Accumulation of flowering gene transcripts in Citrus sinensis during floral bud induction and. Guayaquil, Ecuador. For Peer Review

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1 Page of Accumulation of flowering gene transcripts in Citrus sinensis during floral bud induction and initiation: Water deficit and cool temperatures effects EDUARDO J. CHICA,, and L. GENE ALBRIGO, Citrus Research and Education Center, University of Florida, Lake Alfred, FL 0, USA Present address: Carrera de Ingeniería Agrícola y Biológica, Escuela Superior Politécnica del Litoral, Guayaquil, Ecuador. Corresponding author. Tel.: address: albrigo@ufl.edu Keywords: FLOWERING LOCUS T, APETALA, LEAFY, SUPRESSOR OF OVEREXPRESSION OF CONSTANS, qrt-pcr. Running head: FLOWERING GENE TRANSCRIPTS DURING FLORAL INDUCTION Concise summary. In this work, we characterized the patterns of transcript accumulation of four floweringrelated genes (CsFT, CsSL, CsAP and CsLFY ) from Citrus sinensis during floral induction by water deficit and cool temperature. We found that water deficit induced patterns of accumulation of the investigated genes similar to those reported in response to cool temperatures by other groups. In addition we found that the level of accumulation of transcripts of these genes is increased when trees are exposed to water deficit and cool temperatures simultaneously and that expression of putative floral identity genes CsAP and CsLFY is suppressed until water deficit is relieved. Summary Citrus trees are induced to flower by exposure to either low temperatures or water deficit. In the last decade, several genes considered to be involved in the regulation of flowering have been isolated and their expression characterized in response to low temperature in Citrus. However, reports on the effect of floralinductive water deficit on the expression of flowering-related genes are lacking. In this work, the patterns of transcript accumulation of four flowering-related genes (CsFT, CsSL, CsAP and CsLFY ) from Citrus sinensis were characterized during floral induction by water deficit and cool temperature. Exposure to water

2 Page of deficit increased the accumulation of CsFT in leaves whereas transcript levels of CsSL, CsAP and CsLFY were only slightly reduced for the duration of the treatment. When the water deficit was relieved, however, the accumulation of CsFT decreased sharply; and the accumulation of CsSL, CsAP and CsLFY transcripts increased. When floral-inductive water deficit and cool temperature occurred at the same time, the increase in the accumulation of CsFT, CsAP and CsLFY was larger than when either occurred separately, this response was not observed in CsSL transcripts. These results indicate that floral-inductive water deficit and low temperatures cause a similar response in the accumulation of CsFT transcript and subsequently other flowering-related genes suggesting that these genes could be the ultimate targets of flowering signals initiated by both environmental stimuli that promote flowering in C. sinensis and possibly in other subtropical and tropical tree crops. Citrus trees are induced to flower after exposure to at least weeks of either temperatures below 0 C or water deficit (Moss, ; Cassin et al., ). In recent years, several genes that hypothetically regulate flowering in citrus species have been identified based on their similarity to flowering related genes in Arabidopsis (Pillitteri et al., 00a,b; Nishikawa et al., 00; Tan and Swain, 00). Changes in transcript levels of these genes have already been characterized in response to floral inductive temperatures (Pillitteri et al., 00a; Nishikawa et al., 00, 00), but little is known about their pattern of expression in response to floral-inductive water deficits. Floral-inductive water deficits are the primary source of floral induction of citrus trees growing in regions with tropical climates (Cassin et al., ) and are an important floral inductive stimulus of floral induction in humid subtropical climates where they can complement floral-inductive cool temperatures during the Fall and Winter (Albrigo et al., 00, Chica, 00). Water deficit can also be the primary stimulus of floral induction for many other species growing in tropical and subtropical climates (Albrigo and Galen-Saúco, 00). In this study we investigated the transcript accumulation of Citrus flowering genes in response to water deficit. We hypothesized that citrus FLOWERING LOCUS T (CsFT) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS (CsSL) transcripts accumulate in response to flowering signals initiated by floral-inductive water deficits. CsFT is the putative Citrus ortholog of Arabidopsis s FLOWERING

