Re-Evaluation of Florigen Transport Kinetics with Separation of Functions by Mutations That Uncouple Flowering Initiation and Long-Distance Transport

Re-Evaluation of Florigen Transport Kinetics with Separation of Functions by Mutations That... Abstract In many plants, timing of flowering is regulated by day length. In Arabidopsis, florigen, FLOWERING LOCUS T (FT) protein, is synthesized in leaf phloem companion cells in response to long days and is transported to the shoot apical meristem (SAM) through the phloem. The temporal aspects of florigen transportation have been studied in various plants by physiological experiments. Nevertheless, little is known about how FT protein transportation is regulated in Arabidopsis. In this study, we performed heat shock-based transient FT induction in a single leaf blade and detected the FT protein in the shoot apex by 2D-PAGE. We demonstrated that detectable amounts of FT were transported from the leaf to the shoot apex within 8 h, and subsequent FT-induced target gene expression was detected within 8–12 h. Furthermore, we identified three amino acid residues (V70, S76 and R83) where missense mutations led to reduced mobility. Interestingly, these FT variants lost only their transportation ability, but retained their flowering promotion capacity, suggesting that discrete amino acids are involved in flowering regulation and transport regulation. Since the interaction with FT-INTERACTING PROTEIN 1 (FTIP1) was not affected in these FT variants, we hypothesize that the three amino acid residues are not involved in the FTIP1-mediated pathway of uploading, but rather in the subsequent step(s) of FT transport. Introduction Flowering in the appropriate season is a crucial strategy for plant reproduction. To achieve efficient temporal regulation of flowering, many plant species use photoperiod signals as indicators of seasonal changes (Song et al. 2013). Inductive day lengths perceived by leaves induce the production of the flowering hormone, florigen, in phloem companion cells (Turck et al. 2008, Liu et al. 2013). Phloem-loaded florigen is then transported from the source to sink tissues, including the shoot apical meristem (SAM), where it promotes flowering. Many photoperiodic flowering plants are classified based on the responses to different photoperiod, e.g. short-day plants and long-day plants. Since short-day plants are more sensitive to environmental light changes such as an interruption of night-time darkness (‘night break’) or photoperiod changes, a considerable number of studies have been carried out on short-day plants including Japanese morning glory and soybean (Imamura 1967, Thomas and Vince-Prue 1997). From these studies, physiological features of florigen transportation, such as the velocity of florigen transportation, have been estimated. The florigen transportation rate in phloem was originally estimated to be about 3 mm h–1 (Imamura and Takimoto 1955, Zeevaart 1962, Zeevaart 2006), but this was later re-estimated to be as fast as 30–50 cm h–1 (Takeba and Takimoto 1966, King et al. 1968), which is almost the same speed as phloem flux (Canny 1975, Windt et al. 2006). In contrast to the rapid florigen transportation, however, florigen uploading and/or unloading steps take considerably more time. In Japanese morning glory (a short-day plant), for example, flowering was fully induced by a 14 h short-day treatment. However, when the leaf was immediately cut after the 14 h short-day treatment, the induced flowering was completely abrogated. When the leaf was cut after an 18 h short-day treatment, flowering was fully induced (Zeevaart 1962), which is to say that transport is fast, while unloading and induction are relatively slow. In the facultative long-day plant Arabidopsis thaliana, the FLOWERING LOCUS T (FT) protein is known to be the major component of florigen (Abe et al. 2005, Wigge et al. 2005, Corbesier et al. 2007, Jaeger and Wigge 2007, Mathieu et al. 2007, Notaguchi et al. 2008). Previous studies have demonstrated that FT protein can be transported from the leaf to the shoot apex and from the stock to the scion in grafting, as has been observed in other plant species (Corbesier et al. 2007, Notaguchi et al. 2008, S.J. Yoo et al. 2013). However, we have only begun to understand how FT protein transport from the leaf to the shoot apex is regulated. Two factors which play sequential, but independent roles in FT transport are known. One is an endoplasmic reticulum membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1), which was identified as a regulator of FT protein uploading from phloem companion cells to sieve elements (Liu et al. 2012). The other is a heavy metal-associated plant protein, SODIUM POTASSIUM ROOT DEFECTIVE1 (NaKR1), which interacts with FT and is required for FT transport in the phloem stream (Zhu et al. 2016). Recent works revealed mechanisms for co-ordinated production and transport of FT. A Myb transcription factor FE/ALTERED PHLOEM DEVELOPMENT(FE/APL) activates transcription of FT, FTIP1 and NaKR1 (Abe et al. 2015, Shibuta and Abe 2017), thereby promoting both production and transport of florigen. NaKR1 also plays an important role in FT expression through microRNA156 (miR156) and miR156-targeted transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), besides its role in FT transport (Negishi et al. 2018). However, little is known about the temporal aspect of FT transport and the structural property of FT important for transportation. In this work, we show that detectable amounts of FT protein are loaded onto phloem within 8 h after transient FT induction in a leaf, and FT protein subsequently accumulates in the SAM within 12 h after induction. Furthermore, FT-regulated APETALA1 (AP1), SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and FRUITFULL (FUL) (Schmid et al. 2003, Abe et al. 2005, Wigge et al. 2005, Torti et al. 2012) expression is induced within 8–12 h after FT induction. From alanine scanning mutagenesis of FT, we have identified three FT variants that display severely reduced transportation. Nonetheless, these FT variants can still induce flowering when they are ectopically expressed in the shoot apex, indicating that specific amino acid residues in FT carry out separate functions. These three amino acid residues are located on the surface of FT protein, but they are not crucial for the interaction with FTIP1, suggesting that they are not involved in the FTIP1-mediated pathway of uploading. Taken together with previous work which demonstrated phloem transport of an FT variant harboring all three of these amino acid substitutions in pumpkin (S.C. Yoo et al. 2013), we hypothesize that these three amino acid residues may be involved in the subsequent unloading step. Results Estimation of time for florigen uploading To elucidate the timeline of florigen transportation in terms of time required for each step, we utilized a local transient florigen induction system in Arabidopsis, as described previously (Notaguchi et al. 2008, Abe et al. 2015). In this system, transportable FT-T7 can be transiently expressed by the HEAT SHOCK PROTEIN 18.2 (HSP) promoter in the ft-1 background (HSP::FT-T7), and FT-T7 protein accumulation was observed 1 h after the end of heat treatment. We first estimated how quickly the FT-T7 is exported from a leaf blade after heat shock induction. Plants were grown under long-day conditions for 18 d and then subjected to a single-leaf heat shock (SLHS) treatment (38–39°C) at Zeitgeber time (ZT) 13 to ZT15. In parallel experiments, the FT-T7-induced leaf was then cut off 0, 4, 8, 12 and 24 h after the heat shock treatment, and the total leaf number was counted when the first flower bud opened. In response to the SLHS treatments, plants showed an early-flowering phenotype compared with the untreated control (Fig. 1). Furthermore, the slopes of the flowering rate curves were parallel when untreated, SLHS treated and the uncut groups were compared, suggesting that induced FT-T7 was the major determinant of flowering acceleration, and other factors such as age and heat shock stress were largely negligible (Fig. 1A). Among the SLHS-treated plants, using total leaf number as a proxy for flowering timing, a significant early-flowering phenotype was observed in plants when the treated leaf was cut off at any time between 8 and 24 h after the heat shock (Fig. 1B). Another indicator of flowering time, i.e. days to flower after heat shock treatment, also gave similar results (Supplementary Fig. S1A, D). Since phloem flux is relatively fast (>50 cm h–1), we assumed that only a brief time would be required for transport of florigen in the phloem from the induced leaf to the shoot apex. Therefore, we estimated that a sufficient quantity of florigen to affect flowering time was loaded onto the phloem within 8 h after transient FT induction. We also detected a slight acceleration of flowering even when the treated leaf was cut off after 4 h (Fig. 1; Supplementary Fig. S1), although the differences are not statistically significant. The scant difference between the ‘uncut’ control and plants with the heat-shocked leaf cut off after 24 h suggests that uploading was essentially completed in 24 h. The flowering time response to the SLHS induction of FT-T7 was similar even under short days, indicating that florigen transportation, uploading and unloading processes are independent of day length (Supplementary Fig. S1B, C, E, F). Fig. 1 View largeDownload slide The effect of heat shock induction of HSP::FT-T7 on flowering time under long-day conditions. Untreated, no heat shock induction and no cutting off of the leaf; Cut, no heat shock induction and cutting off of the leaf; 0 h to 24 h, SLHS induction and cutting off of the leaf 0–24 h after induction; Uncut, SLHS induction and no cutting off of the leaf. (A) Percentage of flowering plants with the indicated number of leaves (x-axis). This is a common proxy for timing the onset of flowering; early-flowering plants have fewer leaves at the onset of flowering. (B) Average time of flowering (number of leaves at the onset of flowering) after heat shock induction