Tissue/organ-specific temperature effects on phyB signaling

PBs have been characterized extensively using transgenic lines expressing phyB-FP36,39,40,44,45,46,54,55, and therefore to examine the dynamics of phyB-FP PBs induced by temperature changes, we went back to the phyB-FP lines, including PBG (phyB-GFP), which expresses functional phyB-GFP to complement the null phyB-5 mutant in the Landsberg erecta (Ler) background36, and PBC (phyB-CFP), which expresses phyB-CFP to complement the null phyB-9 mutant in Col-046,55. Because both PBG and PBC overexpress phyB-FP36,55, we first examined whether they retain the same temperature effects as their respective ecotype background. In response to temperature increases from 12 °C to 27 °C, Ler and Col-0 seedlings grown in continuous 10 µmol m−2 s−1 R light exhibited gradual increases in hypocotyl length in a 2.4- and 4.5-fold dynamic range, respectively (Fig. 1a, b)56. PBG and PBC lines showed reduced hypocotyl responses with a 1.4-fold dynamic range for both lines. However, they retained similar temperature effects on phyB signaling as Ler and Col-0, respectively—that is, warmer temperatures attenuated the function of phyB or enhanced hypocotyl elongation in all four lines (Fig. 1a, b). The reduced temperature responses in hypocotyl elongation in PBG and PBC are likely due to overexpression of phyB-FPs. Increasing the flux of phyB signaling in Ler and Col-0 did not lead to a similar effect as overexpression of phyB-FPs. For example, Ler and Col-0 seedlings grown in a higher light intensity, 50 µmol m−2 s−1 R light (R50), showed even greater dynamic ranges of the temperature response (Fig. 1a, b), suggesting that the dynamic range of the temperature response in hypocotyl elongation relies on the level of phyB. Together, these results indicate that PBG and PBC maintain a reduced but similar warm temperature-dependent antagonistic effect on phyB-dependent inhibition of hypocotyl elongation as Ler and Col-0, respectively.

Fig. 1: Temperature imposes opposite effects on phyB functions in hypocotyl and cotyledon. a, b Warmer temperatures attenuate the function of phyB signaling in inhibiting hypocotyl growth. Hypocotyl length measurements of 4-day-old seedlings of Ler and PBG (a) and Col-0 and PBC (b) grown under 10 or 50 (R50) µmol m−2 s−1 R light at 12, 16, 21, and 27 °C. c, d Warmer temperatures enhance the function of phyB signaling in promoting cotyledon expansion. Cotyledon size measurements of 4-day-old seedlings of Ler and PBG (a) and Col-0 and PBC (b) grown under 10 or 50 (R50) µmol m−2 s−1 R light at 12, 16, 21, and 27 °C. For a–d, the fold changes of the median values of either hypocotyl length or cotyledon area between 27 and 12 °C are shown. For the box and whisker plots, the boxes represent from the 25th to the 75th percentile and the bars equal the median values. Samples with different letters exhibit statistically significant differences (ANOVA, Tukey’s HSD, P ≤ 0.05, n ≥ 33). The source data underlying the hypocotyl (a, b) and cotyledon (c, d) measurements are provided in the Source Data file. Full size image

We next examined the effect of temperature on cotyledon expansion. Seedlings of a variety of Arabidopsis ecotypes, including Ler and Col-0, accelerate cotyledon expansion when moved from low intensity (i.e., below 10 µmol m−2 s−1) to high intensity (i.e., above 50 µmol m−2 s−1) of R light, and this cotyledon response depends on phyB29,57. Therefore, in contrast to restricting hypocotyl growth, phyB promotes light-dependent cotyledon expansion29. To our surprise, warmer temperatures further enhanced phyB-dependent cotyledon expansion in Ler and Col-0 in permissive 50 µmol m−2 s−1 R light (Fig. 1c, d). We did not observe a linear trend for the temperature effects on cotyledon expansion in 10 µmol m−2 s−1 R light, despite a slight reduction of cotyledon size from 16 to 27 °C (Fig. 1c, d). Interestingly, PBG and PBC grown in 10 µmol m−2 s−1 R light also showed increases in cotyledon area with temperature in a similar manner as their respective ecotype-background lines grown in 50 µmol m−2 s−1 R light (Fig. 1c, d). These results suggest that the high levels of phyB-FP in PBG and PBC lines could compensate for the low-light conditions and enable the temperature-dependent cotyledon expansion response. Therefore, we conclude that PBG and PBC retain the same temperature effects in promoting phyB-dependent cotyledon expansion as Ler and Col-0, respectively.