3 Page of LOCUS T (FT) (Kobayashi et al., ; Nishikawa et al., 00). In Arabidopsis, the protein encoded by FT is a mobile flowering signal originating in leaves in response to floral-inductive photoperiods and is transported to the shoot apical meristem where it up-regulates the expression of floral identity genes (Samach et al., 000; Corbesier et al., 00; Abe et al., 00). In Citrus unshiu, the expression patterns of the putative FT ortholog (CsFT) support the hypothesis of this gene being involved in the regulation of flowering in citrus (Nishikawa et al., 00, 00). In addition, constitutive expression of CsFT in citrus close relative Poncirus trifoliata resulted in extremely early flowering which further supports a role of citrus FT orthologs as regulators of flowering (Endo et al., 00). CsSL is the putative ortholog of Arabidopsis SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC) (Tan and Swain, 00). In Arabidopsis, SOC is a key integrator of flowering signals initiated by multiple stimuli such as photoperiod, vernalization, gibberellins and endogenous developmental signals (Lee and Lee, 0). Expression patterns of CsSL in citrus in response to floral inductive stimuli have not been described, but introducing CsSL in Arabidopsis soc mutants induced early flowering compared to the wildtype and the late-flowering soc mutant (Tan and Swain, 00). These observations, support the conservation of the role of the citrus SOC ortholog in the regulation of flowering. We also investigated whether the pattern of transcript accumulation of floral identity genes CsAP and CsLFY in trees exposed to floral-inductive water deficit was similar to the pattern of transcript accumulation for these genes in trees exposed to floral-inductive cool temperatures. In Arabidopsis, up-regulation of AP and LFY expression promotes the initiation of floral organs (Mandel and Yanofsky, ; Weigel and Nilsson, ). Orthologs of AP and LFY had been isolated in C. sinensis (Pillitteri et al., 00b) and overexpression of these genes in C. unshiu resulted in accelerated flowering, suggesting a role of these in genes in the regulation of flowering in citrus. In C. sinensis trees exposed to floral induction by low temperatures, transcript accumulation of CsAP and CsLFY remained unchanged from initial levels until the floral-inductive treatment was over and trees were transferred to growth promoting conditions when levels of CsAP and CsLFY transcripts increased (Pillitteri et al., 00a). We hypothesized that a similar pattern of accumulation of CsAP and CsLFY transcripts would be induced by exposure to floral-inductive water

4 Page of deficit. Our results support a role of CsFT as a dynamic integrator of signals initiated by floral-inductive stimuli in C. sinensis. We also show that up-regulation of floral identity genes CsAP and CsLFY did not occur until the floral-inductive stimuli were removed, suggesting that floral induction and floral differentiation may not occur simultaneously. These results represent one of the earliest reports characterizing the effects of water-deficit on the expression of flowering genes in evergreen trees. Materials and Methods Experimental Conditions To determine the patterns of CsFT, CsSL, CsAP and CsLFY transcript accumulation during floralinductive water deficits, transcript levels of these genes were measured in - year-old potted Washington Navel rooted cuttings kept under water deficit for 0 days in a growth room at C illuminated with fluorescent lights (00µmolesm - s - at canopy level) with a /h (day/night) regime. Water deficit was imposed by withholding irrigation until the desired levels of water stress was reached; then, this level of water deficit was maintained by irrigating the trees daily with a volume of water that matched the daily weight loss of the trees. The water status of the trees was monitored using the midday stem water potential (SWP) measured by the pressure chamber method using covered leaves (McCutchan and Shackel, ; Scholander et al., ). Midday SWP of trees under water deficit were -.0±0.MPa whereas midday SWP in well irrigated (control) trees were -.±0.MPa. This level of water deficit was reached between day and 0 from the beginning of the experiment. On day 0 of the experiments, water deficit was relieved by irrigating all trees to soil saturation to promote growth; irrigation was the continued as in the well irrigated control trees. Samples were collected every - day from day 0 until day. This experiment was conducted using a completely randomized design with tree replicates. Differences in transcript accumulation of the selected genes between well irrigated and water deficit trees were analyzed using a repeated measurements model. Differences in accumulation of CsSL, CsAP and CsLFY

5 Page of transcripts after re-irrigation (day and ) between trees that had received normal irrigation or water deficit were analyzed using the t-test. New growth composition was characterized in all the - nodes long shoots formed during the previous year and currently present on the trees. Differences in the composition of the new growth between well irrigated and water deficit trees were analyzed using the t-test. To determine the patterns of CsFT, CsSL, CsAP and CsLFY transcript accumulation during floralinductive water deficit at floral-inductive temperatures, transcript levels of these genes were measured in - year-old Washington Navel rooted cuttings kept under water deficit (SWP = -.0MPa) for 0 days in a growth room at C with the same light and photoperiod conditions as previously indicated. The trees had been kept in a growth room at C for about month before transfer to the room at C. Water deficit, as described above, was imposed and monitored starting days before the transfer to the room at C while control trees remained well-irrigated. Forty days after transfer to the growth room at C, the trees were transferred back to the room at C and the water deficit was relieved. Samples were collected every - days from day 0 until day ( days after the end of the water deficit/low temperature treatment). This experiment was conducted using a completely randomized design with tree replicates. Differences in transcript accumulation of the selected genes between well irrigated and water deficit trees were analyzed using a repeated measurements model. Differences in accumulation of CsSL, CsAP and CsLFY transcripts after re-irrigation and transfer to C (day ) between trees that had received normal irrigation or water deficit were analyzed using the t-test. New growth was characterized in all the shoots - nodes long formed during the previous year on each tree. Differences in the composition of the new growth between well irrigated and water deficit trees were analyzed using the t-test. To determine the patterns of CsFT, CsSL, CsAP and CsLFY transcript accumulation in mature trees exposed to floral inductive conditions in the field, transcript levels of these genes were measured in mature Valencia sweet orange trees grafted on Carrizo citrange in an orchard at the University of Florida s Citrus Research and Education Center in Lake Alfred, Florida ( N, W) during the fall/winter of 00-. Samples were taken from trees under either normal irrigation (like neighboring commercial groves) or water deficit from mid-november to late-january. Water deficit was imposed in field trees by interrupting irrigation