of HSP::FT-T7. Mean ± SEM (n ≥ 20). The P-values with respect to a control group (Cut or Pressed) were estimated by Dunnett’s test. Asterisks indicate statistical significance at P < 0.001. Fig. 1 View largeDownload slide The effect of heat shock induction of HSP::FT-T7 on flowering time under long-day conditions. Untreated, no heat shock induction and no cutting off of the leaf; Cut, no heat shock induction and cutting off of the leaf; 0 h to 24 h, SLHS induction and cutting off of the leaf 0–24 h after induction; Uncut, SLHS induction and no cutting off of the leaf. (A) Percentage of flowering plants with the indicated number of leaves (x-axis). This is a common proxy for timing the onset of flowering; early-flowering plants have fewer leaves at the onset of flowering. (B) Average time of flowering (number of leaves at the onset of flowering) after heat shock induction of HSP::FT-T7. Mean ± SEM (n ≥ 20). The P-values with respect to a control group (Cut or Pressed) were estimated by Dunnett’s test. Asterisks indicate statistical significance at P < 0.001. To corroborate these observations, we also detected FT-T7 protein accumulation in the shoot apex 24 h after the SLHS treatment, by 2D-PAGE (Notaguchi et al. 2008). Long-day-grown plants were subjected to SLHS induction, and the treated leaf blade was cut off after 0, 4, 8, 12 and 24 h (Fig. 2). Compared with the untreated and ‘pressed’ (clamped, but unheated) controls, markedly greater amounts of FT-T7 protein were detected in the shoot apex samples from plants when the treated leaf was cut off 24 h after the heat shock. Even when the treated leaf was removed 8 h after the induction, detectable amounts of FT-T7 protein accumulated in the shoot apex. Since a good correlation between flowering induction after SLHS and detectable FT-T7 amount at the shoot apex can be seen, we concluded that sufficient florigen to alter the flowering time was exported from the heat shock-treated leaf within 8 h. Fig. 2 View largeDownload slide 2D gel-based time-course analysis of FT-T7 protein export from the heat shock-induced leaf under long-day conditions. At 0, 4, 8, 12 and 24 h after the induction in 30 HSP::FT-T7 plants, the heat-shocked leaves were cut off and the 30 shoot apices were collected after 24 h of heat shock induction. Untreated, no heat shock induction and no excision of the leaf; Pressed, the leaf was just sandwiched into the unheated heating plate and the leaf was not cut off. Red circles show the FT-T7 spot, and blue circles show reference spots for precise alignment of the gel images. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 2 View largeDownload slide 2D gel-based time-course analysis of FT-T7 protein export from the heat shock-induced leaf under long-day conditions. At 0, 4, 8, 12 and 24 h after the induction in 30 HSP::FT-T7 plants, the heat-shocked leaves were cut off and the 30 shoot apices were collected after 24 h of heat shock induction. Untreated, no heat shock induction and no excision of the leaf; Pressed, the leaf was just sandwiched into the unheated heating plate and the leaf was not cut off. Red circles show the FT-T7 spot, and blue circles show reference spots for precise alignment of the gel images. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Estimation of the time required for florigen unloading We then estimated how rapidly the phloem-transported FT-T7 is unloaded onto the SAM. SLHS treatment was performed in long-day-grown HSP::FT-T7 plants, in a similar manner to that described above, except that the heat-shocked leaf was left on, and the shoot apex was collected 0, 4, 8, 12 and 24 h after the transient induction of FT-T7. The FT-T7 protein was detected 12 and 24 h after the induction, suggesting that florigen was accumulating in the shoot apex within 12 h (Fig. 3). Previous studies have demonstrated that florigen can be transported not only to the shoot apex but also to other organs (Zeevaart 1958, Navarro et al. 2011, Niwa et al. 2013). Hence, by 2D-PAGE, we analyzed FT-T7 accumulation in other leaves (Fig. 4). In the heat-treated leaf, FT-T7 was readily detected even 24 h after induction. To be able to detect FT-T7 in younger and older leaves, we loaded 10 times the amount of total protein onto the gel compared with that of the heat-treated leaf. We only detected a trace of FT-T7 in younger leaves, whereas we could not detect FT-T7 signals in older leaves and the untreated control with this experimental condition. These observations indicate that efficient FT unloading did not occur in neighboring leaves. The FT unloading efficiency is distinct at least in a leaf and in a shoot apex, implying that FT transportation is an active (targeted) process rather than a passive (non-targeted) process. Fig. 3 View largeDownload slide Time-course detection of FT-T7 protein accumulation in the shoot apex under long-day conditions. At 0, 4, 8, 12 and 24 h after induction of HSP::FT-T7, 30 shoot apices were collected and analyzed by 2D electrophoresis. The time points indicate the time after heat shock induction. Red circles show the FT-T7 spot, and blue circles show reference spots for precise alignment of the gel images. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 3 View largeDownload slide Time-course detection of FT-T7 protein accumulation in the shoot apex under long-day conditions. At 0, 4, 8, 12 and 24 h after induction of HSP::FT-T7, 30 shoot apices were collected and analyzed by 2D electrophoresis. The time points indicate the time after heat shock induction. Red circles show the FT-T7 spot, and blue circles show reference spots for precise alignment of the gel images. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 4 View largeDownload slide FT-T7 protein movement from source to sink leaves under long days. At 24 h after induction of HSP::FT-T7, proteins were extracted from pools of treated leaves (TL, four leaves), younger leaves (YL, seventh and eighth leaves) (40 leaves), older leaves (OL, first and second leaves) (40 leaves) and untreated leaves (20 leaves), respectively. Subsequently, 2D electrophoresis was performed. Red circles indicate the FT-T7 spot. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 4 View largeDownload slide FT-T7 protein movement from source to sink leaves under long days. At 24 h after induction of HSP::FT-T7, proteins were extracted from pools of treated leaves (TL, four leaves), younger leaves (YL, seventh and eighth leaves) (40 leaves), older leaves (OL, first and second leaves) (40 leaves) and untreated leaves (20 leaves), respectively. Subsequently, 2D electrophoresis was performed. Red circles indicate the FT-T7 spot. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. To confirm whether FT-T7 was unloaded onto the SAM within 12 h, we then monitored downstream gene expression. Florigen in the SAM induces several flowering-related genes such as AP1, SOC1 and FUL. Previous studies have demonstrated rapid increases of SOC1 expression in response to florigen (Borner et al. 2000, Samach et al. 2000, Schmid et al. 2003, Wigge et al. 2005, Searle et al. 2006). To detect the initiation of the expression of these genes in response to the heat shock induction, we applied quadruple-leaf heat shock (QLHS) treatment to produce enough florigen by a single shot to induce downstream genes. Samples from plants subjected to whole-plant heat shock (WPHS) treatment and untreated plants were included as controls. Gene expression in the shoot apex was then analyzed by reverse transcription followed by quantitative real-time PCR (RT–qPCR) (Fig. 5A). The expression of AP1 gradually increased 12 h after the QLHS, compared with endogenous circadian oscillation of AP1. The expression levels of SOC1 and FUL increased earlier than that of AP1, with a significant SOC1 response detectable 8 h after the QLHS. In the QLHS plants, these differences in induction times may be accounted for by the differential responsiveness of the respective genes to the FT-T7, because the WPHS treatment that induces florigen in the shoot apex has a distinctly different effect on the induction of these genes. We also examined ACTIN2 (ACT2) as a negative control and demonstrated that heat shock did not affect its gene expression profile. Fig. 5 View largeDownload slide Floral gene expression in response to heat shock induction under long days. Shoot apices of HSP::FT-T7 were harvested 0, 4, 8, 12 and 24 h after heat shock induction. Untreated, no heat shock induction; QLHS, quadruple-leaf heat shock; WPHS, whole-plant heat shock. (A) The samples were analyzed by relative quantification using RT–qPCR following MIQE recommendations (Bustin et al. 2009). RNA extraction was performed three times independently. Mean ± SEM (n = 3). Data points represent geometrical means (Vandesompele et al. 2002). Genes analyzed were AP1, SOC1, FUL and ACT2. ASPARTIC PROTEINASE A1 (APA1), ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 (IPP2) and TUBULIN BETA CHAIN 2 (TUB2) were used as controls (Endo et al. 2007, Michael et al. 2008, Hazen et al. 2005). (B) RNA in situ hybridization of longitudinal sections hybridized with either an AP1 or SOC1 probe. Scale bars = 200 µm. Fig. 5 View largeDownload slide Floral gene expression in response to heat shock induction under long days. Shoot apices of HSP::FT-T7 were harvested 0, 4, 8, 12 and 24 h after heat shock induction. Untreated, no heat shock induction; QLHS, quadruple-leaf heat shock; WPHS, whole-plant heat shock. (A) The samples were analyzed by relative quantification using RT–qPCR following MIQE recommendations (Bustin et al. 2009). RNA extraction was performed three times independently. Mean ± SEM (n = 3). Data points represent geometrical means (Vandesompele et al. 2002). Genes analyzed were AP1, SOC1, FUL and ACT2. ASPARTIC PROTEINASE A1 (APA1), ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 (IPP2) and TUBULIN BETA CHAIN 2 (TUB2) were used as controls (Endo et al. 2007, Michael et al. 2008, Hazen et al. 2005). (B) RNA in situ hybridization of longitudinal sections hybridized with either an AP1 or SOC1 probe. Scale bars = 200 µm. To confirm that AP1 and SOC1 