were transferred to a room at 23 °C and irrigated to saturation. Symbols represent the means of four single-tree replicates ± se (six shoots/tree). Transcript accumulation is relative to the levels of CsGAPDH and to the level of each gene in “well-irrigated” trees on Day 0.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 2; 10.21273/JASHS.138.2.88

Levels of CsSL1 were initially reduced relative to initial levels both in irrigated trees and trees under water deficit (Fig. 2). In trees under water deficit, levels of CsSL1 remained below initial levels (≈0.75-fold) until trees were transferred back to the greenhouse and irrigation was restored; at this point, accumulation of CsSL1 transcripts returned to pre-treatment levels. The latter observation contrasts with the small (≈1.5-fold) but significant increase in the accumulation of CsSL1 transcripts measured after reirrigation in the experiment at 23 °C. In irrigated trees, accumulation of CsSL1 transcripts was initially reduced but later increased from Day 20 onward reaching ≈2-fold increase relative to initial levels by the end of the experiment; 2 d after transfer to the greenhouse, levels of CsSL1 were still ≈2-fold higher than initial levels. These results along with results from the experiment at 23 °C indicate that between cool temperature and water deficit, only cool temperature induced the accumulation of CsSL1 transcripts while the stimulus was present.

Levels of CsAP1 and CsLFY transcript accumulation in buds were reduced to between 0.25- and 0.75-fold after transfer to the growth room at 12 °C in both irrigated trees and trees under water deficit (Fig. 2). The accumulation of transcripts of both genes remained below initial levels until the trees were transferred back to the greenhouse and water deficit was interrupted. After transfer to the greenhouse and interruption of the water deficit, the accumulation of transcripts of both genes increased ≈4-fold in trees previously exposed to water deficit and ≈3-fold in the irrigated trees also previously held at 12 °C. A reduction of the accumulation of CsAP1 and CsLFY transcripts while trees were under floral inductive conditions was in agreement with results from the experiment conducted at 23 °C. The increased accumulation of transcripts after floral induction was interrupted was also in agreement with the results of the experiment at 23 °C and reports in the literature (Pillitteri et al., 2004a). Higher accumulation of CsAP1 and CsLFY transcripts in buds of trees that underwent water deficit than in buds of well-irrigated trees and flowering data indicate that the overall level of accumulation of CsAP1 and CsLFY was also an indicator of the overall level of induction of the trees.

Discussion

In citrus, flowering is only induced by prolonged exposure to either cool (less than 20 °C) temperatures or water deficit (Cassin et al., 1969; Moss, 1969), and only two other factors, gibberellin applications and the presence of fruit, have well-demonstrated effects negatively regulating the intensity of floral induction perceived by the trees (Monselise et al., 1964; Moss, 1971). In recent years, the patterns of expression of putative citrus orthologs of arabidopsis flowering genes have been characterized in response to cool temperatures (Li et al., 2010; Nishikawa et al., 2007, 2009; Pillitteri et al., 2004a; Zhang et al., 2009, 2011), gibberellins (Muñoz Fambuena et al., 2012a), and the presence of fruit (Muñoz Fambuena et al., 2011, 2012b). In this work, we investigated the pattern of expression (measured as transcript accumulation) of a set of four putative flowering genes (CsFT, CsSL1, CsAP1, and CsLFY) during water deficit in potted sweet orange trees.

Increased accumulation of CsFT transcripts during water deficit reported in Figure 1 indicates that, similar to cool temperatures (Nishikawa et al., 2007), water deficit also upregulated the expression of this gene. This increased accumulation of CsFT was correlated to more inflorescences being formed in trees under water deficit than in well-irrigated trees (Table 1). Thus, the level of accumulation of citrus FT ortholog transcripts seems to be sensitive to all four factors, cool temperature, gibberellin application, fruit load, and water deficit (Muñoz Fambuena et al., 2011, 2012a, 2012b; Nishikawa et al., 2007, 2009) known to have a clear effect on citrus flowering with consistent correlations with flowering response. In arabidopsis, FT is a key integrator of floral promoting/inhibiting signals from the photoperiod, vernalization, autonomous, and gibberellin pathways (Amasino, 2010; Turck et al., 2008), which suggests that the role of FT orthologs as integrators of endogenous and exogenous signals regulating flowering likely is conserved in citrus and arabidopsis.