6 Page of and covering the soil underneath their canopies with a sheet of impermeable material (Tyvek, DuPont) for the period indicated. The average temperature during this period was. C and warmer growth-promoting temperatures did not occur until late-february; bloom occurred in mid-march. The water status of the trees was monitored as before using the midday SWP. Midday SWP in trees under water deficit was -.±0.MPa whereas in well irrigated trees it was -.±0.MPa. By the end of January, the soil covers were removed and irrigation was resumed. This experiment was conducted using a completely randomized design with tree replicates. Differences in transcript accumulation of the selected genes between well irrigated and water deficit trees were analyzed using a repeated measurements model. T-tests were used to compare levels of accumulation of transcripts of the selected genes in well-irrigated and water deficit trees at selected points of interest. New growth was characterized in shoots - nodes long formed during the previous year on each tree. In all the experiments, accumulation of CsFT transcripts was quantified in leaf samples whereas accumulation of CsSL, CsAP and CsLFY transcripts was quantified in bud samples. This choice of tissues was made based on the most likely spatial domain of gene expression and protein activity according to reports in other species (Corbesier et al., 00; Wigge et al., 00). Leaf and bud samples consisted of a pool of at least leaves or buds at the sub-apical position from separate shoots on each tree replicate. Samples were collected from the sub-apical position because of the higher probability of developing flowers at this position than other positions (Sauer, ; Valiente and Albrigo, 00). All samples were collected at :00 local standard time (UTC-). qrt-pcr Total RNA was extracted by a phenol-chloroform precipitation method and purified on silica membranes with on-column DNase digestion (Qiagen). Five hundred nanograms of total RNA were used for cdna synthesis in a 0µl reaction with oligo dt primers (SuperScriptIII ;, Invitrogen). One microliter of the synthesized cdna was used for qpcr in a 0µl reaction (SYBR ; Premix ExTaq II, Takara) on an Applied Biosystems 00 FAST real-time PCR system (Life Technologies) using optimized two-step qpcr assays ( C

7 Page of denaturation for seconds and 0 C for minute annealing and extension). Primers for qpcr were: - CGGCGGAAGGACTATGAC- and -TGTGAGAAAGCCAGAGAGGAA- (CsFT), - CAGCCAGAGAATCTAACAAACG- and -TCAGTTTTGTGGTGGTATTGCC- (CsSL), - CCCTGGAGTGCAACAACCT- and -CTGATGTGTTTGAGAGCGGT- (CsAP), and - TCTTGATCCAGGTCCAGAACATC- and -TAGTCACCTTGGTTGGGCATT- (CsLFY ). CsGAPDH was used as the reference gene ( -GGAAGGTCAAGATCGCAATCAA- and - CGTCCCTCTGCAAGATGACTCT- ). All qpcr assays were validated for specific amplification and optimized for amplification efficiencies between. and.0 with a linear dynamic range of log cycles. The sequence of the primers to amplify CsLFY was obtained from Nishikawa et al. (00) whereas all other primer sequences were designed in-house from published sequences available in the GenBank (Benson et al. 00). Relative gene expression was calculated as a fold change ratio using Pfaffl s method (Pfaffl, 00) with sliding-window efficiencies calculated for each reaction using the sliwin function in the qpcr R package (Ritz and Spiess, 00). Data Analysis Mean fold change of transcript levels were transformed to a logarithmic scale (log) for statistical analysis but the untransformed data are presented in the graphs. Unless noted otherwise, all differences reported are statistically significant (p<0.0). All statistical analyses were executed in R (R Development Core Team, 0). Results Water deficit effects on the accumulation of CsFT, CsSL, CsAP and CsLFY transcripts. Exposure to the -MPa water deficit for 0 days induced flowering in the potted Washington Navel trees. Between and days after water deficit was relieved by re-irrigation, the trees produced a flush of growth composed of a mixture of inflorescences and vegetative shoots whereas the well-irrigated control