expression was induced only in the shoot apex, we then examined RNA expression patterns by an RNA in situ hybridization (Fig. 5B). Clear SOC1 signals were detected in the shoot apex 1 d (24 h) after QLHS treatment. We could not detect AP1 signals in the shoot apex 1 d after QLHS treatment but could detect signals 2 d after QLHS in nascent floral meristems. The sequential expression of SOC1 and AP1 was similar in both RT–qPCR and the RNA in situ hybridization assays. Therefore, we concluded that substantial FT-T7 accumulated in the SAM within 12 h after induction. Amino acid residues of FT related to florigen transport Our results suggest that FT transport is actively regulated. If this is indeed the case, it is expected that some FT variants are defective in transport from the leaf to the SAM. To screen for such point mutations, we focused on differences between FT and TERMINAL FLOWER 1 (TFL1). Both FT and TFL1 proteins are members of the PEBP family and are both mobile signal molecules (Conti and Bradley 2007, Lin et al. 2007, Tamaki et al. 2007, Turck et al. 2008). However, TFL1 is a flowering repressor (Ratcliffe et al. 1998) and only has short-distance mobility in the SAM (Conti and Bradley 2007). From these facts, we hypothesized that the non-conserved amino acid residues between FT and TFL1 may be important for flowering induction and/or long-distance mobility (Hanzawa et al. 2005, Ahn et al. 2006, Ho and Weigel 2014). An amino acid sequence alignment of FT and TFL1 pointed to 47 residues to be tested (Supplementary Fig. S2A). To assess their functional significance, we substituted most of the residues with alanine, whereas G97 and A135 were replaced by aspartic acid and glycine, respectively. We first tested if these single amino acid-substituted FT variants (hereafter referred to as mFT) could induce flowering in response to the WPHS. As in the HSP::FT-T7 line, the 47 FT mutants with a T7-tag were driven by the HSP promoter (HSP::mFT-T7). We selected the first-generation transformants (T1) for each mutant FT, subjected them to WPHS treatment and the flowering time was scored (Supplementary Fig. S2B). Most of the transgenic plants flowered as early as the wild-type HSP::FT-T7 line in response to the WPHS, indicating that most of the substituted FT mutants still retained function as florigen, as previously reported (Ho and Weigel 2014). A FT 3D structure places a part of the region encoded by the second exon (V68–W88) and external loop (L128–N141) on the surface of the protein (Ahn et al. 2006) (Supplementary Fig. S3), suggesting that amino acids in these regions may engage in protein–protein interactions. Previous studies also demonstrated that the external loop was involved in florigen function as a flowering inducer (Hanzawa et al. 2005, Ahn et al. 2006). Therefore, we first focused on the second exon (V70A, S76A, S78A, H81A and R83A). For comparison, we analyzed three FT variants in the external loop (L128A, Y134A and W138A) and one FT variant in segment C (N152A). W138A is reported to abolish florigen function in sugar beets (Pin et al. 2010), and N152A may affect external loop structure and is required for florigen activity (Ahn et al. 2006, Ho and Weigel 2014). To evaluate the effects of these FT variants on flowering, we then investigated whether transiently expressed mFT-T7 in a leaf blade was able to reach the shoot apex within 24 h. TWIN SISTER OF FT (TSF) is also active under long-day conditions (Yamaguchi et al. 2005, Hiraoka et al. 2013); therefore, we performed these experiments under non-inductive short-day conditions to make the mFT-T7 effects clear. It allowed us clearly to observe the promotion of flowering by induction of florigen production and to detect FT-T7 transport in the shoot apex within 24 h (Supplementary Fig. S4). All transgenic plants expressing these FT variants showed similar flowering times under untreated conditions (Fig. 6). The V70A, S76A, S78A, H81A, R83A and N152A mutants were able to induce flowering in response to the WPHS treatment, as was the wild-type FT-T7 (designated as ‘FT’). However, L128A, Y134A and W138A did not respond to the WPHS treatment and flowered as late as the untreated control, suggesting that these amino acids are important for flowering induction. Interestingly, V70A, S76A, H81A, R83A and N152A could not fully respond to the SLHS treatment, whereas wild-type FT-T7 and S78A showed similar flowering times to the WPHS treatment. Fig. 6 View largeDownload slide The effect of V70A, S76A, S78A, H81A, R8A, L128A, Y134A, N152A and W138A on flowering time under short days after SLHS and WPHS treatments. FT indicates non-substituted HSP::FT-T7, and the other labels indicate the FT variants with the respective single amino acid substitutions. Mean ± SEM (n ≥ 10). Fig. 6 View largeDownload slide The effect of V70A, S76A, S78A, H81A, R8A, L128A, Y134A, N152A and W138A on flowering time under short days after SLHS and WPHS treatments. FT indicates non-substituted HSP::FT-T7, and the other labels indicate the FT variants with the respective single amino acid substitutions. Mean ± SEM (n ≥ 10). To test if these FT variants in the second exon failed to move from the leaf to the shoot apex, we tried to detect mFT-T7 protein in the shoot apex 24 h after the SLHS treatment (Fig. 7). In all transgenic plants, significant mFT-T7 signal was detected in the shoot apex in response to the WPHS treatment, indicating that these FT mutations had little effect on heat-induced expression and/or protein stability. As shown above, FT-T7 was able to move from the heat-treated leaf blade to the shoot apex, and we could detect the corresponding signal after the SLHS treatment. Interestingly, a significant reduction of signal was observed for the V70A, S76A and R83A variants, suggesting that these amino acid residues are important for the transport from the leaf to the shoot apex. In contrast, mFT-T7 signal was detected in the S78A, H81A and W138A variant lines, indicating that these amino acid residues did not affect florigen transport. Taken together with the results from Fig. 6, we concluded that V70A, S76A and R83A have a defect in transport but not in the promotion of flowering at the SAM. H81A partially lost flowering promotion ability and W138A fully lost it, although both retained mobility. S78A affected neither transport nor flowering promotion ability. Fig. 7 View largeDownload slide Mobility of mFT-T7 variants from the leaf to the shoot apex under short days. At 24 h after induction of HSP::mFT-T7, proteins were extracted from 30 shoot apices and analyzed by 2D electrophoresis. Red circles indicate the FT-T7 spot. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 7 View largeDownload slide Mobility of mFT-T7 variants from the leaf to the shoot apex under short days. At 24 h after induction of HSP::mFT-T7, proteins were extracted from 30 shoot apices and analyzed by 2D electrophoresis. Red circles indicate the FT-T7 spot. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. The mobility defect of the three FT variants cannot be explained by the loss of interaction with FTIP1 FTIP1 was reported as an essential regulator required for FT protein transport, possibly acting via protein interaction in Arabidopsis (Liu et al. 2012). Since V70A, S76A and R83A displayed reduced mobility, we tested if these amino acid substitutions disrupted the interaction with FTIP1. We tested the physical interaction between mFT and FTIP1 by a yeast two-hybrid assay (Supplementary Fig. S5). Wild-type FT interacted with the N-terminal fragment of FTIP1 as previously reported (Liu et al. 2012). All FT variants tested, including a FT variant in which all three amino acid residues, V70, S76 and R83, were replaced by alanine [FT(vsr)], still interacted with FTIP1 in yeast cells. These results suggest that the defect in transport of the three FT mutants is not due to a defect in the FTIP1-mediated step of transport. Discussion Since the florigen hypothesis was proposed, extensive physiological analyses have been performed to estimate the time required for florigen transportation (Zeevaart 2006). These estimations were for transport of a ‘conceptual’ florigen based on transmission of the florigenic activity. However, after the identification of FT protein as the florigen molecule, only a few studies have addressed this issue, although many studies have demonstrated that FT and FT orthologous proteins are actually transported from leaves to the shoot apex (Corbesier et al. 2007, Jaeger and Wigge 2007, Lin et al. 2007, Mathieu et al. 2007, Tamaki et al. 2007, Notaguchi et al. 2008, S.C. Yoo et al. 2013). In a previous report, we demonstrated that FT-T7 protein was transported from the stock to the scion within 24–48 h of induction (Notaguchi et al. 2008). However, due to the low time resolution, those results could not be directly compared with classical physiological studies. Here, using a SLHS technique, we performed 4 h resolution measurements of FT transport and showed that FT-T7 was loaded onto phloem within 8 h after transient induction in a leaf (Figs. 1, 2). Previous reports also estimated the time of phloem uploading. For example, when a Japanese morning glory (a short-day plant) leaf was subjected to 12 h of darkness, flowering was slightly induced; moreover, leaf excision 0 or 2 h after treatment inhibited the flowering promotion, but excision 4 h after treatment did not affect flowering promotion, suggesting that florigen was loaded onto phloem within 4 h (Takeba and Takimoto 1966). Using a synchronous induction system by a single long-day (22 h) treatment of short-day-grown Arabidopsis, it was estimated that movement of the floral stimulus started 20–24 h after the start of a 22 h long day and was completed about 16 h later (Corbesier et al. 1996). Our results are quite consistent with these past observations, demonstrating that Arabidopsis with SLHS treatment is a useful tool that can integrate the classical view of physiological experiments and current knowledge of molecular genetics. We also estimated that the time required for FT unloading was not a protracted process. As