Higher accumulation of CsFT transcripts (Fig. 2) and more inflorescences (Table 2) forming in trees exposed to water deficit than in well-irrigated trees at 12 °C indicate similar and probably additive effects from these two variables, suggesting that the level of accumulation of CsFT transcripts could be an estimator of the level of induction perceived by the trees. Contrary to arabidopsis, in which the inflorescence develops from a single shoot meristem at the end of the rosette resulting in a rather discrete flowering response, in citrus and many other tree crops, inflorescences develop from many apical and axillary meristems resulting in a more continuous distribution of possible flowering responses (Valiente and Albrigo, 2004). In arabidopsis, the protein encoded by FT is a mobile signal synthesized in the phloem of leaves (An et al., 2004; Mathieu et al., 2007) and transported to the shoot apical meristem (Corbesier et al., 2007) where, along with FD (Abe et al., 2005), it activates the expression of floral meristem identity genes (Michaels et al., 2005; Wigge et al., 2005). Like in our results (Figs. 1 and 2), in arabidopsis, accumulation of FT transcripts increases as the plants are exposed for a longer time to a floral-inductive treatment and is sharply reduced after the inductive stimulus is removed (Corbesier et al., 2007). In arabidopsis, the main consequence of the overexpression of FT is early flowering (Kobayashi et al., 1999) measured typically as days until bolting or the number of rosette leaves emitted before bolting. In transgenic Poncirus trifoliata, a close relative of citrus, ectopic expression of the Citrus unshiu FT ortholog resulted in extreme early flowering [weeks vs. years (Endo et al., 2005)]. Similarly, Pyrus communis and arabidopsis plants overexpressing CiFT also flowered earlier than untransformed plants (Kobayashi et al., 1999; Matsuda et al., 2009). In citrus, flowering intensity can be evaluated quantitatively and qualitatively and is related to the duration and intensity of floral inductive conditions (Moss, 1969; Southwick and Davenport, 1986). Quantitatively, the number of buds developing into inflorescences is proportional to the intensity of floral induction. Qualitatively, the leaf:flower ratio of inflorescences is inversely proportional to floral induction intensity (Moss, 1969). Our results indicate that the level of accumulation of CsFT could be a predictor to comparatively estimate flowering intensity in terms of the number of inflorescences induced. However, our results do not support a correlation among the level of accumulation of CsFT transcripts, intensity of floral induction, and the leaf:flower ratio of the inflorescences induced, because no differences were detected in the number of leaf-deficient or leafless inflorescences despite differences in the level of accumulation of CsFT transcripts. A shift toward leaf-deficient and leafless inflorescences would be expected at higher perceived levels of induction (Moss, 1969). In bud samples, the level of accumulation of CsAP1 and CsLFY transcripts was correlated with the number of inflorescences induced and also with the level of accumulation of CsFT transcripts at the end of the inductive treatment. Because the bud samples in which CsAP1 and CsLFY transcripts were quantified consisted of a pool of six buds, the increased accumulation of CsAP1 and CsLFY transcripts in induced buds could be interpreted as higher expression of the genes in individual buds or more buds activating the expression of these genes.

Reduced accumulation of CsSL1 transcripts in trees under water deficit at any temperature but increased accumulation of CsSL1 transcripts in well-irrigated trees at 12 °C (Figs. 1 and 2) indicates that water deficit acts as a negative regulator of the expression of this gene, whereas cool temperatures act as a promoter of its expression. In arabidopsis, SOC1 (CsSL1 putative ortholog) integrates signals from the main four flowering pathways (Lee and Lee, 2010). SOC1 can act downstream of FT or in a FT-independent manner in the shoot meristem to promote the expression of LFY in conjunction with AGL24 (Lee et al., 2008). Thus, assuming a correlation between transcript accumulation and protein transport and activity of CsFT, the negative effect of water deficit on the accumulation of CsSL1 could be a consequence of a water-deficit-specific signal integrated between CsFT and CsSL1 or simply a CsFT-independent signal integrated at CsSL1 to inhibit its expression.

