An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis

Sanghwa Lee; Ling Zhu; Enamul Huq

doi:10.1371/journal.pgen.1009595

. 2021 Jun 1;17(6):e1009595. doi: 10.1371/journal.pgen.1009595

An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis

Sanghwa Lee ¹, Ling Zhu ^1,^¤, Enamul Huq ^1,^*

Editor: Philip Anthony Wigge²

PMCID: PMC8195427 PMID: 34061850

Abstract

Plant growth and development are acutely sensitive to high ambient temperature caused in part due to climate change. However, the mechanism of high ambient temperature signaling is not well defined. Here, we show that HECATEs (HEC1 and HEC2), two helix-loop-helix transcription factors, inhibit thermomorphogenesis. While the expression of HEC1 and HEC2 is increased and HEC2 protein is stabilized at high ambient temperature, hec1hec2 double mutant showed exaggerated thermomorphogenesis. Analyses of the four PHYTOCHROME INTERACTING FACTOR (PIF1, PIF3, PIF4 and PIF5) mutants and overexpression lines showed that they all contribute to promote thermomorphogenesis. Furthermore, genetic analysis showed that pifQ is epistatic to hec1hec2. HECs and PIFs oppositely control the expression of many genes in response to high ambient temperature. PIFs activate the expression of HECs in response to high ambient temperature. HEC2 in turn interacts with PIF4 both in yeast and in vivo. In the absence of HECs, PIF4 binding to its own promoter as well as the target gene promoters was enhanced, indicating that HECs control PIF4 activity via heterodimerization. Overall, these data suggest that PIF4-HEC forms an autoregulatory composite negative feedback loop that controls growth genes to modulate thermomorphogenesis.

Author summary

Global warming caused by climate change affects all terrestrial life forms including crop yield. Proper response to high ambient temperature is crucial for survival of plants. However, our understanding of how plants respond to high ambient temperature is still rudimentary. Here we show that the HECATE and the PHYTOCHROME INTERACTING FACTORs (PIFs) family of basic helix loop helix proteins antagonistically regulate thermomorphogenesis. PIFs transcriptionally activate the expression of HECs in response to high ambient temperature. HEC2 in turn physically heterodimerizes with PIF4 and inhibit its function. Because PIF4 controls its own expression, this PIF-HEC negative feedback loop fine tunes thermomorphogenesis in a temperature-dependent manner. Our data contribute to a deeper understanding of how plants respond to high ambient temperature and are expected to help develop next generation crops that are resilient to global warming.

Introduction

Temperature is one of the most influential environmental factors affecting all terrestrial life forms. As such, a small increase in ambient temperature due to global warming can have profound impacts on disease resistance, crop yield and ultimately ecological balance [1–3]. High ambient temperature-mediated regulation of plant development is termed thermomorphogenesis, which is characterized by elongated hypocotyl, petiole and root, hyponastic growth, and early flowering [4,5]. These morphological changes help plants adapt to a new climate and complete reproduction.

Plants use diverse mechanisms to perceive high ambient temperature [6,7]. In the model plant Arabidopsis thaliana, the red-light photoreceptor phytochrome B (phyB) senses temperature by a process called thermal relaxation, where the active Pfr form of phyB is converted back to the inactive Pr form by high ambient temperature [8,9]. A prion-like domain in EARLY FLOWERING 3 (ELF3) is necessary for the conversion of the soluble active form to an inactive phase-separated form of ELF3 at high ambient temperature [10]. In addition, similar to the bacterial RNA thermometer, an RNA thermoswitch within the 5’-untranslated region of PHYTOCHROME INTERCTING FACTOR 7 (PIF7) acts as a temperature sensor to regulate the translational efficiency of PIF7 mRNA [11].