Temperature-dependent PB dynamics in the hypocotyl

To investigate how ambient temperature influences PB morphology, we first characterized the steady-state patterns of phyB-GFP PBs in epidermal-cell nuclei of the top one-third of the hypocotyl in 4-day-old PBG seedlings grown at 12, 16, 21, and 27 °C. We chose to grow seedlings in 10 µmol m−2 s−1 R light because it has been previously shown that in this light condition phyB-FP localizes only to PBs (as opposed to some small foci as well)40,55. We quantified the number and volume of PBs using three-dimensional imaging analysis (see Methods). To our surprise, we did not observe a shade-like transition from PBs to small foci with temperature increases. Instead, phyB-GFP localizes to PBs in all four temperatures (Fig. 2a, top panels). Intriguingly, the average number of PBs per nucleus decreased progressively with temperature increases from four-to-five at 12 °C to two-to-three at 27 °C (Fig. 2b). We found that PBs can be categorized into two types based on their relative positions to the nucleolus: one type at the nucleolar periphery, referred hereafter as nucleolar-associated PB (Nuo-PB), and the other type away from the nucleolus, referred hereafter as non-nucleolar-associated PB (nonNuo-PB). The numbers of both Nuo-PBs and nonNuo-PBs decreased with temperature increases (Fig. 2b). The average number of Nuo-PBs per nucleus dropped from two at 12 °C to one at 27 °C (Fig. 2a, b). Similarly, the number of nonNuo-PBs declined from two-to-three per nucleus at 12 °C to one per nucleus at 27 °C (Fig. 2a, b). Consistently, the percentage of nuclei containing two or more either Nuo-PBs or nonNuo-PBs reduced gradually with temperature increases (Fig. 2c, d). For instance, at 12 °C, 87 ± 3% of the nuclei had two or more nonNuo-PBs, whereas at 27 °C, the majority of the nuclei (67 ± 5%) had either only one or zero nonNuo-PB (Fig. 2a, d). The volume of PB became larger with temperature increases (Fig. 2e). Interestingly, the amount of phyB-GFP in PBG increased with temperature and was more than tripled at 27 °C compared to that at 12 °C (Fig. 2f). Therefore, the increases in PB size with temperature could be due to the redistribution of phyB-GFP to fewer PBs and/or the enhanced phyB accumulation. Because the expression of phyB-GFP was under the control of the 35S constitutive promoter36, the accumulation of phyB-GFP is likely due to a mechanism at the post-transcriptional level. Together, these results show that increases in temperature progressively reduce the number of PBs in the nuclei of hypocotyl epidermal cells. Not all PBs reacted to temperature equally, while some thermo-unstable PBs disappeared in warmer temperatures, certain PBs appear to be thermostable even at 27 °C.