we showed in Fig. 3, FT-T7 was unloaded onto the SAM within 12 h whereas FT-T7 was transported to the shoot apex within 8 h, although our time resolution was not more refined than the 4 h windows. This time estimation was also supported by downstream AP1 and SOC1 gene expression (Fig. 5). In our study, a clear increase of SOC1 expression in shoot apices was detected by 8 h, and AP1 expression was detected 12 h after FT induction in leaves. The early induction of SOC1 was consistent with previous reports that SOC1 is expressed in the SAM within 8–10 h after photoperiodic induction of FT expression (16–18 h after shift from short days to long days) (Borner et al. 2000, Samach et al. 2000). Compared with significant FT-T7 accumulation in the shoot apex, FT-T7 accumulation in younger leaves appeared to be low, suggesting that florigen is predominantly transported to targeted tissues such as the SAM, axillary meristems and stolon tips (Navarro et al. 2011, Niwa et al. 2013). Interestingly, phloem proteins from pumpkin (Cucurbita moschata CmPP16-1 and CmPP16-2) selectively controlled their own long-distance transportation in phloem (Aoki et al. 2005). It has been hypothesized that simple diffusion of FT protein might be insufficient for transport of FT from the leaf to the SAM (Giakountis and Coupland 2008). To achieve a detailed understanding of active FT transportation, appropriate 3D conformation of FT or protein–protein interactions may be needed. Consistent with this aim, we have identified three amino acid residues that are required for efficient FT protein accumulation in the SAM (V70A, S76A and R83A), but are not essential for the promotion of flowering per se (Figs. 6, 7). We cannot, however, exclude the possibility that these three residues have some contribution to flowering promotion activity. These three amino acid residues are located in close proximity on the surface of FT protein, but they are not crucial for the interaction with FTIP1 (Supplementary Fig. S5), suggesting that they are not involved in the FTIP1-mediated uploading pathway. Consistent with this view, the FT variant harboring all three of these amino acid substitutions [FT(vsr)] was detected in phloem sap collected immediately below the shoot apex, when expressed in a leaf by a viral vector in pumpkin, implying that the FT(vsr) was successfully uploaded onto sieve elements and transported in phloem, but the subsequent unloading step might be disturbed (S.C. Yoo et al. 2013). Hence, it is likely that FT(vsr) is not defective in the NaKR1-mediated transport in phloem. Unfortunately, we were not able consistently to demonstrate protein interaction between FT and NaKR1 in our yeast two-hybrid assay, and this possibility could not be explored further. In Arabidopsis FT, the importance of the second exon and the fourth exon including the region for the external loop has been demonstrated (Hanzawa et al. 2005, Ahn et al. 2006, Ho and Weigel 2014). These regions are involved in the FT vs. TFL1 distinction, and include the binding interface for 14-3-3 protein (common to FT and TFL1) for complex formation with a bZIP transcription factor FD in a phosphorylation-dependent manner (Taoka et al. 2011, Niwa et al. 2013, Ho and Weigel 2014; Kawamoto et al. 2015). Although Y85 in the second exon constitutes a critical difference between FT and TFL1 (Hanzawa et al. 2005, Ahn et al. 2006, Ho and Weigel 2014), little is known about the importance of the three amino acid residues in the second exon examined in our study. These three amino acid residues are located at a different surface position, implying that important structural domain(s) for transportation are distinct from those that function in floral regulation. Recent work provides support for this view. By extensive grafting experiments, Jin et al. (2015) showed that TSF has a lesser ability to move than FT, which may in part be explained by the reported inability of TSF to interact with NaKR1 (Zhu et al. 2016). Interestingly, ‘region II’ of FT (residues 28–98), which differs from that of TSF in six positions, can confer mobility on TSF in a TSF/FT/TSF chimeric protein. The three amino acid residues in the second exon, which are conserved between FT and TSF, are located in the latter half of ‘region II’ (Jin et al. 2015). We also identified N152 as another candidate amino acid implicated in FT transportation. However, phloem companion cell-specific overexpression of this FT variant, driven by the SUC2 promoter, displayed an early flowering phenotype, indicating that N152A could be unloaded successfully in the SAM (Ho and Weigel 2014). Other candidate amino acid residues important for FT transportation are D17 and V18, which are conserved between FT and TFL1. D17K and V18A expressed by the SUC2 promoter displayed a severely reduced ability to rescue the ft mutant (Ho and Weigel 2014). Whether these residues in a different region from the three amino acid residues discussed above are involved in different step(s) in transport is an interesting question to be addressed in the future. In conclusion, our work confirms the temporal kinetics of florigen transport. A detailed florigen transportation profile will enable us to perform transcriptome or metabolome analyses just before and after florigen arrival at the SAM. In addition, we have presented evidence that two functions of FT, one for flowering promotion and the other for transportation, are separable. These findings will stimulate further study to identify key residues and/or structural features of FT protein and interacting factors important for florigen transportation. Materials and Methods Plant materials and growth conditions HSP::FT-T7 was described previously (Matsuhara et al. 2000, Notaguchi et al. 2008). Mutant versions of HSP::FT-T7 constructs were prepared by artificial gene synthesis. All mutant and transgenic lines were in the A. thaliana ft-1 background (Col-0). Plants were grown on soil for 18 d under long days (16 h light 8 h dark, 60 µmol m–2 s–1) at 21°C, and SLHS induction was performed at ZT13–ZT15 of day 19. Plants were grown for 3 weeks under short days (8 h light 16 h dark, 100 µmol m–2 s–1) at 21°C and subjected to heat shock induction at ZT1–ZT3. Heat shock induction of FT-T7 protein and 2D electrophoresis WPHS and SLHS induction, and 2D electrophoresis were performed according to previous studies (Notaguchi et al. 2008, Abe et al. 2015). For the ‘single leaf’ or ‘quadruple leaf’ heat treatment, the leaf blade of well-expanded leaf/leaves was exposed to 38–39°C for 2 h by placing it in the water-filled space between a heated copper plate and a glass slide. The copper plate was heated using a silicon rubber plate heater fixed under the plate, and was connected to a temperature controller (SHM-CONT2; Asahi Techno Glass). For the ‘whole-plant’ heat treatment, plants were incubated for 2 h at 38–39°C in an incubator. Isoelectric focusing was performed using Immobiline DryStrip pH 3–10 NL (GE Healthcare). RNA in situ hybridization For in situ hybridization, plants were grown for 18 d under long days, and QLHS treatment was carried out for 2 h at ZT13–ZT15. After 24 and 48 h, shoot apices were collected and rapidly fixed. RNA in situ hybridization was performed according to Takada et al. (2001). The AP1 and SOC1 probes have been described previously (Liu et al. 2007, Karim et al. 2009). The AP1 probe was transcribed using T7 RNA polymerase (Roche), and the SOC1 probe was transcribed using T3 RNA polymerase (Roche). Hybridization was performed at 50°C. Western Blue (Promega) was used as the substrate for signal detection. Yeast two-hybrid assay The Clontech Matchmaker two-hybrid system was used to analyze the protein–protein interaction in yeast. The cDNAs corresponding to full-length FT and the mutant version of FT were cloned into the bait vector. The cDNA corresponding to the N-terminal region of FTIP1 was cloned into the prey vector. The plasmids were transformed into yeast strain AH109. All the experiments were carried out following the Yeast Protocol Handbook (Clontech). Supplementary Data Supplementary data are available at PCP online. Funding This work was partially supported by the Ministry of Education, Culture, Sports, Science & Technology, Japan [Grants-in-Aid for Scientific Research on Priority Areas 19060012 and 19060016 (to T.A.), Grant-in-Aid for Scientific Research on Innovative Areas 25113005 (to T.A.) and 16H01228 (to M.A.), and Grants-in-Aids for Scientific Research (B) 15H04390 (to T.A.)]. Acknowledgments We thank Y. Tomita and A. Watanabe-Taneda for technical assistance, and J.A. Hejna for English proofreading. Disclosures The authors have no conflicts of interest to declare. References Abe M. , Kaya H. , Watanabe-Taneda A. , Shibuta M. , Yamaguchi A. , Sakamoto T. ( 2015 ) FE, a phloem-specific Myb-related protein, promotes flowering through transcriptional activation of FLOWERING LOCUS T and FLOWERING LOCUS T INTERACTING PROTEIN 1 . Plant J. 83 : 1059 – 1068 . Google Scholar CrossRef Search ADS PubMed Abe M. , Kobayashi Y. , Yamamoto S. , Daimon Y. , Yamaguchi A. , Ikeda Y. , et al. 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( 2016 ) NaKR1 regulates long-distance movement of FLOWERING LOCUS T in Arabidopsis . Nat. Plants 2 : 16075 . Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations ACT2 ACTIN2 AP1 APETALA1 APA1 ASPARTIC PROTEINASE A1 FE/APL FE/ALTERED PHLOEM DEVELOPMENT FT FLOWERING LOCUS T FTIP1 FT-INTERACTING PROTEIN 1 FUL FRUITFULL HSP HEAT SHOCK PROTEIN 18.2 IPP2 ISOPENTENYL PYROPHOSPHATE, DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 miR156 microRNA156 NaKR1 SODIUM POTASSIUM ROOT DEFECTIVE1 QLHS quadruple-leaf heat shock RT–qPCR reverse trnascription–quantitative real-time PCR SAM shoot apical meristem SOC1 SUPPRESSOR OF OVEREXPRESSION OF CO1 SPL3 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 SLHS single-leaf heat shock TFL1 TERMINAL FLOWER 1 TSF TWIN SISTER OF FT TUB2 TUBULIN BETA CHAIN 2 WPHS whole-plant heat shock ZT Zeitgeber time © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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Re-Evaluation of Florigen Transport Kinetics with Separation of Functions by Mutations That Uncouple Flowering Initiation and Long-Distance Transport