The signaling pathway regulating thermomorphogenesis involves multiple factors [4]. Among these, PIF4 acts as a crucial hub transcription factor regulating this process [12]. Both the expression and stability of PIF4 is upregulated by high ambient temperature, which in turn controls downstream genes involved in auxin signaling and biosynthesis to promote hypocotyl elongation [12–16]. Furthermore, several factors have been shown to control either the PIF4 abundance and/or activity to regulate thermomorphogenesis. Among the positive regulators of PIF4, HEMERA (HMR) stabilizes PIF4 to promote thermomorphogenesis [17]. BBX18/23 promotes PIF4 activity through inhibition of ELF3 function [18]. TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) transcription factors directly control the expression as well as the activity of PIF4 by physical interaction to promote thermomorphogenesis [19,20]. The cold response regulator CBF1 and MYB30 directly control the expression as well as the stability of PIF4 by inhibiting phyB-PIF4 interaction [21,22]. SHORT HYPOCOTYL UNDER BLUE1 associates with the clock components CCA1/LHY, which directly binds to the PIF4 promoter and activate PIF4 transcription [23]. The CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)-DEETIOLATED 1 (DET1)-ELONGATED HYPOCOTYL 5 (HY5) module also controls PIF4 both transcriptionally and post- translationally [24–27]. Finally, SUPPRESSOR OF PHYA-105 (SPA) family of genes have been shown to regulate thermomorphogenesis in part via controlling the phyB-PIF4 module in part through its kinase activity [28].

Among the negative regulators of PIF4, the blue-light receptor Cryptochrome 1 (Cry1) directly interacts with PIF4 in a blue light-dependent manner to inhibit thermomorphogenesis [29]. Ultraviolet-B light (UV-B) perceived by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8) attenuates thermomorphogenesis either by repressing the expression of PIF4 via UVR8-COP1 complex and/or UV-B-mediated stabilization of HFR1, which suppresses PIF4 activity at high ambient temperature [30]. The RNA-binding protein FCA inhibits PIF4 promoter occupancy by direct physical interaction with PIF4 and attenuates thermomorphogenesis [31]. TIMING OF CAB EXPRESSION 1 (TOC1) suppresses thermoresponsive growth in the evening by inhibiting PIF4 activity [32]. EARLY FLOWERING 4 (ELF4) stabilizes ELF3 function, which inhibits PIF4 activity independent of the circadian clock to regulate thermomorphogenesis [10,33,34]. Furthermore, GIGANTEA (GI) represses thermomorphogenesis by stabilizing REPRESSOR OF ga1-3 (RGA), which inhibits PIF4 activity [35]. While these factors control the activity of PIF4 to regulate thermomorphogenesis, here we report a unique autoregulatory mechanism where PIF4 and HECs control their own expression. Moreover, HECs form a composite negative feedback loop with PIF4 that controls growth genes to modulate thermomorphogenesis.

Results

HECATEs (HECs) inhibit thermomorphogenesis

HECATE (HEC) genes were originally reported not only as essential components in the reproductive tissue development, but also as negative regulators of photomorphogenesis [36–38]. In our recent RNA-seq analysis of thermo-regulated gene expression [28], we found that HECATE 1 and 2 are both induced after high ambient temperature exposure (S1 Fig). Therefore, qPCR was conducted for independent verification of the RNA-seq result using 6-day-old seedlings grown at 22°C or transferred to 28°C for additional 24 hours under continuous white light. As expected, the transcript levels of both HEC1 and HEC2 were increased at high ambient temperature (Fig 1A). To investigate the potential temperature regulation of HEC2 at the post-translational level, we examined HEC2 protein level at 22°C and 28°C using HEC2-GFP overexpression line driven by the constitutively active cauliflower mosaic virus (CaMV) 35S promoter. The result shows that HEC2 protein level was accumulated at high ambient temperature without alteration of the transcript level (Figs 1B and S2), indicating that high ambient temperature stabilizes HEC2 post-translationally. To investigate whether HEC1 and HEC2 play any role in regulating thermomorphogenesis, we examined the phenotype of hec1hec2 double mutant and 35S:HEC2-GFP overexpression line at high ambient temperature. Intriguingly, hec1hec2 double mutant showed longer hypocotyl and petiole length, whereas 35S:HEC2-GFP showed shorter hypocotyl and petiole length compared to the wild-type at high ambient temperature (Figs 1C, 1D and S3A). Furthermore, hec1 hec2 mutant showed early flowering while 35S:HEC2-GFP and Col-0 remained the same at high ambient temperature (S3B Fig). Overall, these data show that HECs are induced at high ambient temperature both transcriptionally and post-translationally, and act as negative regulators of thermomorphogenesis.