Although water deficit reduced the accumulation of CsSL1 transcripts during the treatments, the small increase in the accumulation of CsSL1 transcripts after reirrigation (and transfer to 23 °C in the second experiment) suggests a carryover effect of water deficit on the expression of this gene. This response was also observed (even more clearly) in the accumulation of CsAP1 and CsLFY transcripts in buds of trees previously exposed to water deficit (Figs. 1 and 2). In arabidopsis, AP1 and LFY act as floral meristem identity genes, and their activation triggers floral development (Mandel and Yanofsky, 1995; Weigel and Nilsson, 1995). Expression of AP1 and LFY is activated by FT and SOC1 and later reinforced by each other to maintain the floral identity of the meristem (Liljegren et al., 1999; Wagner et al., 1999; Wigge et al., 2005). Expression of SOC1 not only was required for upregulation of CsLFY, but also for the activation of B and C floral organ identity genes during flower development (Liu et al., 2009). In arabidopsis, consistent upregulation of AP1 has been reported to occur ≈16 h after transfer to floral-inductive photoperiods marking floral determination (Hempel et al., 1997). Contrary to model species and most deciduous trees in which floral inductive and growth-promoting conditions occur simultaneously, in citrus, floral meristem differentiation and determination are only identifiable when growth-promoting conditions resume, which implies that floral-inductive conditions are no longer present or are less intense when growth starts (Lord and Eckard, 1985; Moss, 1976). Increased accumulation of CsAP1 and CsLFY transcripts has been reported in citrus after floral induction by cool temperatures (Pillitteri et al., 2004a) providing molecular support for the hypothesis of floral differentiation occurring only after floral induction but not simultaneously in citrus. Our results using water deficit induction (Fig. 1) are also consistent with the latter observation.

In recent years, much interest has arisen over the conservation of the genetic framework regulating flowering among species. Although knowledge about the genetic–molecular mechanisms regulating flowering in model species has contributed significantly to the identification of potential regulators of flowering in crop species, evolution and adaptation to different environments have likely resulted in important modifications to these mechanisms. In citrus, floral induction is practically photoperiod-insensitive, gibberellin treatments induce responses opposite to those induced in arabidopsis, and the role of cool temperatures is not comparable to the flowering-enabling role of vernalization. Our results and those of others (Muñoz Fambuena et al., 2011, 2012a; Nishikawa et al., 2007; Pillitteri et al., 2004a) support the involvement of CsFT as an integrator of signals regulating flowering in citrus and probably a defining factor for the number of inflorescences to be formed after induction as well as the likely conservation of CsAP1 and CsLFY function as floral meristem identity genes. Nonetheless, the characterization of the genetic framework upstream of these genes regulating their expression remains mostly unexplored in citrus. Determining the composition and topology of the network regulating flowering in citrus will be useful to understand how citrus and other species perceive developmental signals from the environment and to engineer mechanisms to manipulate the reproductive biology of these species for horticultural purposes.

Literature Cited

  • AbbotC.E.1935Blossom–bud differentiation in citrus treesAmer. J. Bot.22476485

  • AbeM.KobayashiY.YamamotoS.DaimonY.YamaguchiA.IkedaY.IchinokiH.NotaguchiM.GotoK.ArakiT.2005FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apexScience30910521056

  • AlbrigoL.G.Galen-SaúcoV.2004Flower bud induction, flowering and fruit-set of some tropical and subtropical fruit tree crops with special reference to citrusActa Hort.6328191

  • AlbrigoL.G.ValienteJ.I.Van Parys de WitC.2006Influence of winter and spring weather on year-to-year citrus fruit set and yield variation in São Paulo, BrazilProc. Intl. Soc. Citricult.1263267

  • AmasinoR.2010Seasonal and developmental timing of floweringPlant J.6110011013

  • AnH.RoussotC.Suarez-LopezP.CorbesierL.VincentC.PineiroM.HepworthS.MouradovA.JustinS.TurnbullC.CouplandG.2004Constans acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of ArabidopsisDevelopment13136153626

  • BalasubramanianS.SureshkumarS.LempeJ.WeigelD.2006Potent induction of Arabidopsis thaliana flowering by elevated growth temperaturePLoS Genet.2e106

  • BlázquezM.A.AhnJ.H.WeigelD.2003A thermosensory pathway controlling flowering time in Arabidopsis thalianaNat. Genet.33168171

  • BlázquezM.A.GreenR.NilssonO.SussmanM.R.WeigelD.1998Gibberellins promote flowering of arabidopsis by activating the Leafy promoterPlant Cell10791800