8 Page of trees produced only a few isolated inflorescences (Table ). Accumulation of CsFT transcripts increased with time in trees under water deficit whereas in well-irrigated trees accumulation of CsFT transcripts remained mostly unchanged from initial levels (Fig. ). Differences in the accumulation of CsFT transcripts between well-irrigated and water deficit trees was evident starting on day from the beginning of the treatment. Three days after the re-watering, the accumulation of CsFT transcripts in trees previously exposed to water deficit decreased to the initial control level and remained at this level for at least another days. There were no changes in the accumulation of CsSL transcripts in buds during water deficit but after re-irrigation, CsSL transcripts increased transiently. The accumulation of CsAP and CsLFY transcripts in buds was slightly reduced while trees were under water deficit. Three days after re-irrigation, CsAP and CsLFY transcripts increased transiently and returned to initial levels days later. Accumulation of CsFT, CsSL, CsAP and CsLFY transcripts under water deficit at cool floralinductive temperatures Trees exposed to water deficit at C produced more inflorescences and flowers than trees that received normal irrigation at the same temperature (Table ). Both groups of trees developed a flush of a mixture of inflorescences and vegetative growth between and 0 days after irrigation was re-established and/or trees were transferred to C. Accumulation of CsFT transcripts increased with time during water deficit and/or cool temperatures and returned to control/initial levels when both stimuli were removed (Fig. ). Accumulation of CsFT transcripts was significantly higher in trees under water deficit than in well-irrigated trees and the net fold change in CsFT transcripts after 0 days of treatment was higher than the fold change after exposing trees to only cool temperatures (Fig. ) or to water deficit at C (Fig. ). The pattern of CsSL, CsAP and CsLFY transcript accumulation in trees under water deficit at C was similar to the pattern observed at C (Fig. ). There was a slight reduction in the amount of transcripts of

9 Page of these genes while under water deficit/cool temperatures that was followed by an sharp increase after both stimuli were removed. In well-irrigated trees at C, accumulation of CsSL transcripts increased from day 0 until transfer to C. Levels of CsAP and CsLFY were slightly lower than initial levels while at C but also increased after transfer to C. After re-irrigation and transfer to C, levels of CsSL, CsAP and CsLFY transcripts were higher in trees that had previously been under water deficit than in continuously well-irrigated trees. Accumulation of flowering-related genes transcripts in field-grown trees exposed to floral-inductive conditions Trees exposed to water deficit during the fall/winter in 00- produced more flowers and inflorescences than shoots that received normal irrigation during the same period (Table ). Well-irrigated trees bloomed in mid-march whereas trees previously exposed to water deficit bloomed about days later. Accumulation of CsFT transcripts was not statistically different between trees under water deficit and wellirrigated trees except for one sampling date (Fig. ). Two discrete instances of increasing accumulation of CsFT transcripts were detected in November and from early-december until mid-january. These two periods of increasing CsFT transcript accumulation coincided with periods of consistent cool weather, whereas the end of these periods of increasing CsFT transcript accumulation coincided with periods of warmer weather. In well-irrigated trees, the accumulation of CsSL transcripts followed a similar pattern as the accumulation of CsFT transcripts, with two discrete periods of increasing accumulation of CsSL transcripts coinciding with those reported before for CsFT (Fig. ). In trees under water deficit, however, accumulation of CsSL transcripts was slightly lower than initial levels and remained mostly constant during the experiment. In well-irrigated trees, accumulation of CsAP transcripts remained unchanged from initial levels except for two peaks that occurred following periods of relatively warm weather and/or precipitation (similar to those reported earlier for CsFT and CsSL transcripts, Fig. ). Accumulation of CsLFY transcripts was slightly reduced below control levels for most of the experiment and had a single peak in expression that coincided

10 Page of with the first peak reported for CsAP (Fig. ). In trees under water deficit, accumulation of CsAP transcripts remained mostly unchanged while accumulation of CsLFY transcripts was slightly reduced but was more variable than CsAP accumulation during the experiment. Discussion Increased accumulation of CsFT transcripts was a common response to floral induction in C. sinensis Increasing accumulation of CsFT transcripts in leaves of trees exposed to water deficit (Fig. ) indicated that the mechanism regulating CsFT expression is responsive to signals initiated by water deficit and cool temperature as has been reported elsewhere (Nishikawa et al., 00). In Arabidopsis, the protein encoded by FT is a mobile flowering signal generated in the leaves that activate, along with FD, the expression of floral identity genes in the apex, initiating as a consequence floral development (Samach et al., 000; Abe et al., 00; Corbesier et al., 00). Increased expression of CsFT in response to cool temperatures has been reported before in Citrus (Nishikawa et al., 00) but there had been no reports of the response of CsFT to water deficit, the only other factor known to induce flowering in Citrus. Expression of FT is up-regulated by exposure to long days (Corbesier et al., 00; Valverde et al., 00) in several long day plants primarily by the activity of CONSTANS (CO), a transcription factor whose peak of accumulation occurs at the end of the day and is stabilized by light (Suárez-Lopez et al., 00; Valverde et al., 00). The mechanism regulating flowering in response to long days in Arabidopsis has been investigated extensively and key processes in this mechanism have been devised (Turck et al., 00). However, little is known about the potential mechanisms promoting flowering through FT in response to other stimuli (Helliwell et al., 00; Balasubramanian et al., 00; Kim et al., 00; Tiwari et al., 0). Since flowering in C. sinensis is not considered to be dependent on photoperiod but initiated only in response to cool temperatures (<0 C) and water deficit (Cassin et al., ; Moss, ); thus,