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Abstract

Abstract In many plants, timing of flowering is regulated by day length. In Arabidopsis, florigen, FLOWERING LOCUS T (FT) protein, is synthesized in leaf phloem companion cells in response to long days and is transported to the shoot apical meristem (SAM) through the phloem. The temporal aspects of florigen transportation have been studied in various plants by physiological experiments. Nevertheless, little is known about how FT protein transportation is regulated in Arabidopsis. In this study, we performed heat shock-based transient FT induction in a single leaf blade and detected the FT protein in the shoot apex by 2D-PAGE. We demonstrated that detectable amounts of FT were transported from the leaf to the shoot apex within 8 h, and subsequent FT-induced target gene expression was detected within 8–12 h. Furthermore, we identified three amino acid residues (V70, S76 and R83) where missense mutations led to reduced mobility. Interestingly, these FT variants lost only their transportation ability, but retained their flowering promotion capacity, suggesting that discrete amino acids are involved in flowering regulation and transport regulation. Since the interaction with FT-INTERACTING PROTEIN 1 (FTIP1) was not affected in these FT variants, we hypothesize that the three amino acid residues are not involved in the FTIP1-mediated pathway of uploading, but rather in the subsequent step(s) of FT transport. Introduction Flowering in the appropriate season is a crucial strategy for plant reproduction. To achieve efficient temporal regulation of flowering, many plant species use photoperiod signals as indicators of seasonal changes (Song et al. 2013). Inductive day lengths perceived by leaves induce the production of the flowering hormone, florigen, in phloem companion cells (Turck et al. 2008, Liu et al. 2013). Phloem-loaded florigen is then transported from the source to sink tissues, including the shoot apical meristem (SAM), where it promotes flowering. Many photoperiodic flowering plants are classified based on the responses to different photoperiod, e.g. short-day plants and long-day plants. Since short-day plants are more sensitive to environmental light changes such as an interruption of night-time darkness (‘night break’) or photoperiod changes, a considerable number of studies have been carried out on short-day plants including Japanese morning glory and soybean (Imamura 1967, Thomas and Vince-Prue 1997). From these studies, physiological features of florigen transportation, such as the velocity of florigen transportation, have been estimated. The florigen transportation rate in phloem was originally estimated to be about 3 mm h–1 (Imamura and Takimoto 1955, Zeevaart 1962, Zeevaart 2006), but this was later re-estimated to be as fast as 30–50 cm h–1 (Takeba and Takimoto 1966, King et al. 1968), which is almost the same speed as phloem flux (Canny 1975, Windt et al. 2006). In contrast to the rapid florigen transportation, however, florigen uploading and/or unloading steps take considerably more time. In Japanese morning glory (a short-day plant), for example, flowering was fully induced by a 14 h short-day treatment. However, when the leaf was immediately cut after the 14 h short-day treatment, the induced flowering was completely abrogated. When the leaf was cut after an 18 h short-day treatment, flowering was fully induced (Zeevaart 1962), which is to say that transport is fast, while unloading and induction are relatively slow. In the facultative long-day plant Arabidopsis thaliana, the FLOWERING LOCUS T (FT) protein is known to be the major component of florigen (Abe et al. 2005, Wigge et al. 2005, Corbesier et al. 2007, Jaeger and Wigge 2007, Mathieu et al. 2007, Notaguchi et al. 2008). Previous studies have demonstrated that FT protein can be transported from the leaf to the shoot apex and from the stock to the scion in grafting, as has been observed in other plant species (Corbesier et al. 2007, Notaguchi et al. 2008, S.J. Yoo et al. 2013). However, we have only begun to understand how FT protein transport from the leaf to the shoot apex is regulated. Two factors which play sequential, but independent roles in FT transport are known. One is an endoplasmic reticulum membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1), which was identified as a regulator of FT protein uploading from phloem companion cells to sieve elements (Liu et al. 2012). The other is a heavy metal-associated plant protein, SODIUM POTASSIUM ROOT DEFECTIVE1 (NaKR1), which interacts with FT and is required for FT transport in the phloem stream (Zhu et al. 2016). Recent works revealed mechanisms for co-ordinated production and transport of FT. A Myb transcription factor FE/ALTERED PHLOEM DEVELOPMENT(FE/APL) activates transcription of FT, FTIP1 and NaKR1 (Abe et al. 2015, Shibuta and Abe 2017), thereby promoting both production and transport of florigen. NaKR1 also plays an important role in FT expression through microRNA156 (miR156) and miR156-targeted transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), besides its role in FT transport (Negishi et al. 2018). However, little is known about the temporal aspect of FT transport and the structural property of FT important for transportation. In this work, we show that detectable amounts of FT protein are loaded onto phloem within 8 h after transient FT induction in a leaf, and FT protein subsequently accumulates in the SAM within 12 h after induction. Furthermore, FT-regulated APETALA1 (AP1), SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and FRUITFULL (FUL) (Schmid et al. 2003, Abe et al. 2005, Wigge et al. 2005, Torti et al. 2012) expression is induced within 8–12 h after FT induction. From alanine scanning mutagenesis of FT, we have identified three FT variants that display severely reduced transportation. Nonetheless, these FT variants can still induce flowering when they are ectopically expressed in the shoot apex, indicating that specific amino acid residues in FT carry out separate functions. These three amino acid residues are located on the surface of FT protein, but they are not crucial for the interaction with FTIP1, suggesting that they are not involved in the FTIP1-mediated pathway of uploading. Taken together with previous work which demonstrated phloem transport of an FT variant harboring all three of these amino acid substitutions in pumpkin (S.C. Yoo et al. 2013), we hypothesize that these three amino acid residues may be involved in the subsequent unloading step. Results Estimation of time for florigen uploading To elucidate the timeline of florigen transportation in terms of time required for each step, we utilized a local transient florigen induction system in Arabidopsis, as described previously (Notaguchi et al. 2008, Abe et al. 2015). In this system, transportable FT-T7 can be transiently expressed by the HEAT SHOCK PROTEIN 18.2 (HSP) promoter in the ft-1 background (HSP::FT-T7), and FT-T7 protein accumulation was observed 1 h after the end of heat treatment. We first estimated how quickly the FT-T7 is exported from a leaf blade after heat shock induction. Plants were grown under long-day conditions for 18 d and then subjected to a single-leaf heat shock (SLHS) treatment (38–39°C) at Zeitgeber time (ZT) 13 to ZT15. In parallel experiments, the FT-T7-induced leaf was then cut off 0, 4, 8, 12 and 24 h after the heat shock treatment, and the total leaf number was counted when the first flower bud opened. In response to the SLHS treatments, plants showed an early-flowering phenotype compared with the untreated control (Fig. 1). Furthermore, the slopes of the flowering rate curves were parallel when untreated, SLHS treated and the uncut groups were compared, suggesting that induced FT-T7 was the major determinant of flowering acceleration, and other factors such as age and heat shock stress were largely negligible (Fig. 1A). Among the SLHS-treated plants, using total leaf number as a proxy for flowering timing, a significant early-flowering phenotype was observed in plants when the treated leaf was cut off at any time between 8 and 24 h after the heat shock (Fig. 1B). Another indicator of flowering time, i.e. days to flower after heat shock treatment, also gave similar results (Supplementary Fig. S1A, D). Since phloem flux is relatively fast (>50 cm h–1), we assumed that only a brief time would be required for transport of florigen in the phloem from the induced leaf to the shoot apex. Therefore, we estimated that a sufficient quantity of florigen to affect flowering time was loaded onto the phloem within 8 h after transient FT induction. We also detected a slight acceleration of flowering even when the treated leaf was cut off after 4 h (Fig. 1; Supplementary Fig. S1), although the differences are not statistically significant. The scant difference between the ‘uncut’ control and plants with the heat-shocked leaf cut off after 24 h suggests that uploading was essentially completed in 24 h. The flowering time response to the SLHS induction of FT-T7 was similar even under short days, indicating that florigen transportation, uploading and unloading processes are independent of day length (Supplementary Fig. S1B, C, E, F). Fig. 1 View largeDownload slide The effect of heat shock induction of HSP::FT-T7 on flowering time under long-day conditions. Untreated, no heat shock induction and no cutting off of the leaf; Cut, no heat shock induction and cutting off of the leaf; 0 h to 24 h, SLHS induction and cutting off of the leaf 0–24 h after induction; Uncut, SLHS induction and no cutting off of the leaf. (A) Percentage of flowering plants with the indicated number of leaves (x-axis). This is a common proxy for timing the onset of flowering; early-flowering plants have fewer leaves at the onset of flowering. (B) Average time of flowering (number of leaves at the onset of flowering) after heat shock induction of HSP::FT-T7. Mean ± SEM (n ≥ 20). The P-values with