Fig. 2: Temperature-dependent photobody dynamics in hypocotyl epidermal cells. a Maximum-projection, deconvolved fluorescence microscopy images showing representative steady-state patterns of phyB-GFP (green) or phyB-CFP (cyan) PBs in hypocotyl epidermal-cell nuclei of 4-day-old PBG or PBC seedlings, respectively, grown under 10 µmol m−2 s−1 R light at 12, 16, 21 °C, and 27 °C. Nuclei were labeled by DAPI (blue), and the boundaries of the nucleoli are traced by red dashed lines. Nuo-PBs and nonNuo-PBs are indicated by white and orange arrowheads, respectively. The percentage of nuclei including s.e. with the indicated PB pattern is shown in each image; n indicates the total number of nuclei analyzed. Scale bars represent 5 μm. b Quantification of the numbers of total, Nuo, and nonNuo PBs per nucleus in hypocotyl epidermal-cell nuclei of the PBG and PBC seedlings described in a. c Quantification of the percentage of nuclei in hypocotyl epidermal-cell nuclei of the PBG and PBC seedlings described in a with two or more Nuo-PBs. d Quantification of the percentage of nuclei in hypocotyl epidermal-cell nuclei of the PBG and PBC seedlings described in a with two or more nonNuo-PBs. For c, d, error bars represent s.e. calculated from groups of seedlings. Different letters denote statistically significant differences (ANOVA, Tukey’s HSD, P ≤ 0.05, n = 3). e Quantification of the volume of PBs in hypocotyl epidermal-cell nuclei of the PBG and PBC seedlings described in a. For b, e, the boxes represent from the 25th to the 75th percentile and the bars equal the median values. Samples with different letters exhibit statistically significant differences (ANOVA, Tukey’s HSD, P ≤ 0.05, n ≥ 9). f Immunoblot results showing the levels of phyB-GFP or phyB-CFP in 4-day-old PBG or PBC seedlings grown under 10 µmol m−2 s−1 R light in the indicated temperatures. phyB-FPs were detected by anti-phyB antibodies. RPN6 was used as a loading control, and phyB-9 was used as a negative control. The relative levels of phyB-FPs, normalized against the corresponding levels of RPN6, are shown below the phyB-FP immunoblot. The source data underlying the PB measurements in b–e, and the immunoblots in f are provided in the Source Data file. Full size image

To test whether the temperature-dependent PB dynamics vary in different Arabidopsis ecotypes, we characterized the PBs in the PBC line. Similar to PBG, the numbers of total, Nuo, and nonNuo PBs in the nuclei of hypocotyl epidermal cells in PBC decreased with temperature increases (Fig. 2a, b). The percentage of nuclei containing two or more either Nuo-PBs or nonNuo-PBs declined with temperature increases (Fig. 2a–d). The PBs also became larger with temperature increases in PBC, so as the level of phyB-CFP (Fig. 2e, f). The main difference between PBC and PBG was that PBC had significantly more PBs per nucleus than PBG. This difference was particularly obvious in lower temperatures. For example, at 12 °C, while PBG contained four to five total PBs per nucleus, PBC had almost twice as many (Fig. 2a, b). Interestingly, while the numbers of Nuo-PBs of PBG and PBC differed only slightly, the number of nonNuo-PBs per nucleus in PBC was more than doubled compared with that in PBG—that is, two to three nonNuo-PBs in PBG vs. six to eight in PBC (Fig. 2a, b). Therefore, the differences in PB numbers between PBC and PBG were mainly contributed by the numbers of nonNuo-PBs. The differences in PB number between PBG and PBC were unlikely caused by a difference in the amount of phyB-FP because the levels of phyB-GFP and phyB-CFP are comparable between these two lines across the temperature range examined (Fig. 2f). Intriguingly, the dynamic changes in PB number by temperature were also mainly contributed by the variations in nonNuo-PBs (Fig. 2b), suggesting that nonNuo-PBs are more dynamically regulated by both environmental and genetic factors. Together, these results show that temperature changes trigger similar PB dynamics in the hypocotyl epidermal cells in Ler and Col, despite variations in the precise number of PBs per nucleus between the two ecotypes. These results suggest that individual PBs vary in thermostability, and temperature changes trigger dynamic assembly/disassembly of selective thermo-unstable PBs, while thermostable PBs can persist even in elevated temperatures.