11 Page of the model currently accepted (Turck et al., 00) for the regulation of photoperiodic flowering in other species cannot be directly extrapolated to explain flowering in Citrus. Assuming CsFT is also a mobile flowering signal in C. sinensis, its increased expression during floral induction must be regulated by factors responsive to cool temperatures and water deficit. Accumulation of CsFT transcripts in leaves exposed to simultaneous cool temperatures and water deficit was higher than in leaves exposed to either of the stimuli separately (Figs. and ). Thus, the mechanism regulating CsFT transcript accumulation responded at least in a positive additive way to signals initiated by both stimuli. The effects of temperature and the water status on flowering responses in C. sinensis have been reported to be interactive with positive additive effects at specific combinations of levels of both factors (Chica, 00). A common link between floral induction by cool temperatures and water deficit has unsuccessfully been sought after by other researchers (Southwick and Davenport, ; Koshita and Takahara, 00). Such a common link could be an indicator of the total overall level of floral induction in Citrus. Our results show that the pattern of accumulation of CsFT transcripts could be a common indicator of the level of floral induction of C. sinensis trees regardless of whether the signals originated from low temperature or water deficits. Further support for this hypothesis comes from the accumulation of CsFT transcripts that increased as the duration of the floral inductive treatments increased which is consistent with the correlation between the duration of floral induction and the number of inflorescences formed after induction (Moss, ; Southwick and Davenport,, Valiente and Albrigo, 00). Accumulation of CsSL transcripts was responsive to cool temperature signals but not water deficit signals during floral induction CsSL is the putative ortholog of Arabidopsis SOC and also seems to be involved in flowering in Citrus (Tan and Swain, 00). In Arabidopsis, SOC is responsive to signals from all known floral-promoting pathways (Lee and Lee, 0) and promotes the up-regulation of CsAP and CsLFY in the meristem (Samach et al., 000). Our results show that in C. sinensis, cool temperatures but not water deficit induced higher accumulation of CsSL transcripts during floral induction (Figs. and ). Furthermore, water deficit

12 Page of could also have an inhibited the accumulation of CsSL transcripts since accumulation of CsSL transcripts in trees under water deficit at C remained whereas it increased in well-irrigated trees with time (Fig. ). A similar response was also observed in field trees exposed to water deficit during the winter (Fig. ). Thus, different from Arabidopsis, the putative C. sinensis SOC ortholog, CsSL, maybe a common integrator of floral-promoting signals initiated by different stimuli, but in the case of water deficit it is only operative at the beginning of floral differentiation after water stress has been relieved. Increased accumulation of CsAP and CsLFY transcripts occurred only after floral induction was over In Arabidopsis, up-regulation of AP and LFY in the meristem marks the shift from vegetative to reproductive development (Mandel and Yanofsky, ; Weigel and Nilsson, ). CsAP and CsLFY are the C. sinensis putative orthologs of Arabidopsis AP and LFY (Pillitteri et al., 00b). An increase in the accumulation of CsAP and CsLFY transcripts in C. sinensis trees exposed to cool floral-inductive temperatures only occurred towards the end of the floral inductive treatment (Pillitteri et al. 00a). Our results show a similar response when water deficit was used to induce flowering (Figs. ) and also when trees were exposed to both water deficit and cool temperatures (Figs. ). Assuming that the function of AP and LFY is conserved in Arabidopsis and C. sinensis, up-regulation of the expression of CsAP and CsLFY could be an indicator of floral determination (Hempel et al., ). In C. sinensis trees growing in subtropical climates, flowering is induced during the fall/winter and floral bud differentiation is not visibly evident until late in the winter (Abbot, ; Lord and Eckard, ). This is different from deciduous species where floral buds are formed before the winter. Nishikawa et al. (00) reported that accumulation of FT and LFY orthologs transcripts in C. unshiu and P. trifoliata was correlated with the periods of floral induction and differentiation even though C. unshiu is an evergreen and P. trifoliata is deciduous. However, no apparent correlation between the pattern of accumulation of the AP ortholog was found (Nishikawa et al., 00). In our experiments with potted trees (Figs. and ), both AP and