  • CassinJ.BourdeuetJ.FougueA.FuronV.GaillandJ.P.BourdellesJ.MontaguadG.MoreuilC.1969The influence of climate upon the blooming of citrus in tropical areas. Proc. First Intl. Citrus Symp. 1:315–323

  • ChomczynskiP.SacchiN.1987Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extractionAnal. Biochem.162156159

  • CorbesierL.VincentC.JangS.FornaraF.FanQ.SearleI.GiakountisA.FarronaS.GissotL.TurnbullC.CouplandG.2007FT protein movement contributes to long-distance signaling in floral induction of ArabidopsisScience31610301033

  • EndoT.ShimadaT.FujiiH.KobayashiY.ArakiT.OmuraM.2005Ectopic expression of an FT homolog from citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.)Transgenic Res.14703712

  • FurrJ.R.CooperW.C.ReeceP.C.1947An investigation of flower formation in adult and juvenile citrus treesAmer. J. Bot.3418

  • García-LuísA.KandušerM.1995Changes in dormancy and sensitivity to vernalization in axillary buds of Satsuma mandarin examined in vitro during the annual cycleAnn. Bot. (Lond.)76451455

  • García-LuisA.P.SantamarinaP.GuardiolaJ.L.1989Flower formation from Citrus unshiu buds cultured in vitroAnn. Bot. (Lond.)64515519

  • HempelF.WeigelD.MandelM.DittaG.ZambryskiP.FeldmanL.YanofskyM.1997Floral determination and expression of floral regulatory genes in ArabidopsisDevelopment12438453853

  • KimD.-H.DoyleM.R.SungS.AmasinoR.M.2009Vernalization: Winter and the timing of flowering in plantsAnnu. Rev. Cell Dev. Biol.25277299

  • KobayashiY.KayaH.GotoK.IwabuchiM.ArakiT.1999A pair of related genes with antagonistic roles in mediating flowering signalsScience28619601962

  • LeeJ.LeeI.2010Regulation and function of SOC1, a flowering pathway integratorJ. Expt. Bot.6122472254

  • LeeJ.OhM.ParkH.LeeI.2008SOC1 translocated to the nucleus by interaction with AGL24 directly regulates LeafyPlant J.55832843

  • LiZ.-M.ZhangJ.-Z.MeiL.DengX.-X.HuC.-G.YaoJ.-L.2010PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plantsPlant Mol. Biol.74129142

  • LiljegrenS.J.Gustafson-BrownC.PinyopichA.DittaG.S.YanofskyM.F.1999Interactions among Apetala, Leafy, and Terminal Flower1 specify meristem fatePlant Cell1110071018

  • LiuC.XiW.ShenL.TanC.YuH.2009Regulation of floral patterning by flowering time genesDev. Cell16711722

  • LordE.M.EckardK.J.1985Shoot development in Citrus sinensis L. (Washington Navel orange). I. Floral and inflorescence ontogenyBot. Gaz.146320326

  • MandelM.A.YanofskyM.1995A gene triggering flower formation in ArabidopsisNature377522524

  • MathieuJ.WarthmannN.KüttnerF.SchmidM.2007Export of FT protein from phloem companion cells is sufficient for floral induction in ArabidopsisCurr. Biol.1710551060

  • MatsudaN.IkedaK.KurosakaM.TakashinaT.IsuzugawaK.EndoT.OmuraM.2009Early flowering phenotype in transgenic pears (Pyrus communis L.) expressing the CiFT geneJ. Jpn. Soc. Hort. Sci.78410416

  • McCutchanH.ShackelK.1992Stem water potential as a sensitive indicator of water stress in prune trees (Prunus domestica L. cv. French)J. Amer. Soc. Hort. Sci.117607611

  • MichaelsS.D.HimelblauE.KimS.Y.SchomburgF.M.AmasinoR.M.2005Integration of flowering signals in winter-annual arabidopsisPlant Physiol.137149156

  • MonseliseS.P.GorenR.HalevyA.1964Chemical inhibition and promotion of citrus flower bud inductionProc. Amer. Soc. Hort. Sci.84141146

  • MossG.I.1969Influence of temperature and photoperiod on flower induction and inflorescence development in sweet orange (Citrus sinensis L. Osbeck)J. Hort. Res.44311320