Temperature-dependent PB dynamics in the cotyledon

PB dynamics have been characterized mainly in epidermal cells of the hypocotyl. It remains unclear whether PB dynamics are influenced by tissue/organ type. Given temperature elicits opposing effects on the function of phyB signaling in hypocotyl elongation and cotyledon expansion, we asked whether temperature induces similar or distinct PB dynamics in the cotyledon. To that end, we characterized the steady-state PB patterns under the same series of ambient temperatures in the epidermal cells on the adaxial side of the cotyledon. Interestingly, similar to hypocotyl epidermal cells, temperature increases from 12 to 27 °C led to progressive decreases in the total number of PBs in the cotyledon epidermal nuclei in both PBG and PBC (Fig. 3a–c). Notably, the cotyledon nuclei contained fewer PBs compared with hypocotyl nuclei. For example, the cotyledon nuclei in PBG grown at 12 °C had mostly two-to-three PBs vs. four-to-five PBs in the hypocotyl nuclei (Figs. 2b and 3b). Similarly, the cotyledon nuclei of PBC grown at 12 °C contained five-to-six PBs compared to nine-to-ten PBs in the hypocotyl nuclei (Figs. 2b and 3c). In PBC, the numbers of both Nuo-PBs and nonNuo-PBs in cotyledon cells also declined with temperature increases (Fig. 3c); the percentage of nuclei with two or more of either Nuo-PBs or nonNuo-PBs decreased dramatically from 12 to 27 °C (Fig. 3a, d). Comparing the PB numbers between PBG and PBC, we found that the cotyledon epidermal nuclei in PBC had more PBs than those in PBG (Fig. 3a–c), this trend is the same as the hypocotyl (Fig. 2a, b). Interestingly, we found that the majority of the nuclei in PBG cotyledon epidermal nuclei contained only two PBs—one Nuo-PB and one nonNuo-PB—at 12 °C (68 ± 3%), 16 °C (64 ± 3%), and 21 °C (63 ± 3%) (Fig. 3a, e). In striking contrast, at 27 °C, the nonNuo-PB disappeared in 67 ± 3% of the cotyledon epidermal nuclei in PBG, leaving only one Nuo-PB (Fig. 3a, b, e). More than 85% of cotyledon epidermal nuclei in PBG maintained one Nuo-PB across the temperature range between 12 and 27 °C (Fig. 3e), indicating distinct thermostabilities between the thermosensitive nonNuo-PB and the thermo-insensitive Nuo-PB. The reduction of PB number with temperature increases suggests that the thermostability of PBs is determined by the stability of the Pfr form of phyB, which is attenuated by warmer temperatures18. Consistent with this hypothesis, increasing light intensity from 10 to 50 µmol m−2 s−1 R light, which stabilizes the Pfr, increased the numbers of PBs in the cotyledon cells of both PBG and PBC lines at 27 °C (Fig. 3a–d). Similar to hypocotyl PBs, the size of the cotyledon PBs also increased with temperature increases (Fig. 3f). Together, these results further support the notion that individual PBs exhibit distinct thermostabilities. The number of PBs can be influenced by tissue/organ-specific factors. The fact that cotyledon and hypocotyl epidermal cells display similar PB dynamics suggests that the opposing effects of temperature on phyB signaling might be caused by tissue/organ-specific signaling circuitry downstream of PB dynamics or independent of phyB.

Fig. 3: Temperature-dependent photobody dynamics in cotyledon epidermal cells. a Maximum-projection, deconvolved fluorescence microscopy images showing representative steady-state patterns of phyB-GFP (green) or phyB-CFP (cyan) PBs in cotyledon epidermal-cell nuclei of 4-day-old PBG or PBC seedlings, respectively, grown under 10 µmol m−2 s−1 R light at 12, 16, 21, and 27 °C, as well as under 50 µmol m−2 s−1 R light at 27 °C (R50). Nuclei were labeled by DAPI (blue), and the boundaries of nucleoli are traced by red dashed lines. Nuo-PBs and nonNuo-PBs are indicated by white and orange arrowheads, respectively. The percentage (with s.e.) of nuclei with the indicated PB pattern is shown in each image; n indicates the total number of nuclei analyzed. Scale bars represent 5 μm. b, c Quantification of the numbers of total, Nuo, and nonNuo PBs per nucleus in cotyledon epidermal-cell nuclei of the PBG (b) and PBC (c) seedlings described in a. d Quantification of the percentages of nuclei in cotyledon epidermal-cell nuclei with two or more either Nuo- or nonNuo-PBs in the PBC seedlings described in a. e Quantification of the percentage of nuclei in cotyledon epidermal-cell nuclei containing one or more either Nuo- or nonNuo-PB in the PBG seedlings described in a. For d, e, error bars represent s.e. calculated from groups of seedlings. Different letters denote statistically significant differences (ANOVA, Tukey’s HSD, P ≤ 0.05, n = 3). f Quantification of the volume of PBs in cotyledon epidermal-cell nuclei of the PBG and PBC seedlings described in a. For b, c, f, the boxes represent from the 25th to the 75th percentile, and the bars equal the median values. Samples with different letters exhibit statistically significant differences in PB number (ANOVA, Tukey’s HSD, P ≤ 0.05, n ≥ 9). The source data underlying the PB measurements in b–f are provided in the Source Data file. Full size image