13 Page of LFY transcripts accumulated to higher levels after the floral inductive treatments and their pattern of transcript accumulation was similar throughout the experiment. In field trees during the winter (Figs. ), however, the accumulation of CsAP and CsLFY was slightly different from each other but in both cases it correlated with warmer and rainier weather which promotes floral differentiation (Valiente, 00). Similar to the response of CsSL transcripts, water deficit also seems to reduce the accumulation of CsAP and CsLFY transcripts. In both growth chamber experiments (Figs. and ), accumulation of CsAP and CsLFY transcripts during induction was lower in trees under water deficit than in well-irrigated trees. In the field, levels of CsAP and CsLFY transcripts remained low in trees under water deficit and did not increase as in well-irrigated trees in response to warmer weather. Thus, it is possible that floral-inductive conditions negatively regulated the onset of floral differentiation or that warmer temperatures and moisture are required for its initiation. Interestingly, from our results in the growth chamber, accumulation of CsAP and CsLFY transcripts after induction was increased and was highest in trees exposed to both water deficit and cool temperatures, indicating that the intensity at which floral induction occurred could be related to the level of up-regulation of CsAP and CsLFY following induction. Conclusions Water deficit induced increasing accumulation of CsFT transcripts in C. sinensis leaves similar to previous reports in response to cool temperatures (Nishikawa et. al. 00). Furthermore, when water deficit and cool temperatures were applied simultaneously to C. sinensis trees, the accumulation of CsFT transcripts in leaves was higher than when either water deficit or cool temperatures were applied separately. Thus, CsFT could be a common integrator of flowering signals initiated by these two stimuli in C. sinensis. Conversely, accumulation of the putative floral identity gene transcripts (CsAP, CsLFY) was reduced or remained unchanged while the trees were exposed to water deficit and cool temperatures and increased only after these stimuli were removed and the opposite conditions (moisture and warmer temperatures) occur, suggesting that floral induction and floral initiation are promoted by opposite stimuli.

14 Page of References Abbot, C. E... Blossom-bud differentiation in citrus trees. Amer. J. Bot. :. Abe, M., Y. Kobayashi, S. Yamamoto, Y. Daimon, A. Yamaguchi, Y. Ikeda, H. Ichinoki, M. Notaguchi, K. Goto, and T. Araki. 00. FD, a bzip protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 0:. Albrigo, L. G. and V. Galen-Saúco. 00. Flower bud induction, flowering and fruit-set of some tropical and subtropical fruit tree crops with special reference to citrus. Acta Hort. :. Albrigo, L. G., J. I. Valiente, and C. Van Parys de Wit. 00. Influence of winter and spring weather on year-to-year citrus fruit set and yield variation in São Paulo, Brazil. In: A. El-Otmani, M; Ait-Oubahou (ed.) Proceedings of the International Society of Citriculture, volume, pp.. Agadir, Morocco. Balasubramanian, S., S. Sureshkumar, J. Lempe, and D. Weigel. 00. Potent induction of Arabidopsis thaliana flowering by elevated growth temperature. PLoS Genetics :e. Benson, D. A.; I. Karsch-Mizrachi; D. J. Lipman, D. J.; J. Ostell and D. L. Wheeler. 00. GenBank. Nucleic Acids Res. :D-D. Cassin, J., J. Bourdeuet, A. Fougue, V. Furon, J. P. Gailland, J. Bourdelles, G. Montaguad, and C. Moreuil.. The influence of climate upon the blooming of citrus in tropical areas. In: H. D. Chapman (ed.) Proceedings of the First International Citrus Symposium, volume, pp.. Riverside, California (USA). Chica, E Studies on Some Factors Affecting Flower Bud Induction in Sweet Orange (Citrus sinensis Osbeck): Cold, Drought and Removal of Terminal Buds. University of Florida, Gainesville. Master s thesis. Corbesier, L., C. Vincent, S. Jang, F. Fornara, Q. Fan, I. Searle, A. Giakountis, S. Farrona, L. Gissot,

15 Page of C. Turnbull, and G. Coupland. 00. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science :0. Endo, T., T. Shimada, H. Fujii, Y. Kobayashi, T. Araki, and M. Omura. 00. Ectopic expression of an FT homolog from citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). Transgenic Research :0. Helliwell, C. A., C. C. Wood, M. Robertson, W. James Peacock, and E. S. Dennis. 00. The arabidopsis FLC protein interacts directly in vivo with SOC and FT chromatin and is part of a high-molecular-weight protein complex. Plant J. :. Hempel, F., D. Weigel, M. Mandel, G. Ditta, P. Zambryski, L. Feldman, and M. Yanofsky.. Floral determination and expression of floral regulatory genes in Arabidopsis. Development :. Kim, S.-G., S.-Y. Kim, and C.-M. Park. 00. A membrane-associated NAC transcription factor regulates salt-responsive flowering via FLOWERING LOCUS T in Arabidopsis. Planta :. Kobayashi, Y., H. Kaya, K. Goto, M. Iwabuchi, and T. Araki.. A pair of related genes with antagonistic roles in mediating flowering signals. Science :0. Koshita, Y. and T. Takahara. 00. Effect of water stress on flower-bud formation and plant hormone content of Satsuma mandarin (Citrus unshiu Marc.). Sci. Hortic. :0 0. Lee, J. and I. Lee. 0. Regulation and function of SOC, a flowering pathway integrator. J. Exp. Bot. :. Lord, E. M. and K. J. Eckard.. Shoot development in citrus sinensis l. (washington navel orange). i. floral and inflorescence ontogeny. Bot. Gaz. :0. Mandel, M. A. and M. Yanofsky.. A gene triggering flower formation in arabidopsis. Nature :.