respect to a control group (Cut or Pressed) were estimated by Dunnett’s test. Asterisks indicate statistical significance at P < 0.001. Fig. 1 View largeDownload slide The effect of heat shock induction of HSP::FT-T7 on flowering time under long-day conditions. Untreated, no heat shock induction and no cutting off of the leaf; Cut, no heat shock induction and cutting off of the leaf; 0 h to 24 h, SLHS induction and cutting off of the leaf 0–24 h after induction; Uncut, SLHS induction and no cutting off of the leaf. (A) Percentage of flowering plants with the indicated number of leaves (x-axis). This is a common proxy for timing the onset of flowering; early-flowering plants have fewer leaves at the onset of flowering. (B) Average time of flowering (number of leaves at the onset of flowering) after heat shock induction of HSP::FT-T7. Mean ± SEM (n ≥ 20). The P-values with respect to a control group (Cut or Pressed) were estimated by Dunnett’s test. Asterisks indicate statistical significance at P < 0.001. To corroborate these observations, we also detected FT-T7 protein accumulation in the shoot apex 24 h after the SLHS treatment, by 2D-PAGE (Notaguchi et al. 2008). Long-day-grown plants were subjected to SLHS induction, and the treated leaf blade was cut off after 0, 4, 8, 12 and 24 h (Fig. 2). Compared with the untreated and ‘pressed’ (clamped, but unheated) controls, markedly greater amounts of FT-T7 protein were detected in the shoot apex samples from plants when the treated leaf was cut off 24 h after the heat shock. Even when the treated leaf was removed 8 h after the induction, detectable amounts of FT-T7 protein accumulated in the shoot apex. Since a good correlation between flowering induction after SLHS and detectable FT-T7 amount at the shoot apex can be seen, we concluded that sufficient florigen to alter the flowering time was exported from the heat shock-treated leaf within 8 h. Fig. 2 View largeDownload slide 2D gel-based time-course analysis of FT-T7 protein export from the heat shock-induced leaf under long-day conditions. At 0, 4, 8, 12 and 24 h after the induction in 30 HSP::FT-T7 plants, the heat-shocked leaves were cut off and the 30 shoot apices were collected after 24 h of heat shock induction. Untreated, no heat shock induction and no excision of the leaf; Pressed, the leaf was just sandwiched into the unheated heating plate and the leaf was not cut off. Red circles show the FT-T7 spot, and blue circles show reference spots for precise alignment of the gel images. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 2 View largeDownload slide 2D gel-based time-course analysis of FT-T7 protein export from the heat shock-induced leaf under long-day conditions. At 0, 4, 8, 12 and 24 h after the induction in 30 HSP::FT-T7 plants, the heat-shocked leaves were cut off and the 30 shoot apices were collected after 24 h of heat shock induction. Untreated, no heat shock induction and no excision of the leaf; Pressed, the leaf was just sandwiched into the unheated heating plate and the leaf was not cut off. Red circles show the FT-T7 spot, and blue circles show reference spots for precise alignment of the gel images. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Estimation of the time required for florigen unloading We then estimated how rapidly the phloem-transported FT-T7 is unloaded onto the SAM. SLHS treatment was performed in long-day-grown HSP::FT-T7 plants, in a similar manner to that described above, except that the heat-shocked leaf was left on, and the shoot apex was collected 0, 4, 8, 12 and 24 h after the transient induction of FT-T7. The FT-T7 protein was detected 12 and 24 h after the induction, suggesting that florigen was accumulating in the shoot apex within 12 h (Fig. 3). Previous studies have demonstrated that florigen can be transported not only to the shoot apex but also to other organs (Zeevaart 1958, Navarro et al. 2011, Niwa et al. 2013). Hence, by 2D-PAGE, we analyzed FT-T7 accumulation in other leaves (Fig. 4). In the heat-treated leaf, FT-T7 was readily detected even 24 h after induction. To be able to detect FT-T7 in younger and older leaves, we loaded 10 times the amount of total protein onto the gel compared with that of the heat-treated leaf. We only detected a trace of FT-T7 in younger leaves, whereas we could not detect FT-T7 signals in older leaves and the untreated control with this experimental condition. These observations indicate that efficient FT unloading did not occur in neighboring leaves. The FT unloading efficiency is distinct at least in a leaf and in a shoot apex, implying that FT transportation is an active (targeted) process rather than a passive (non-targeted) process. Fig. 3 View largeDownload slide Time-course detection of FT-T7 protein accumulation in the shoot apex under long-day conditions. At 0, 4, 8, 12 and 24 h after induction of HSP::FT-T7, 30 shoot apices were collected and analyzed by 2D electrophoresis. The time points indicate the time after heat shock induction. Red circles show the FT-T7 spot, and blue circles show reference spots for precise alignment of the gel images. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 3 View largeDownload slide Time-course detection of FT-T7 protein accumulation in the shoot apex under long-day conditions. At 0, 4, 8, 12 and 24 h after induction of HSP::FT-T7, 30 shoot apices were collected and analyzed by 2D electrophoresis. The time points indicate the time after heat shock induction. Red circles show the FT-T7 spot, and blue circles show reference spots for precise alignment of the gel images. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 4 View largeDownload slide FT-T7 protein movement from source to sink leaves under long days. At 24 h after induction of HSP::FT-T7, proteins were extracted from pools of treated leaves (TL, four leaves), younger leaves (YL, seventh and eighth leaves) (40 leaves), older leaves (OL, first and second leaves) (40 leaves) and untreated leaves (20 leaves), respectively. Subsequently, 2D electrophoresis was performed. Red circles indicate the FT-T7 spot. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 4 View largeDownload slide FT-T7 protein movement from source to sink leaves under long days. At 24 h after induction of HSP::FT-T7, proteins were extracted from pools of treated leaves (TL, four leaves), younger leaves (YL, seventh and eighth leaves) (40 leaves), older leaves (OL, first and second leaves) (40 leaves) and untreated leaves (20 leaves), respectively. Subsequently, 2D electrophoresis was performed. Red circles indicate the FT-T7 spot. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. To confirm whether FT-T7 was unloaded onto the SAM within 12 h, we then monitored downstream gene expression. Florigen in the SAM induces several flowering-related genes such as AP1, SOC1 and FUL. Previous studies have demonstrated rapid increases of SOC1 expression in response to florigen (Borner et al. 2000, Samach et al. 2000, Schmid et al. 2003, Wigge et al. 2005, Searle et al. 2006). To detect the initiation of the expression of these genes in response to the heat shock induction, we applied quadruple-leaf heat shock (QLHS) treatment to produce enough florigen by a single shot to induce downstream genes. Samples from plants subjected to whole-plant heat shock (WPHS) treatment and untreated plants were included as controls. Gene expression in the shoot apex was then analyzed by reverse transcription followed by quantitative real-time PCR (RT–qPCR) (Fig. 5A). The expression of AP1 gradually increased 12 h after the QLHS, compared with endogenous circadian oscillation of AP1. The expression levels of SOC1 and FUL increased earlier than that of AP1, with a significant SOC1 response detectable 8 h after the QLHS. In the QLHS plants, these differences in induction times may be accounted for by the differential responsiveness of the respective genes to the FT-T7, because the WPHS treatment that induces florigen in the shoot apex has a distinctly different effect on the induction of these genes. We also examined ACTIN2 (ACT2) as a negative control and demonstrated that heat shock did not affect its gene expression profile. Fig. 5 View largeDownload slide Floral gene expression in response to heat shock induction under long days. Shoot apices of HSP::FT-T7 were harvested 0, 4, 8, 12 and 24 h after heat shock induction. Untreated, no heat shock induction; QLHS, quadruple-leaf heat shock; WPHS, whole-plant heat shock. (A) The samples were analyzed by relative quantification using RT–qPCR following MIQE recommendations (Bustin et al. 2009). RNA extraction was performed three times independently. Mean ± SEM (n = 3). Data points represent geometrical means (Vandesompele et al. 2002). Genes analyzed were AP1, SOC1, FUL and ACT2. ASPARTIC PROTEINASE A1 (APA1), ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 (IPP2) and TUBULIN BETA CHAIN 2 (TUB2) were used as controls (Endo et al. 2007, Michael et al. 2008, Hazen et al. 2005). (B) RNA in situ hybridization of longitudinal sections hybridized with either an AP1 or SOC1 probe. Scale bars = 200 µm. Fig. 5 View largeDownload slide Floral gene expression in response to heat shock induction under long days. Shoot apices of HSP::FT-T7 were harvested 0, 4, 8, 12 and 24 h after heat shock induction. Untreated, no heat shock induction; QLHS, quadruple-leaf heat shock; WPHS, whole-plant heat shock. (A) The samples were analyzed by relative quantification using RT–qPCR following MIQE recommendations (Bustin et al. 2009). RNA extraction was performed three times independently. Mean ± SEM (n = 3). Data points represent geometrical means (Vandesompele et al. 2002). Genes analyzed were AP1, SOC1, FUL and ACT2. ASPARTIC PROTEINASE A1 (APA1), ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 (IPP2) and TUBULIN BETA CHAIN 2 (TUB2) were used as controls (Endo et al. 2007, Michael et al. 2008, Hazen et al. 2005). (B) RNA in situ hybridization of longitudinal sections hybridized with either an AP1 or SOC1 probe. Scale bars = 200 µm. To confirm that AP1 and SOC1 expression was induced only in the shoot apex, we then