Fig 1 — (A) The expression of *HEC1* and *HEC2* is upregulated by high ambient temperature. RT-qPCR samples were from Col-0 whole seedlings grown for 5 days at 22°C and then either kept at 22°C or transferred to 28°C for 24 hours. Four biological repeats were performed. Relative gene expression levels were normalized using *ACT7*. (B) Western blot shows the level of HEC2-GFP from *35S*:*HEC2-GFP* whole seedlings grown for 5 days at 22°C and either kept at 22°C or transferred to 28°C for 4 hours. Coomassie staining was used as a loading control. The experiments were repeated three times with similar results as indicated in the bar graph in lower panel. (C) Photograph showing seedling phenotypes of *35S*:*HEC2-GFP* and *hec1hec2* in normal and high ambient temperature. Seedlings were grown for two days in continuous white light at 22°C and then either kept at 22°C or transferred to 28°C for additional four days before being photographed. The scale bar represents 5 mm. (D) Box plot shows the hypocotyl lengths grown under the conditions described in (C). More than 10 seedlings were measured for each experiment and was repeated 3 times. The letters a-d indicate statistically significant differences based on one-way ANOVA analysis with Tukey’s HSD test. Tukey’s box plot was used with median as a center value. The experiments were repeated more than three times with similar results.

PIF family members contribute to thermomorphogenesis

Among all PIFs, only PIF4 and PIF7 have been shown to regulate thermomorphogenesis [11,12,39]. Because PIF family members interact with HECs [37], we examined whether other PIFs also contribute to regulating thermomorphogenesis. Therefore, we tested high ambient temperature mediated hypocotyl and petiole elongation phenotypes using the higher order pif mutants and 35S:PIF-overexpression lines (PIF1, PIF3, PIF4 and PIF5). The results show that pif single, double, triple and quadruple mutants display attenuated or no response at 28°C compared to 22°C, and all four PIF-overexpression lines display elongated hypocotyl at high ambient temperature (Figs 2A–2C and S4A and S4B). Among pif single mutants, pif4 displayed the strongest attenuated response as previously shown [12]; however, other PIFs, such as PIF1, PIF3, and PIF5 also contribute to regulating thermomorphogenesis at high ambient temperature (Fig 2A–2C). Taken together, these data suggest that PIF family members collectively contribute to regulating thermomorphogenesis.

Fig 2 — (A) Photograph showing seedling phenotypes of high order *pif*s at normal and high ambient temperature conditions. Seedlings were grown for two days under continuous white light at 22°C and then either kept at 22°C or transferred to 28°C for additional six days before being photographed. The scale bar represents 5 mm. (B and C) Dot plot shows the hypocotyl and petiole lengths of seedlings grown under conditions described in (A). (D) Western blot shows the level of phyB from whole seedlings grown for 5 days in 22°C and either kept at 22°C or transferred to 28°C for 4 hours. Coomassie staining was used as a control. Red number indicates the quantitation value from anti-phyB divided by the control. (E) Bar graph shows the relative amount of phyB (n = 3). Asterisks indicate statistically significant difference using Student’s t-test; *p < 0.05 and **p < 0.01. The experiments were repeated three times with similar results. (F) Photograph shows the seedling phenotypes of *phyB*, *pifQphyB* and *pifQ* at normal and high ambient temperature. Seedlings were grown as described in (A). (G) Box plot shows the hypocotyl lengths for seedlings described in (F). (H) Photograph showing seedling phenotypes of WT, *hec1hec2*, *hec1hec2 pifQ* and *pifQ* in normal and high ambient temperature. More than 10 seedlings were measured for each experiment and was repeated 3 times. (I) Box plot shows the hypocotyl length of seedlings grown under conditions described in (A). The letters a-g indicate statistically significant differences based on one-way ANOVA analysis with Tukey’s HSD test. Tukey’s box plot was used with median as a center value. (J) Photograph showing seedling phenotypes of WT, *hec1hec2*, *35S*::*PIF4-myc* in either Col-0 or *hec1hec2* background at high ambient temperature. Conditions are the same as described in (A). (K) Box plot shows the hypocotyl lengths of seedlings grown under conditions described in (J). More than 10 seedlings were measured for each experiment and was repeated 3 times. The letters a-f indicate statistically significant differences based on one-way ANOVA analysis with Tukey’s HSD test. Tukey’s box plot was used with median as a center value.