16 Page of McCutchan, H. and K. Shackel.. Stem water potential as a sensitive indicator of water stress in prune trees (Prunus domestica L. cv. French). J. Amer. Soc. Hort. Sci. :0. Moss, G. I... Influence of temperature and photoperiod on flower induction and inflorescence development in sweet orange (Citrus sinensis l. osbeck). J. Hort. Res. : 0. Nishikawa, F., T. Endo, T. Shimada, H. Fujii, T. Shimizu, and M. Omura. 00. Differences in seasonal expression of flowering genes between deciduous trifoliate orange and evergreen Satsuma mandarin. Tree Physiol :. Nishikawa, F., T. Endo, T. Shimada, H. Fujii, T. Shimizu, M. Omura, and Y. Ikoma. 00. Increased CiFT abundance in the stem correlates with floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.). J. Exp. Bot. :. Pfaffl, M. W A new mathematical model for relative quantification in real-time RT-PCR. Nucl. Acids Res. :e. Pillitteri, L. J., C. J. Lovatt, and L. L. Walling. 00a. Isolation and characterization of a TERMINAL FLOWER homolog and its correlation with juvenility in citrus. Plant Physiol. :0. Pillitteri, L. J., C. J. Lovatt, and L. L. Walling. 00b. Isolation and characterization of LEAFY and APETALA homologues from Citrus sinensis L. Osbeck Washington. J. Amer. Soc. Hort. Sci. :. R Development Core Team. 0. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN Ritz, C. and A. N. Spiess. 00. qpcr: an R package for sigmoidal model selection in quantitative realtime polymerase chain reaction analysis. Bioinformatics :. Samach, A., H. Onouchi, S. E. Gold, G. S. Ditta, Z. Schwarz-Sommer, M. F. Yanofsky, and G. Coupland Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science

17 Page of :. Sauer, M. R.. Flowering in the sweet orange. Aust. J. Agric. Res. :-. Scholander, P. F., A. D. Hammel, A. D. Bradstreet, and E. A. Hemmingsen.. Sap pressure in vascular plants. Science :. Southwick, S. M. and T. L. Davenport.. Characterization of water stress and low temperature effects on flower induction in citrus. Plant Physiol. :. Suárez-Lopez, P., K. Wheatley, F. Robson, H. Onouchi, F. Valverde, and G. Coupland. 00. CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. Nature :. Tan, F.-C. and S. M. Swain. 00. Functional characterization of AP, SOC and WUS homologues from citrus (Citrus sinensis). Physiol. Plant. :. Tiwari, S. B., Y. Shen, H.-C. Chang, Y. Hou, A. Harris, S. F. Ma, M. McPartland, G. J. Hymus, L. Adam, C. Marion, A. Belachew, P. P. Repetti, T. L. Reuber, and O. J. Ratcliffe. 0. The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element. New Phytologist :. Turck, F., F. Fornara, and G. Coupland. 00. Regulation and identity of florigen: Flowering locus t moves center stage. Annual Review of Plant Biology :. Valiente, J. I Timing and intensity of flowering of sweet orange [Citrus sinensis (L.) Osbeck] as a function of local weather factors and crop under central Florida conditions. University of Florida, Gainesville. Ph.D. thesis. Valiente, J. I. And L. G. Albrigo. 00. Flower bud induction of sweet orange trees [Citrus sinensis (L.) Osbeck]: effect of temperature, crop load and bud age. J. Amer. Soc. Hort. Sci., :.

18 Page of Valverde, F., A. Mouradov, W. Soppe, D. Ravenscroft, A. Samach, and G. Coupland. 00. Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 0:0 0. Weigel, D. and O. Nilsson.. A developmental switch sufficient for flower initiation in diverse. Nature : 00. Wigge, P. A., M. C. Kim, K. E. Jaeger, W. Busch, M. Schmid, J. U. Lohmann, and D. Weigel. 00. Integration of spatial and temporal information during floral induction in arabidopsis. Science 0:.