examined RNA expression patterns by an RNA in situ hybridization (Fig. 5B). Clear SOC1 signals were detected in the shoot apex 1 d (24 h) after QLHS treatment. We could not detect AP1 signals in the shoot apex 1 d after QLHS treatment but could detect signals 2 d after QLHS in nascent floral meristems. The sequential expression of SOC1 and AP1 was similar in both RT–qPCR and the RNA in situ hybridization assays. Therefore, we concluded that substantial FT-T7 accumulated in the SAM within 12 h after induction. Amino acid residues of FT related to florigen transport Our results suggest that FT transport is actively regulated. If this is indeed the case, it is expected that some FT variants are defective in transport from the leaf to the SAM. To screen for such point mutations, we focused on differences between FT and TERMINAL FLOWER 1 (TFL1). Both FT and TFL1 proteins are members of the PEBP family and are both mobile signal molecules (Conti and Bradley 2007, Lin et al. 2007, Tamaki et al. 2007, Turck et al. 2008). However, TFL1 is a flowering repressor (Ratcliffe et al. 1998) and only has short-distance mobility in the SAM (Conti and Bradley 2007). From these facts, we hypothesized that the non-conserved amino acid residues between FT and TFL1 may be important for flowering induction and/or long-distance mobility (Hanzawa et al. 2005, Ahn et al. 2006, Ho and Weigel 2014). An amino acid sequence alignment of FT and TFL1 pointed to 47 residues to be tested (Supplementary Fig. S2A). To assess their functional significance, we substituted most of the residues with alanine, whereas G97 and A135 were replaced by aspartic acid and glycine, respectively. We first tested if these single amino acid-substituted FT variants (hereafter referred to as mFT) could induce flowering in response to the WPHS. As in the HSP::FT-T7 line, the 47 FT mutants with a T7-tag were driven by the HSP promoter (HSP::mFT-T7). We selected the first-generation transformants (T1) for each mutant FT, subjected them to WPHS treatment and the flowering time was scored (Supplementary Fig. S2B). Most of the transgenic plants flowered as early as the wild-type HSP::FT-T7 line in response to the WPHS, indicating that most of the substituted FT mutants still retained function as florigen, as previously reported (Ho and Weigel 2014). A FT 3D structure places a part of the region encoded by the second exon (V68–W88) and external loop (L128–N141) on the surface of the protein (Ahn et al. 2006) (Supplementary Fig. S3), suggesting that amino acids in these regions may engage in protein–protein interactions. Previous studies also demonstrated that the external loop was involved in florigen function as a flowering inducer (Hanzawa et al. 2005, Ahn et al. 2006). Therefore, we first focused on the second exon (V70A, S76A, S78A, H81A and R83A). For comparison, we analyzed three FT variants in the external loop (L128A, Y134A and W138A) and one FT variant in segment C (N152A). W138A is reported to abolish florigen function in sugar beets (Pin et al. 2010), and N152A may affect external loop structure and is required for florigen activity (Ahn et al. 2006, Ho and Weigel 2014). To evaluate the effects of these FT variants on flowering, we then investigated whether transiently expressed mFT-T7 in a leaf blade was able to reach the shoot apex within 24 h. TWIN SISTER OF FT (TSF) is also active under long-day conditions (Yamaguchi et al. 2005, Hiraoka et al. 2013); therefore, we performed these experiments under non-inductive short-day conditions to make the mFT-T7 effects clear. It allowed us clearly to observe the promotion of flowering by induction of florigen production and to detect FT-T7 transport in the shoot apex within 24 h (Supplementary Fig. S4). All transgenic plants expressing these FT variants showed similar flowering times under untreated conditions (Fig. 6). The V70A, S76A, S78A, H81A, R83A and N152A mutants were able to induce flowering in response to the WPHS treatment, as was the wild-type FT-T7 (designated as ‘FT’). However, L128A, Y134A and W138A did not respond to the WPHS treatment and flowered as late as the untreated control, suggesting that these amino acids are important for flowering induction. Interestingly, V70A, S76A, H81A, R83A and N152A could not fully respond to the SLHS treatment, whereas wild-type FT-T7 and S78A showed similar flowering times to the WPHS treatment. Fig. 6 View largeDownload slide The effect of V70A, S76A, S78A, H81A, R8A, L128A, Y134A, N152A and W138A on flowering time under short days after SLHS and WPHS treatments. FT indicates non-substituted HSP::FT-T7, and the other labels indicate the FT variants with the respective single amino acid substitutions. Mean ± SEM (n ≥ 10). Fig. 6 View largeDownload slide The effect of V70A, S76A, S78A, H81A, R8A, L128A, Y134A, N152A and W138A on flowering time under short days after SLHS and WPHS treatments. FT indicates non-substituted HSP::FT-T7, and the other labels indicate the FT variants with the respective single amino acid substitutions. Mean ± SEM (n ≥ 10). To test if these FT variants in the second exon failed to move from the leaf to the shoot apex, we tried to detect mFT-T7 protein in the shoot apex 24 h after the SLHS treatment (Fig. 7). In all transgenic plants, significant mFT-T7 signal was detected in the shoot apex in response to the WPHS treatment, indicating that these FT mutations had little effect on heat-induced expression and/or protein stability. As shown above, FT-T7 was able to move from the heat-treated leaf blade to the shoot apex, and we could detect the corresponding signal after the SLHS treatment. Interestingly, a significant reduction of signal was observed for the V70A, S76A and R83A variants, suggesting that these amino acid residues are important for the transport from the leaf to the shoot apex. In contrast, mFT-T7 signal was detected in the S78A, H81A and W138A variant lines, indicating that these amino acid residues did not affect florigen transport. Taken together with the results from Fig. 6, we concluded that V70A, S76A and R83A have a defect in transport but not in the promotion of flowering at the SAM. H81A partially lost flowering promotion ability and W138A fully lost it, although both retained mobility. S78A affected neither transport nor flowering promotion ability. Fig. 7 View largeDownload slide Mobility of mFT-T7 variants from the leaf to the shoot apex under short days. At 24 h after induction of HSP::mFT-T7, proteins were extracted from 30 shoot apices and analyzed by 2D electrophoresis. Red circles indicate the FT-T7 spot. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. Fig. 7 View largeDownload slide Mobility of mFT-T7 variants from the leaf to the shoot apex under short days. At 24 h after induction of HSP::mFT-T7, proteins were extracted from 30 shoot apices and analyzed by 2D electrophoresis. Red circles indicate the FT-T7 spot. Close-up images of FT-T7 spots are shown in each panel in the upper right corner. pI, the isoelectric point. The mobility defect of the three FT variants cannot be explained by the loss of interaction with FTIP1 FTIP1 was reported as an essential regulator required for FT protein transport, possibly acting via protein interaction in Arabidopsis (Liu et al. 2012). Since V70A, S76A and R83A displayed reduced mobility, we tested if these amino acid substitutions disrupted the interaction with FTIP1. We tested the physical interaction between mFT and FTIP1 by a yeast two-hybrid assay (Supplementary Fig. S5). Wild-type FT interacted with the N-terminal fragment of FTIP1 as previously reported (Liu et al. 2012). All FT variants tested, including a FT variant in which all three amino acid residues, V70, S76 and R83, were replaced by alanine [FT(vsr)], still interacted with FTIP1 in yeast cells. These results suggest that the defect in transport of the three FT mutants is not due to a defect in the FTIP1-mediated step of transport. Discussion Since the florigen hypothesis was proposed, extensive physiological analyses have been performed to estimate the time required for florigen transportation (Zeevaart 2006). These estimations were for transport of a ‘conceptual’ florigen based on transmission of the florigenic activity. However, after the identification of FT protein as the florigen molecule, only a few studies have addressed this issue, although many studies have demonstrated that FT and FT orthologous proteins are actually transported from leaves to the shoot apex (Corbesier et al. 2007, Jaeger and Wigge 2007, Lin et al. 2007, Mathieu et al. 2007, Tamaki et al. 2007, Notaguchi et al. 2008, S.C. Yoo et al. 2013). In a previous report, we demonstrated that FT-T7 protein was transported from the stock to the scion within 24–48 h of induction (Notaguchi et al. 2008). However, due to the low time resolution, those results could not be directly compared with classical physiological studies. Here, using a SLHS technique, we performed 4 h resolution measurements of FT transport and showed that FT-T7 was loaded onto phloem within 8 h after transient induction in a leaf (Figs. 1, 2). Previous reports also estimated the time of phloem uploading. For example, when a Japanese morning glory (a short-day plant) leaf was subjected to 12 h of darkness, flowering was slightly induced; moreover, leaf excision 0 or 2 h after treatment inhibited the flowering promotion, but excision 4 h after treatment did not affect flowering promotion, suggesting that florigen was loaded onto phloem within 4 h (Takeba and Takimoto 1966). Using a synchronous induction system by a single long-day (22 h) treatment of short-day-grown Arabidopsis, it was estimated that movement of the floral stimulus started 20–24 h after the start of a 22 h long day and was completed about 16 h later (Corbesier et al. 1996). Our results are quite consistent with these past observations, demonstrating that Arabidopsis with SLHS treatment is a useful tool that can integrate the classical view of physiological experiments and current knowledge of molecular genetics. We also estimated that the time required for FT unloading was not a protracted