  • MossG.I.1971Effect of fruit on flowering in relation to biennial bearing in sweet orange (Citrus sinensis)J. Hort. Sci.46177184

  • MossG.I.1976Temperature effects on flower initiation in sweet orange (Citrus sinensis)Austral. J. Agr. Res.27399407

  • Muñoz FambuenaN.MesejoC.Carmen González-MasM.Primo-MilloE.AgustíM.IglesiasD.J.2011Fruit regulates seasonal expression of flowering genes in alternate-bearing moncada mandarinAnn. Bot. (Lond.)108511519

  • Muñoz FambuenaN.MesejoC.González-MasM.IglesiasD.Primo-MilloE.AgustíM.2012aGibberellic acid reduces flowering intensity in sweet orange [Citrus sinensis (L.) Osbeck] by repressing CiFT gene expressionJ. Plant Growth Regul.31529536

  • Muñoz FambuenaN.MesejoC.González-MasM.C.Primo-MilloE.AgustíM.IglesiasD.J.2012bFruit load modulates flowering-related gene expression in buds of alternate-bearing Moncada mandarinAnn. Bot. (Lond.)11011091118

  • NishikawaF.EndoT.ShimadaT.FujiiH.ShimizuT.OmuraM.2009Differences in seasonal expression of flowering genes between deciduous trifoliate orange and evergreen Satsuma mandarinTree Physiol.29921926

  • NishikawaF.EndoT.ShimadaT.FujiiH.ShimizuT.OmuraM.IkomaY.2007Increased CiFT abundance in the stem correlates with floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.)J. Expt. Bot.5839153927

  • PfafflM.W.2001A new mathematical model for relative quantification in real-time RT-PCRNucleic Acids Res.29e45

  • PillitteriL.J.LovattC.J.WallingL.L.2004aIsolation and characterization of a Terminal Flower homolog and its correlation with juvenility in citrusPlant Physiol.13515401551

  • PillitteriL.J.LovattC.J.WallingL.L.2004bIsolation and characterization of Leafy and Apetala1 homologues from Citrus sinensis L. Osbeck ‘Washington’J. Amer. Soc. Hort. Sci.129846856

  • R Development Core Team2011R: A language and environment for statistical computing. 3 Jan. 2013. <http://www.r-project.org/>

  • RitzC.SpiessA.N.2008qpcR: An R package for sigmoidal model selection in quantitative real-time polymerase chain reaction analysisBioinformatics2415491551

  • SamachA.OnouchiH.GoldS.E.DittaG.S.Schwarz-SommerZ.YanofskyM.F.CouplandG.2000Distinct roles of Constans target genes in reproductive development of ArabidopsisScience28816131616

  • SambrookJ.FritschE.ManiatisT.1989Molecular cloning A laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press New York NY

  • SauerM.R.1954Flowering in the sweet orangeAustral. J. Agr. Res.5649657

  • ScholanderP.F.HammelA.D.BradstreetA.D.HemmingsenE.A.1965Sap pressure in vascular plantsScience148339346

  • SouthwickS.M.DavenportT.L.1986Characterization of water stress and low temperature effects on flower induction in citrusPlant Physiol.812629

  • TanF.-C.SwainS.M.2007Functional characterization of AP3, SOC1 and WUS homologues from citrus (Citrus sinensis)Physiol. Plant.131481495

  • TurckF.FornaraF.CouplandG.2008Regulation and identity of florigen: Flowering locus t moves center stageAnnu. Rev. Plant Biol.59573594

  • ValienteJ.I.AlbrigoL.G.2004Flower bud induction of sweet orange trees [Citrus sinensis (L.) Osbeck]: Effect of temperature, crop load and bud ageJ. Amer. Soc. Hort. Sci.129158164

  • WagnerD.SablowskiR.W.M.MeyerowitzE.M.1999Transcriptional activation of APETALA1 by LEAFYScience285582584

  • WeigelD.NilssonO.1995A developmental switch sufficient for flower initiation in diverse plantsNature377495500

  • WiggeP.A.KimM.C.JaegerK.E.BuschW.SchmidM.LohmannJ.U.WeigelD.2005Integration of spatial and temporal information during floral induction in arabidopsisScience30910561059