Our previous report showed that stabilized PIF4 is correlated with destabilized phyB at high ambient temperature [28]. Therefore, we conducted immunoblot assays using 5-day-old seedlings of pif4, pifQ and PIF-overexpression lines with or without 28°C treatment for 4 hours. Consistent with their phenotypes, phyB level was up-regulated in the pif4 and more robustly in pifQ mutant background at 22°C (Fig 2D and 2E). However, in response to high ambient temperature, phyB level was reduced in both pif4 and pifQ backgrounds at 28°C (Fig 2D and 2E). Conversely, all four PIFs were stabilized at high ambient temperature (S4C and S4D Fig). In contrast, phyB levels were decreased in the wild-type as well as in four PIF-overexpression lines in response to high ambient temperature (S4E and S4F Fig). Thus, PIFs and phyB are inversely regulated at high ambient temperature as previously observed under red light conditions [40].

To investigate whether the altered protein levels of phyB and PIFs are caused by transcriptional regulation, we conducted qPCR to check the transcript levels of phyB and PIFs in PIF-overexpression lines with or without 28°C treatment for 4 hours. As expected, the transcript level of PHYB and PIF-overexpression lines did not alter in wild-type and PIF-overexpression lines, respectively after high ambient temperature treatment (S5A and S5B Fig). However, examination of the expression level of native PIFs showed that all four PIFs are transcriptionally up-regulated with the PIF4 being at the highest level in response to high ambient temperature (S6 Fig). Thus, PIFs are up-regulated both transcriptionally and post-translationally, while phyB is down-regulated only post-translationally in response to high ambient temperature. The altered phyB-PIF ratio contributes to the regulation of thermomorphogenesis.

Finally, we also examined the phenotype of phyB pifQ to test the genetic relationships between phyB and PIFs at high ambient temperature. Interestingly, phyB pifQ showed an intermediate phenotype in response to high ambient temperature (Fig 2F and 2G), implying other factors (e.g., PIF7 and others) might be involved in this process. Taken together, these data suggest that as a temperature sensor, phyB is acting as a master regulator, while PIFs are acting positively downstream of phyB in promoting thermormoprhogenesis.

Genetic relationships between HECs and PIFs

To examine the genetic relationships between HECs and PIFs, hec1 hec2 pifQ hexuple mutant and PIF4 overexpression line in hec1 hec2 background were generated. At high ambient temperature, hec1 hec2 pifQ showed strongly reduced hypocotyl and petiole elongation phenotype compared to parental genotypes (Figs 2H, 2I and S7A). The hexuple mutant phenotypes are almost similar to that of pifQ. In addition, the thermo-induced flowering time of hec1 hec2 mutant was inhibited in hec1 hec2 pifQ (S7B Fig), indicating that pifQ is epistatic to hec1 hec2 in response to high ambient temperature. Moreover, the elongated phenotype of the PIF4 overexpression line was exaggerated in the hec1 hec2 background (Fig 2J and 2K), suggesting that HECs genetically attenuate PIF4 function. Overall, these data suggest that HECs and PIFs are regulating thermomorphogenesis in an inter-dependent manner.

HECs alter global gene expression at high ambient temperature

High ambient temperature induces global gene expressions to promote thermomorphogenesis [9,18,28]. To examine whether HECs control thermo-induced gene expression, RNA-seq was conducted using wild-type Col-0, hec1 hec2 double mutant and pifQ at both 22°C and 28°C (Fig 3). The results show that 2008, 2209 and 1837 genes were differentially expressed in Col-0, hec1 hec2 and pifQ, respectively in response to high ambient temperature (Fig 3A). Among the three genotypes, 735 genes were co-regulated. Gene Ontology (GO) analysis revealed that 735 temperature-dependent genes are mainly involved in pigment biosynthetic pathways (S8 Fig). Examination of the HEC- and PIF-dependent genes display opposite expression patterns for many of these genes (S9A and S9B Fig). GO analysis of the HEC-dependent genes shows an enrichment of the genes involved in leucine and glucosinolate biosynthesis, plant cell-wall organization, organ morphogenesis and others (Fig 3B), while the PIF-dependent genes show that they are involved in auxin signaling, translational processes and leaf morphogenesis (Fig 3C). These data reinforce previous conclusions that PIFs regulate auxin signaling to modulate organ morphogenesis in response to high ambient temperature. Furthermore, 2008 DEGs in wild-type displayed distinct patterns in hec1 hec2 and pifQ as shown in the heatmap analysis (Fig 3D). Overall, these data suggest that HECs and PIFs control the expression of a large number of genes to regulate thermomorphogenesis.