Temperature-induced rapid disassembly of thermosensitive PBs

The simplicity of the PB pattern in the PBG cotyledon epidermal nuclei allows us to distinguish the two PBs based on their positions to the nucleolus, and therefore provides an opportunity to determine the kinetics of the disappearance of the thermosensitive nonNuo-PB during the 21 to 27 °C transition. A major challenge in determining PB dynamics is that the localization pattern of phyB-FPs can be rapidly and dramatically altered by the excitation light during live-cell fluorescence imaging58. To circumvent this problem, we fixed the seedling samples and characterized the steady-state phyB-GFP PB patterns at selected time points within 12 h after the 21 to 27 °C transition (Fig. 4a). The results show that the percentage of nuclei containing one nonNuo-PB dropped from about 60% at 21 °C at time 0 to 36% after 6 h at 27 °C and maintained at this percentage for the rest of the time course (Fig. 4a, b). In contrast, the percentage of nuclei containing one Nuo-PB did not change (Fig. 4c). Neither the level of phyB-GFP nor the size of PBs varied more than 2-fold (Fig. 4d, e). Together, these results demonstrate that individual PBs could respond to a specific temperature range and temperature increases induce rapid disassembly of phyB-GFP from distinct thermosensitive PBs.

Fig. 4: Kinetics of warm temperature-induced disappearance of the nonNuo-PB in PBG. a Schematic illustration of the 21 to 27 °C transition experiment with maximum-projection, fluorescence microscopy images showing representative steady-state patterns of phyB-GFP PBs in cotyledon epidermal-cell nuclei of 4-day-old PBG seedlings at the indicated time points. Nuclei were labeled by DAPI (blue), and the boundaries of the nucleoli are traced by red dashed lines. Nuo-PBs and nonNuo-PBs are indicated by white and orange arrowheads, respectively. The percentage of nuclei with s.e. showing the indicated PB pattern is included in each image; n indicates the total number of nuclei analyzed. Scale bars represent 5 μm. b Quantification of the percentage of cotyledon epidermal-cell nuclei with one or more nonNuo-PB in the PBG seedlings described in a. c Quantification of the percentage cotyledon epidermal-cell nuclei with one Nuo-PB in the PBG seedlings described in a. For b, c, error bars represent s.e. calculated from groups of seedlings. Different letters denote statistically significant differences (ANOVA, Tukey’s HSD, P ≤ 0.05, n = 3). d Immunoblot results showing the levels of phyB-GFP in 4-day-old PBG seedlings grown under 10 µmol m−2 s−1 R light in the indicated time points during the 21 to 27 °C transition. phyB-GFP was detected by anti-phyB antibodies, and RPN6 was used as a loading control. phyB-9 was used as a negative control. The relative levels of phyB-GFP normalized against the corresponding levels of RPN6 are shown below the phyB-GFP immunoblot. e Quantification of the volume of PBs in cotyledon epidermal-cell nuclei of the PBG seedlings during the 21 to 27 °C transition described in a. The boxes represent from the 25th to the 75th percentile, and the bars equal the median values. Samples with the same letter exhibit no statistically significant difference in PB volume (ANOVA, Tukey’s HSD, P ≤ 0.05, n = 10). The source data underlying the PB measurements in b, c, e, and the immunoblots in d are provided in the Source Data file. Full size image