  • ZhangJ.-Z.AiX.-Y.SunL.-M.ZhangD.-L.GuoW.-W.DengX.-X.HuC.-G.2011Molecular cloning and functional characterization of genes associated with flowering in citrus using an early-flowering trifoliate orange (Poncirus trifoliata L. Raf.) mutantPlant Mol. Biol.76187204

  • ZhangJ.-Z.LiZ.-M.MeiL.YaoJ.-L.HuC.-G.2009PtFLC homolog from trifoliate orange (Poncirus trifoliata) is regulated by alternative splicing and experiences seasonal fluctuation in expression levelPlanta229847859

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Contributor Notes

Current address: Carrera de Ingeniería Agrícola y Biológica, Escuela Superior Politécnica del Litoral, Km. 30.5 vía perimentral, Guayaquil, Ecuador.

Corresponding author. E-mail: albrigo@ufl.edu.

Headings

Figures

  • View in gallery

    Transcript accumulation of CsFT, CsSL1, CsAP1, and CsLFY in two- to three-year-old ‘Washington Navel’ sweet orange cuttings under water deficit at 23 °C. Transcript accumulation was monitored in leaves (CsFT) and buds (CsSL1, CsAP1, and CsLFY) of four-month-old or older shoots using quantitative reverse transcription–polymerase chain reaction. Moderate water deficit (–2 MPa midday stem water potential) was imposed by reducing irrigation frequency and volume. Water deficit was removed on Day 60 (gray line). Symbols represent the means of four single-tree replicates ± se (six shoots/tree). Transcript accumulation is relative to the levels of CsGAPDH and to the level of each gene in “well-irrigated” trees on Day 0.

  • View in gallery

    Transcript accumulation of CsFT, CsSL1, CsAP1, and CsLFY in two- to three-year-old ‘Washington Navel’ sweet orange cuttings under water deficit at 12 °C. Transcript accumulation was monitored in leaves (CsFT) and buds (CsSL1, CsAP1, and CsLFY) of four-month-old or older shoots using quantitative reverse transcription–polymerase chain reaction. Moderate water deficit (–2 MPa midday stem water potential) was imposed by reducing irrigation frequency and volume starting 7 d before transfer from 23 to 12 °C (gray area). Water deficit was removed on Day 40 (darker gray line) and trees were transferred to a room at 23 °C and irrigated to saturation. Symbols represent the means of four single-tree replicates ± se (six shoots/tree). Transcript accumulation is relative to the levels of CsGAPDH and to the level of each gene in “well-irrigated” trees on Day 0.

References

AbbotC.E.1935Blossom–bud differentiation in citrus treesAmer. J. Bot.22476485

AbeM.KobayashiY.YamamotoS.DaimonY.YamaguchiA.IkedaY.IchinokiH.NotaguchiM.GotoK.ArakiT.2005FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apexScience30910521056

AlbrigoL.G.Galen-SaúcoV.2004Flower bud induction, flowering and fruit-set of some tropical and subtropical fruit tree crops with special reference to citrusActa Hort.6328191

AlbrigoL.G.ValienteJ.I.Van Parys de WitC.2006Influence of winter and spring weather on year-to-year citrus fruit set and yield variation in São Paulo, BrazilProc. Intl. Soc. Citricult.1263267

AmasinoR.2010Seasonal and developmental timing of floweringPlant J.6110011013

AnH.RoussotC.Suarez-LopezP.CorbesierL.VincentC.PineiroM.HepworthS.MouradovA.JustinS.TurnbullC.CouplandG.2004Constans acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of ArabidopsisDevelopment13136153626

BalasubramanianS.SureshkumarS.LempeJ.WeigelD.2006Potent induction of Arabidopsis thaliana flowering by elevated growth temperaturePLoS Genet.2e106

BlázquezM.A.AhnJ.H.WeigelD.2003A thermosensory pathway controlling flowering time in Arabidopsis thalianaNat. Genet.33168171

BlázquezM.A.GreenR.NilssonO.SussmanM.R.WeigelD.1998Gibberellins promote flowering of arabidopsis by activating the Leafy promoterPlant Cell10791800

CassinJ.BourdeuetJ.FougueA.FuronV.GaillandJ.P.BourdellesJ.MontaguadG.MoreuilC.1969The influence of climate upon the blooming of citrus in tropical areas. Proc. First Intl. Citrus Symp. 1:315–323