process. As we showed in Fig. 3, FT-T7 was unloaded onto the SAM within 12 h whereas FT-T7 was transported to the shoot apex within 8 h, although our time resolution was not more refined than the 4 h windows. This time estimation was also supported by downstream AP1 and SOC1 gene expression (Fig. 5). In our study, a clear increase of SOC1 expression in shoot apices was detected by 8 h, and AP1 expression was detected 12 h after FT induction in leaves. The early induction of SOC1 was consistent with previous reports that SOC1 is expressed in the SAM within 8–10 h after photoperiodic induction of FT expression (16–18 h after shift from short days to long days) (Borner et al. 2000, Samach et al. 2000). Compared with significant FT-T7 accumulation in the shoot apex, FT-T7 accumulation in younger leaves appeared to be low, suggesting that florigen is predominantly transported to targeted tissues such as the SAM, axillary meristems and stolon tips (Navarro et al. 2011, Niwa et al. 2013). Interestingly, phloem proteins from pumpkin (Cucurbita moschata CmPP16-1 and CmPP16-2) selectively controlled their own long-distance transportation in phloem (Aoki et al. 2005). It has been hypothesized that simple diffusion of FT protein might be insufficient for transport of FT from the leaf to the SAM (Giakountis and Coupland 2008). To achieve a detailed understanding of active FT transportation, appropriate 3D conformation of FT or protein–protein interactions may be needed. Consistent with this aim, we have identified three amino acid residues that are required for efficient FT protein accumulation in the SAM (V70A, S76A and R83A), but are not essential for the promotion of flowering per se (Figs. 6, 7). We cannot, however, exclude the possibility that these three residues have some contribution to flowering promotion activity. These three amino acid residues are located in close proximity on the surface of FT protein, but they are not crucial for the interaction with FTIP1 (Supplementary Fig. S5), suggesting that they are not involved in the FTIP1-mediated uploading pathway. Consistent with this view, the FT variant harboring all three of these amino acid substitutions [FT(vsr)] was detected in phloem sap collected immediately below the shoot apex, when expressed in a leaf by a viral vector in pumpkin, implying that the FT(vsr) was successfully uploaded onto sieve elements and transported in phloem, but the subsequent unloading step might be disturbed (S.C. Yoo et al. 2013). Hence, it is likely that FT(vsr) is not defective in the NaKR1-mediated transport in phloem. Unfortunately, we were not able consistently to demonstrate protein interaction between FT and NaKR1 in our yeast two-hybrid assay, and this possibility could not be explored further. In Arabidopsis FT, the importance of the second exon and the fourth exon including the region for the external loop has been demonstrated (Hanzawa et al. 2005, Ahn et al. 2006, Ho and Weigel 2014). These regions are involved in the FT vs. TFL1 distinction, and include the binding interface for 14-3-3 protein (common to FT and TFL1) for complex formation with a bZIP transcription factor FD in a phosphorylation-dependent manner (Taoka et al. 2011, Niwa et al. 2013, Ho and Weigel 2014; Kawamoto et al. 2015). Although Y85 in the second exon constitutes a critical difference between FT and TFL1 (Hanzawa et al. 2005, Ahn et al. 2006, Ho and Weigel 2014), little is known about the importance of the three amino acid residues in the second exon examined in our study. These three amino acid residues are located at a different surface position, implying that important structural domain(s) for transportation are distinct from those that function in floral regulation. Recent work provides support for this view. By extensive grafting experiments, Jin et al. (2015) showed that TSF has a lesser ability to move than FT, which may in part be explained by the reported inability of TSF to interact with NaKR1 (Zhu et al. 2016). Interestingly, ‘region II’ of FT (residues 28–98), which differs from that of TSF in six positions, can confer mobility on TSF in a TSF/FT/TSF chimeric protein. The three amino acid residues in the second exon, which are conserved between FT and TSF, are located in the latter half of ‘region II’ (Jin et al. 2015). We also identified N152 as another candidate amino acid implicated in FT transportation. However, phloem companion cell-specific overexpression of this FT variant, driven by the SUC2 promoter, displayed an early flowering phenotype, indicating that N152A could be unloaded successfully in the SAM (Ho and Weigel 2014). Other candidate amino acid residues important for FT transportation are D17 and V18, which are conserved between FT and TFL1. D17K and V18A expressed by the SUC2 promoter displayed a severely reduced ability to rescue the ft mutant (Ho and Weigel 2014). Whether these residues in a different region from the three amino acid residues discussed above are involved in different step(s) in transport is an interesting question to be addressed in the future. In conclusion, our work confirms the temporal kinetics of florigen transport. A detailed florigen transportation profile will enable us to perform transcriptome or metabolome analyses just before and after florigen arrival at the SAM. In addition, we have presented evidence that two functions of FT, one for flowering promotion and the other for transportation, are separable. These findings will stimulate further study to identify key residues and/or structural features of FT protein and interacting factors important for florigen transportation. Materials and Methods Plant materials and growth conditions HSP::FT-T7 was described previously (Matsuhara et al. 2000, Notaguchi et al. 2008). Mutant versions of HSP::FT-T7 constructs were prepared by artificial gene synthesis. All mutant and transgenic lines were in the A. thaliana ft-1 background (Col-0). Plants were grown on soil for 18 d under long days (16 h light 8 h dark, 60 µmol m–2 s–1) at 21°C, and SLHS induction was performed at ZT13–ZT15 of day 19. Plants were grown for 3 weeks under short days (8 h light 16 h dark, 100 µmol m–2 s–1) at 21°C and subjected to heat shock induction at ZT1–ZT3. Heat shock induction of FT-T7 protein and 2D electrophoresis WPHS and SLHS induction, and 2D electrophoresis were performed according to previous studies (Notaguchi et al. 2008, Abe et al. 2015). For the ‘single leaf’ or ‘quadruple leaf’ heat treatment, the leaf blade of well-expanded leaf/leaves was exposed to 38–39°C for 2 h by placing it in the water-filled space between a heated copper plate and a glass slide. The copper plate was heated using a silicon rubber plate heater fixed under the plate, and was connected to a temperature controller (SHM-CONT2; Asahi Techno Glass). For the ‘whole-plant’ heat treatment, plants were incubated for 2 h at 38–39°C in an incubator. Isoelectric focusing was performed using Immobiline DryStrip pH 3–10 NL (GE Healthcare). RNA in situ hybridization For in situ hybridization, plants were grown for 18 d under long days, and QLHS treatment was carried out for 2 h at ZT13–ZT15. After 24 and 48 h, shoot apices were collected and rapidly fixed. RNA in situ hybridization was performed according to Takada et al. (2001). The AP1 and SOC1 probes have been described previously (Liu et al. 2007, Karim et al. 2009). The AP1 probe was transcribed using T7 RNA polymerase (Roche), and the SOC1 probe was transcribed using T3 RNA polymerase (Roche). Hybridization was performed at 50°C. Western Blue (Promega) was used as the substrate for signal detection. Yeast two-hybrid assay The Clontech Matchmaker two-hybrid system was used to analyze the protein–protein interaction in yeast. The cDNAs corresponding to full-length FT and the mutant version of FT were cloned into the bait vector. The cDNA corresponding to the N-terminal region of FTIP1 was cloned into the prey vector. The plasmids were transformed into yeast strain AH109. All the experiments were carried out following the Yeast Protocol Handbook (Clontech). Supplementary Data Supplementary data are available at PCP online. Funding This work was partially supported by the Ministry of Education, Culture, Sports, Science & Technology, Japan [Grants-in-Aid for Scientific Research on Priority Areas 19060012 and 19060016 (to T.A.), Grant-in-Aid for Scientific Research on Innovative Areas 25113005 (to T.A.) and 16H01228 (to M.A.), and Grants-in-Aids for Scientific Research (B) 15H04390 (to T.A.)]. Acknowledgments We thank Y. Tomita and A. Watanabe-Taneda for technical assistance, and J.A. Hejna for English proofreading. Disclosures The authors have no conflicts of interest to declare. References Abe M. , Kaya H. , Watanabe-Taneda A. , Shibuta M. , Yamaguchi A. , Sakamoto T. ( 2015 ) FE, a phloem-specific Myb-related protein, promotes flowering through transcriptional activation of FLOWERING LOCUS T and FLOWERING LOCUS T INTERACTING PROTEIN 1 . Plant J. 83 : 1059 – 1068 . Google Scholar CrossRef Search ADS PubMed Abe M. , Kobayashi Y. , Yamamoto S. , Daimon Y. , Yamaguchi A. , Ikeda Y. , et al. 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( 2016 ) NaKR1 regulates long-distance movement of FLOWERING LOCUS T in Arabidopsis . Nat. Plants 2 : 16075 . Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations ACT2 ACTIN2 AP1 APETALA1 APA1 ASPARTIC PROTEINASE A1 FE/APL FE/ALTERED PHLOEM DEVELOPMENT FT FLOWERING LOCUS T FTIP1 FT-INTERACTING PROTEIN 1 FUL FRUITFULL HSP HEAT SHOCK PROTEIN 18.2 IPP2 ISOPENTENYL PYROPHOSPHATE, DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 miR156 microRNA156 NaKR1 SODIUM POTASSIUM ROOT DEFECTIVE1 QLHS quadruple-leaf heat shock RT–qPCR reverse trnascription–quantitative real-time PCR SAM shoot apical meristem SOC1 SUPPRESSOR OF OVEREXPRESSION OF CO1 SPL3 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 SLHS single-leaf heat shock TFL1 TERMINAL FLOWER 1 TSF TWIN SISTER OF FT TUB2 TUBULIN BETA CHAIN 2 WPHS whole-plant heat shock ZT Zeitgeber time © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Mar 19, 2018

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