Fig 3 — (A) Venn diagrams show differentially expressed genes (DEGs) in wild-type (WT) vs *hec1hec2* vs *pifQ* mutant at high ambient temperature. Six-day-old white light-grown seedlings were transferred to 22°C or 28°C for additional 24 hours and total RNA was extracted from three biological replicates for RNA-seq analyses. (B-C) Gene Ontology (GO) analysis of *HEC-*dependent 844 genes (B) and *PIF*-dependent 1067 genes (C). (D) Hierarchical clustering from 2008 DEGs from WT show distinct pattern in *hec1hec2* and *pifQ* mutant at high ambient temperature. (E-F) RT-qPCR analysis using growth genes (E), and PIF inhibitor genes (F). RT-qPCR samples were from whole seedlings grown for 6 days at 22°C and then either kept at 22°C or transferred to 28°C for 24 hours. Three biological repeats were performed. Relative gene expression levels were normalized using the expression levels of *ACT7* for RT-qPCR. The letters a-c indicate statistically significant differences based on one-way ANOVA analysis with Tukey’s HSD test.

To independently verify the RNA-seq data, qPCR assays were performed to examine the transcript level of several thermo-responsive marker genes such as YUC8 and IAA29 (Fig 3E and 3F). Consistent with the RNA-seq data, the transcript level of these genes was upregulated in response to high ambient temperature and correlated with thermomorphogensis as previously shown [15,29,30]. Interestingly, the transcript level of LONG HYPOCOTYL IN FAR-RED 1 (HFR1) and PIF3-LIKE 1 (PIL1), which are known inhibitors of PIFs in photomorphogenesis, thermomorphogenesis and shade avoidance responses [41–46], were up-regulated in the absence of HECs (Fig 3F). These data suggest that similar to HEC1 and HEC2, genes encoding the inhibitors of PIFs are also induced under high ambient temperature responses and perhaps participate in the negative feedback loop to regulate PIF level and activity at high ambient temperature. Taken together, these data also suggest that HECs play an important role in thermomorphogenesis via transcriptional regulation.

HECs alter phyB-PIF4 level at high ambient temperature

It has been shown that the protein level of PIF4 is crucial in regulating thermomorphogenesis due to its transcriptional regulation of growth genes via direct promoter binding [13,15]. Furthermore, we recently showed that the thermosensor phyB and the hub transcription factor PIF4 levels are oppositely regulated where low phyB level is correlated with high PIF4 level and vice versa at high ambient temperature [28]. To investigate whether phyB as well as PIF4 levels are controlled by HECs, Western blot analyses were conducted using hec1 hec2 mutant and 35S:HEC2-GFP seedlings grown at 22°C for 5 days and kept in 22°C or transferred to 28°C for 4 hours. Consistent with the phenotype (Fig 1C and 1D), hec1 hec2 mutant displayed reduced level of phyB at both 22°C and 28°C compared to that of wild-type (Fig 4A and 4B). The 35S:HEC2-GFP transgenic plant showed higher level of phyB compared to that of the wild-type at both temperatures, indicating that HECs contribute to stabilize phyB level. In contrast, PIF4 level is strongly stabilized in the 35S:HEC2-GFP transgenic plant at both temperature and modestly stabilized in the hec1 hec2 mutant compared to wild type at high ambient temperature (Fig 4A and 4B). While the phyB level in hec mutants correlates with the thermomorphogenesis phenotype, the PIF4 level did not correlate with the phenotype in hec mutants. These data suggest that HECs not only alter the phyB-PIF4 level at high ambient temperature, but also might regulate the activity of PIF4 to modulate thermomorphogenesis.

Fig 4 — (A) Western blot shows the level of phyB or PIF4 in WT, *35S*:*HEC2-GFP* and *hec1hec2* mutant. Whole seedlings were grown for 5 days at 22°C and either kept at 22°C or transferred to 28°C for 4 hours. Coomassie staining was used as a loading control. Red number indicates the quantitation value from anti-phyB or anti-PIF4 levels divided by Coomassie blue staining intensity. (B) Dot plot shows the relative amount of phyB (n = 3) or PIF4 (n = 3 or 4). Asterisks indicate statistically significant differences using Student’s t-test; **p < 0.01.