phyB’s C-terminal module localizes to thermo-insensitive PBs

The structural basis of the light-dependent PB localization of phyB has been extensively investigated. PhyB is a homodimer; PB localization requires the dimeric Pfr form of phyB40,41,43,46,59. Each phyB monomer contains an N-terminal photosensory module and a C-terminal output module20,21. PB localization of phyB is mediated by the dimeric form of phyB’s C-terminal output module, which presumably contains localization signals or sequences for targeting phyB to the nucleus and PBs46,54,55. PhyB’s C-terminal module alone localizes constitutively to PBs54,55. The current model posits that the subcellular targeting activities of the C-terminal output module is masked in the Pr form by the N-terminal module, at least in part, through light-dependent interactions between the two modules, the interaction between N- and C-terminal modules weakens in the Pfr and thereby the PB localization activity of the C-terminal module is unmasked or exposed55. If this model is correct, the C-terminal module alone would be expected to localize to PBs that are temperature insensitive and should show a similar pattern to that of active phyB. To test this hypothesis, we used the BCY line, which expresses the phyB C-terminal module fused with YFP (BCY) in the phyB-9 background55, and tested whether the localization of BCY to PBs in cotyledon-cell nuclei in the dark can be altered by temperature changes between 12 and 27 °C. Supporting our hypothesis, the BCY-containing PBs did not respond to temperature changes. In all four temperatures tested from 12 to 27 °C, most of the nuclei contained more than eight PBs, including at least two Nuo-PBs and six nonNuo-PBs (Fig. 5a, b). The percentage of nuclei with such a PB pattern stayed the same across the temperature range (Fig. 5d). Interestingly, comparing the PB pattern of BCY with that of PBC (both in the phyB-9 background), the pattern of BCY PBs resembles that of PBC PBs at 12 °C, where the Pfr is more stabilized (Fig. 3a, c). The number of BCY PBs, including Nuo-PBs and nonNuo-PBs, also stayed the same between dark- and light-grown BCY lines (Fig. 5a, b, d). These results indicate that the localization of BCY to PBs is not responsive to light and temperature, and also is not influenced by possible heterodimerization with other phys in the light60,61. Consistent with the thermo-insensitivity of BCY PBs, the cotyledon size of BCY did not respond to temperature changes in the dark (Fig. 5e). The sizes of dark-grown BCY cotyledons were at least 5-fold smaller than those of PBC grown in the light, suggesting that phyB’s C-terminal module alone is not sufficient in promoting cotyledon expansion (Figs. 1d and 5e). BCY seedlings grown in the light did show significantly larger cotyledons compared with dark-grown BCY seedlings (Fig. 5e), which is likely due to the actions of other phys. The level of BCY also increased with temperature increases (Fig. 5f). However, in this case, the volume of individual BCY PBs did not change significantly (Fig. 5g), possibly due to the distribution of the additional BCY to the large numbers of PBs. Together, these results indicate that temperature does not affect the PB localization activity of the C-terminal module of phyB per se, and therefore the thermostability of PBs likely depends on phyB’s N-terminal photosensory module18.

Fig. 5: BCY and YHB-YFP display distinct temperature-insensitive PB patterns. a Maximum-projection, deconvolved images showing representative steady-state patterns of phyB C-terminal module fused with YFP (BCY) or YHB-YFP PBs in cotyledon epidermal-cell nuclei of 4-day-old BCY and YHB seedlings, respectively, grown at 12, 16, 21, and 27 °C in the dark or in 10 µmol m−2 s−1 in R light. Nuclei were labeled by DAPI (blue), white arrowheads indicate Nuo-PB, and the boundaries of the nucleoli are traced by red dashed lines. The percentage of nuclei with s.e. showing the representative PB pattern is shown in each image; n indicates the total number of nuclei analyzed. Scale bars represent 5 μm. b, c Quantification of the numbers of total, Nuo, and nonNuo PBs per nucleus in cotyledon epidermal-cell nuclei of the BCY (b) and YHB (c) seedlings described in a. d Quantification of the percentage of nuclei with the indicated number of PBs in cotyledon epidermal-cell nuclei of the BCY and YHB seedlings described in a. Error bars represent s.e. calculated from groups of seedlings. Different letters denote statistically significant differences (ANOVA, Tukey’s HSD, P ≤ 0.05, n = 3). e Cotyledon size measurements of the BCY or YHB seedlings described in a. f Immunoblot results showing the levels of BCY or YHB-YFP in 4-day-old BCY or YHB seedlings grown in the dark or 10 µmol m−2 s−1 R light in the indicated temperatures. BCY and YHB-YFP were detected by anti-phyB antibodies. RPN6 was used as a loading control. The relative levels of BCY and YHB-YFP normalized against the corresponding levels of RPN6 are shown. g Quantification of the volume of BCY and YHB-YFP PBs in cotyledon epidermal-cell nuclei of the BCY and YHB seedlings described in a. For b, c, e, g, the boxes represent from the 25th to the 75th percentile and the bars equal the median values. Samples with different letters exhibit statistically significant differences (ANOVA, Tukey’s HSD, P ≤ 0.05, for b, c, and g, n ≥ 9, for e, n ≥ 78). The source data underlying the PB measurements in b–d, g, cotyledon measurements in e, and immunoblots in f are provided in the Source Data file. Full size image