ChomczynskiP.SacchiN.1987Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extractionAnal. Biochem.162156159

CorbesierL.VincentC.JangS.FornaraF.FanQ.SearleI.GiakountisA.FarronaS.GissotL.TurnbullC.CouplandG.2007FT protein movement contributes to long-distance signaling in floral induction of ArabidopsisScience31610301033

EndoT.ShimadaT.FujiiH.KobayashiY.ArakiT.OmuraM.2005Ectopic expression of an FT homolog from citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.)Transgenic Res.14703712

FurrJ.R.CooperW.C.ReeceP.C.1947An investigation of flower formation in adult and juvenile citrus treesAmer. J. Bot.3418

García-LuísA.KandušerM.1995Changes in dormancy and sensitivity to vernalization in axillary buds of Satsuma mandarin examined in vitro during the annual cycleAnn. Bot. (Lond.)76451455

García-LuisA.P.SantamarinaP.GuardiolaJ.L.1989Flower formation from Citrus unshiu buds cultured in vitroAnn. Bot. (Lond.)64515519

HempelF.WeigelD.MandelM.DittaG.ZambryskiP.FeldmanL.YanofskyM.1997Floral determination and expression of floral regulatory genes in ArabidopsisDevelopment12438453853

KimD.-H.DoyleM.R.SungS.AmasinoR.M.2009Vernalization: Winter and the timing of flowering in plantsAnnu. Rev. Cell Dev. Biol.25277299

KobayashiY.KayaH.GotoK.IwabuchiM.ArakiT.1999A pair of related genes with antagonistic roles in mediating flowering signalsScience28619601962

LeeJ.LeeI.2010Regulation and function of SOC1, a flowering pathway integratorJ. Expt. Bot.6122472254

LeeJ.OhM.ParkH.LeeI.2008SOC1 translocated to the nucleus by interaction with AGL24 directly regulates LeafyPlant J.55832843

LiZ.-M.ZhangJ.-Z.MeiL.DengX.-X.HuC.-G.YaoJ.-L.2010PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plantsPlant Mol. Biol.74129142

LiljegrenS.J.Gustafson-BrownC.PinyopichA.DittaG.S.YanofskyM.F.1999Interactions among Apetala, Leafy, and Terminal Flower1 specify meristem fatePlant Cell1110071018

LiuC.XiW.ShenL.TanC.YuH.2009Regulation of floral patterning by flowering time genesDev. Cell16711722

LordE.M.EckardK.J.1985Shoot development in Citrus sinensis L. (Washington Navel orange). I. Floral and inflorescence ontogenyBot. Gaz.146320326

MandelM.A.YanofskyM.1995A gene triggering flower formation in ArabidopsisNature377522524

MathieuJ.WarthmannN.KüttnerF.SchmidM.2007Export of FT protein from phloem companion cells is sufficient for floral induction in ArabidopsisCurr. Biol.1710551060

MatsudaN.IkedaK.KurosakaM.TakashinaT.IsuzugawaK.EndoT.OmuraM.2009Early flowering phenotype in transgenic pears (Pyrus communis L.) expressing the CiFT geneJ. Jpn. Soc. Hort. Sci.78410416

McCutchanH.ShackelK.1992Stem water potential as a sensitive indicator of water stress in prune trees (Prunus domestica L. cv. French)J. Amer. Soc. Hort. Sci.117607611

MichaelsS.D.HimelblauE.KimS.Y.SchomburgF.M.AmasinoR.M.2005Integration of flowering signals in winter-annual arabidopsisPlant Physiol.137149156

MonseliseS.P.GorenR.HalevyA.1964Chemical inhibition and promotion of citrus flower bud inductionProc. Amer. Soc. Hort. Sci.84141146

MossG.I.1969Influence of temperature and photoperiod on flower induction and inflorescence development in sweet orange (Citrus sinensis L. Osbeck)J. Hort. Res.44311320

MossG.I.1971Effect of fruit on flowering in relation to biennial bearing in sweet orange (Citrus sinensis)J. Hort. Sci.46177184

MossG.I.1976Temperature effects on flower initiation in sweet orange (Citrus sinensis)Austral. J. Agr. Res.27399407