19 Page of Figure captions: Figure : Transcript accumulation of CsFT, CsSL, CsAP and CsLFY in - year old Washington Navel cuttings under water deficit at C. Transcript accumulation was monitored in leaves (CsFT) and buds (CsSL, CsAP and CsLFY ) of months-old or older shoots using qrt-pcr. Water deficit was removed on day 0 (grey line). Symbols represent the means of tree-replicates ± S.E. ( 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. Figure : Transcript accumulation of CsFT, CsSL, CsAP and CsLFY in - year old Washington Navel cuttings under water deficit at C. Transcript accumulation was monitored in leaves (CsFT) and buds (CsSL, CsAP and CsLFY ) of months-old or older shoots using qrt-pcr. Trees were kept at C for 0 days (shaded area). Water deficit was removed on day 0 (grey line) and trees were kept at C afterwards. Symbols represent the means of tree-replicates ± S.E. ( 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. Figure : Transcript accumulation of CsFT, CsSL, CsAP and CsLFY in mature (> year-old) field-grown grafted Valencia trees exposed to water deficit during the fall/winter of 00-. Water deficit was applied to the trees from mid-november to late-january. Symbols represent the means of tree-replicates ± S.E. ( shoots/tree). Transcript accumulation is relative to the levels of CsGAPDH and to the level of each gene in Well irrigated trees at the beginning on November.

20 Page 0 of Table : Characteristics of the new growth on - node shoots in - year-old Washington Navel cuttings exposed to water deficit for 0 days at C and then re-irrigated. La and Ld refer respectively to leaf abundant and leaf deficient inflorescences based on their leaf/flower ratios (La, Ld <). Single refers to single flowers without leaves. Values are means per shoot ± S.E. ( treereplicates, - shoots/tree). The average number of nodes per shoot was.±0. Inflorescences La Ld Leafless Single Vegetative Flowers Irrigated 0. ± ± ± ± ±0.0 0 ±0 0. ±0. Water deficit.0 ±0.. ± ± ± ±0.0. ±0.. ±0.

21 Page of Table : Characteristics of the new growth on - node shoots in - year-old Washington Navel cuttings exposed to water deficit for 0 days at C and then re-irrigated. La and Ld refer respectively to leaf abundant and leaf deficient inflorescences based on their leaf/flower ratios (La, Ld <). Single refers to single flowers without leaves. Values are means per shoot ± S.E. ( treereplicates, - shoots/tree). The average number of nodes per shoot was.±0. Inflorescences La Ld Leafless Single Vegetative Flowers Irrigated. ±0.0.0 ± ± ± ± ±0.. ±0. Water deficit. ±0.. ± ± ± ± ±0.. ±0.

22 Page of Table : Characteristics of the new growth on - node shoots in field-grown Valencia trees exposed to water deficit during the fall/winter in 00-. La and Ld refer respectively to leaf abundant and leaf deficient inflorescences based on their leaf/flower ratios (La, Ld <). Single refers to single flowers without leaves. Figures are means per shoot of tree-replicates± S.E. ( treereplicates, shoots/tree). The average number of nodes per shoot was.±0. Inflorescences La Ld Leafless Single Vegetative Flowers Irrigated.±0. 0.±0.0 0.±0. 0.±0. 0.±0.0 0.±0..0±. Water deficit.±0. 0.±0.0 0.±0..±0. 0.±0.0 0.±0.0.±.

23 Page of Figure : Transcript accumulation of CsFT, CsSL, CsAP and CsLFY in - year old Washington Navel cuttings under water deficit at C. Transcript accumulation was monitored in leaves (CsFT) and buds (CsSL, CsAP and CsLFY ) of months-old or older shoots using qrt-pcr. Water deficit was removed on day 0 (grey line). Symbols represent the means of tree-replicates ± S.E. ( 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. xmm (00 x 00 DPI)

24 Page of Figure : Transcript accumulation of CsFT, CsSL, CsAP and CsLFY in - year old Washington Navel cuttings under water deficit at C. Transcript accumulation was monitored in leaves (CsFT) and buds (CsSL, CsAP and CsLFY ) of months-old or older shoots using qrt-pcr. Trees were kept at C for 0 days (shaded area). Water deficit was removed on day 0 (grey line) and trees were kept at C afterwards. Symbols represent the means of tree-replicates ± S.E. ( 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. xmm (00 x 00 DPI)

25 Page of Figure : Transcript accumulation of CsFT, CsSL, CsAP and CsLFY in mature (> year-old) fieldgrown grafted Valencia trees exposed to water deficit during the fall/winter of 00-. Water deficit was applied to the trees from mid-november to late-january. Symbols represent the means of tree-replicates ± S.E. ( shoots/tree). Transcript accumulation is relative to the levels of CsGAPDH and to the level of each gene in Well irrigated trees at the beginning on November. xmm (00 x 00 DPI)