YHB preferentially localizes to one thermo-insensitive PB

To further examine the structural basis of PB’s thermostability, we turned to the constitutively active phyB mutant YHB, which carries a Y276H mutation in phyB’s chromophore attachment domain and locks phyB in an active form53. It is important to note that although YHB is biologically active, it represents a unique active conformation because YHB is poorly photoactive and stuck mainly in an R light-absorbing conformation—that is, a biologically active Pr form53,62,63,64. Although YHB has been shown to localize constitutively to PBs even in the dark50,53,65, the pattern of YHB PBs has not been characterized in comparison with the wild-type phyB PBs. If the thermostability of PBs is determined by the N-terminal module, a constitutively active phyB is expected to show a similar PB pattern as BCY or phyB-FP in low temperatures. To test this hypothesis, we used a YHB line, which expresses YHB-YFP in the phyB-9 background66. The pattern of YHB-YFP PBs in cotyledon epidermal nuclei in the dark stayed the same between 12 and 27 °C (Fig. 5a, c). However, this pattern was strikingly different from that of BCY. The majority of the nuclei contained only a YHB-YFP Nuo-PB without any nonNuo-PB, and the rest nuclei contained one Nuo-PB and one nonNuo-PB (Fig. 5a, c). The pattern of YHB-YFP PBs is thus similar to that of phyB-CFP at 27 °C with only thermostable PBs (Fig. 3a, c). The pattern of YHB-YFP PBs stayed the same between light- and dark-grown YHB seedlings (Fig. 5a, c–e). The level of YHB-YFP increased with temperature; in this case, we also observed an increase in the size of YHB-YFP PBs at 27 °C (Fig. 5f, g). Together, these results show that disruption of the ability of conformational changes within the N-terminal module of phyB abrogates the temperature responsiveness of PBs, supporting the notion that the thermosensitivity of PBs relies on the N-terminal module. The fact that YHB localizes preferentially to only a couple of thermostable PBs as opposed to multiple PBs like phyB-CFP suggests that either the assembly of thermosensitive PBs might require the photo or thermal reversion of phyB or the specific conformation of YHB only permits its assembly to selective thermostable PBs.

Warm temperature and shade induce distinct PB dynamics

The current model suggests that warm temperature attenuates phyB functions by enhancing the thermal reversion of phyB to its inactive Pr form. Therefore, warm temperature signaling is thought to work in a similar manner as shade, which promotes the photoconversion of phyB to the Pr. Previous studies have shown that an increase in FR light under continuous R light or an end-of-day FR treatment leads to the relocalization of phyB-FP to many small foci and the nucleoplasm40. However, the temperature-dependent PB dynamics we observed are quite different from the published PB response by FR or shade. To confirm this discrepancy between warm temperature and shade in the same cell type, we characterized the localization pattern of phyB-GFP in cotyledon epidermal-cell nuclei of PBG grown at 21 °C in a simulated shade condition with 10 µmol m−2 s−1 R light supplemented by 10 µmol m−2 s−1 FR light. We found that all phyB-GFP-containing PBs disappeared under the shade condition, phyB-GFP localized to many smaller subnuclear foci (Fig. 6a–c). The numbers of both Nuo and nonNuo foci increased in the shade condition compared with the PBs in 10 µmol m−2 s−1 R light at 21 and 27 °C (Fig. 6b). Because it is rather arbitrary to define large PBs and small foci, we looked at the distribution of the sizes of the PBs or foci in the three datasets (Fig. 6c). The results clearly show that the transition to small foci appeared unique to the shade response, as the size distribution of PBs remained the same between 21 and 27 °C (Fig. 6c). These results, therefore, demonstrate that warm temperature and shade induce distinct PB dynamics.