Muñoz FambuenaN.MesejoC.Carmen González-MasM.Primo-MilloE.AgustíM.IglesiasD.J.2011Fruit regulates seasonal expression of flowering genes in alternate-bearing moncada mandarinAnn. Bot. (Lond.)108511519

Muñoz FambuenaN.MesejoC.González-MasM.IglesiasD.Primo-MilloE.AgustíM.2012aGibberellic acid reduces flowering intensity in sweet orange [Citrus sinensis (L.) Osbeck] by repressing CiFT gene expressionJ. Plant Growth Regul.31529536

Muñoz FambuenaN.MesejoC.González-MasM.C.Primo-MilloE.AgustíM.IglesiasD.J.2012bFruit load modulates flowering-related gene expression in buds of alternate-bearing Moncada mandarinAnn. Bot. (Lond.)11011091118

NishikawaF.EndoT.ShimadaT.FujiiH.ShimizuT.OmuraM.2009Differences in seasonal expression of flowering genes between deciduous trifoliate orange and evergreen Satsuma mandarinTree Physiol.29921926

NishikawaF.EndoT.ShimadaT.FujiiH.ShimizuT.OmuraM.IkomaY.2007Increased CiFT abundance in the stem correlates with floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.)J. Expt. Bot.5839153927

PfafflM.W.2001A new mathematical model for relative quantification in real-time RT-PCRNucleic Acids Res.29e45

PillitteriL.J.LovattC.J.WallingL.L.2004aIsolation and characterization of a Terminal Flower homolog and its correlation with juvenility in citrusPlant Physiol.13515401551

PillitteriL.J.LovattC.J.WallingL.L.2004bIsolation and characterization of Leafy and Apetala1 homologues from Citrus sinensis L. Osbeck ‘Washington’J. Amer. Soc. Hort. Sci.129846856

R Development Core Team2011R: A language and environment for statistical computing. 3 Jan. 2013. <http://www.r-project.org/>

RitzC.SpiessA.N.2008qpcR: An R package for sigmoidal model selection in quantitative real-time polymerase chain reaction analysisBioinformatics2415491551

SamachA.OnouchiH.GoldS.E.DittaG.S.Schwarz-SommerZ.YanofskyM.F.CouplandG.2000Distinct roles of Constans target genes in reproductive development of ArabidopsisScience28816131616

SambrookJ.FritschE.ManiatisT.1989Molecular cloning A laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press New York NY

SauerM.R.1954Flowering in the sweet orangeAustral. J. Agr. Res.5649657

ScholanderP.F.HammelA.D.BradstreetA.D.HemmingsenE.A.1965Sap pressure in vascular plantsScience148339346

SouthwickS.M.DavenportT.L.1986Characterization of water stress and low temperature effects on flower induction in citrusPlant Physiol.812629

TanF.-C.SwainS.M.2007Functional characterization of AP3, SOC1 and WUS homologues from citrus (Citrus sinensis)Physiol. Plant.131481495

TurckF.FornaraF.CouplandG.2008Regulation and identity of florigen: Flowering locus t moves center stageAnnu. Rev. Plant Biol.59573594

ValienteJ.I.AlbrigoL.G.2004Flower bud induction of sweet orange trees [Citrus sinensis (L.) Osbeck]: Effect of temperature, crop load and bud ageJ. Amer. Soc. Hort. Sci.129158164

WagnerD.SablowskiR.W.M.MeyerowitzE.M.1999Transcriptional activation of APETALA1 by LEAFYScience285582584

WeigelD.NilssonO.1995A developmental switch sufficient for flower initiation in diverse plantsNature377495500

WiggeP.A.KimM.C.JaegerK.E.BuschW.SchmidM.LohmannJ.U.WeigelD.2005Integration of spatial and temporal information during floral induction in arabidopsisScience30910561059

ZhangJ.-Z.AiX.-Y.SunL.-M.ZhangD.-L.GuoW.-W.DengX.-X.HuC.-G.2011Molecular cloning and functional characterization of genes associated with flowering in citrus using an early-flowering trifoliate orange (Poncirus trifoliata L. Raf.) mutantPlant Mol. Biol.76187204

ZhangJ.-Z.LiZ.-M.MeiL.YaoJ.-L.HuC.-G.2009PtFLC homolog from trifoliate orange (Poncirus trifoliata) is regulated by alternative splicing and experiences seasonal fluctuation in expression levelPlanta229847859

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