HEC2 regulates thermomorphogenesis in part by heterodimerizing with PIF4

HECs are known to interact with PIFs and regulate PIF1-mediated seed germination in a heterodimerization-dependent manner [37]. To examine if HECs regulate thermomorphogenesis through PIF4 in a heterodimerization-dependent manner, we tested the interaction of PIF4 with wild type HEC2 and a dimerization-defective mutant (mHEC2) both in yeast and in transgenic plants. Results show that PIF4 heterodimerizes with wild type HEC2 in yeast-two-hybrid assay, but not the dimerization-defective mutant HEC2 (Fig 5A). In vivo coimmunoprecipitation assay also showed that PIF4 interacts with the wild type HEC2 but not the mutant HEC2 (Fig 5B). Thus, HEC2 and possibly other HECs might regulate thermomorphogenesis by direct heterodimerization with PIF4.

Fig 5 — (A) HEC2 interacts with PIF4. Yeast two hybrid assay using PIF4 a bait and HEC2 or mHEC2 as preys. The error bars represent standard deviation. Three biological replicates were used in this study. (B) HEC2 interacts with PIF4 *in vivo*. *In vivo* coimmunoprecipitation assay was performed using *35S*:*PIF4-myc* with *35S*:*HEC2-GFP* or *mHEC2-GFP* crossed line. 35S:PIF4-myc was used as a control. (C) Photograph showing seedling phenotypes of WT, *35S*:*HEC2-GFP* and *35S*:*mHEC2-GFP* grown at normal and high ambient temperature conditions. Seedlings were grown for two days under continuous white light at 22°C and then either kept at 22°C or transferred to 28°C for additional four days before being photographed. The scale bar represents 5 mm. (D) Box plot shows the hypocotyl length grown under conditions described in (C). More than 10 seedlings were measured for each experiment and was repeated 3 times. The letters a-c indicate statistically significant differences between means of hypocotyl lengths (P<0.05) based on one-way ANOVA analysis with Tukey’s HSD test. Tukey’s box plot was used with median as a center value. n.s. stands for statistically not significant. (E) Western blots show the level of phyB and PIF4 in WT, *35S*:*HEC2-GFP* and *35S*:*mHEC2-GFP*. Coomassie staining was used as a control. Red number indicates the quantitation value from anti-phyB or anti-PIF4 detection divided by Coomassie blue staining intensity. (F) Dot plot shows the relative amount of phyB (top, n = 3) or PIF4 (bottom, n = 3). (G) Western blot shows the level of PIF4-myc from *35S*::*PIF4-myc* in either Col-0 or *hec1hec2* background. Seedlings were grown for 5 days at 22°C and either kept at 22°C or transferred to 28°C for 4 hours. Coomassie staining was used as a control. Red numbers indicate the anti-myc intensity divided from Coomassie control. (H) ChIP-qPCR analysis using promoter regions of *YUC8* and *IAA29*. Simple genomic region scheme is described with PIF4 binding motif in vertical line. *ACT7* was used as a control. ChIP-qPCR samples were from whole seedlings grown for 6 days at 22°C and then either kept at 22°C or transferred to 28°C for 24 hours. Three biological repeats were performed. Relative abundance was normalized from % of input value of *35S*::*PIF4-myc* at 22°C for ChIP-qPCR.

To test the above hypothesis more directly, we examined the phenotype of 35S:mHEC2-GFP plant compared to 35S:HEC2-GFP transgenic plant. The results show that the wild type HEC2 overexpression line (35S:HEC2-GFP) attenuates thermomorphogenesis, while the 35S:mHEC2-GFP plant display hypocotyl lengths similar to that of wild-type (Fig 5C and 5D), suggesting that heterodimerization of HEC2 with PIF4 is necessary for regulating thermomorphogenesis. Both 35S:HEC2-GFP and 35S:mHEC2-GFP did not affect flowering time at high ambient temperature (S10 Fig). We also performed Western blot analysis using phyB and PIF4 antibodies to test whether HEC2 regulates phyB and PIF4 level during thermomorphogenesis. Results show that the wild type HEC2 stabilized both phyB and PIF4 level in response to high ambient temperature, while the 35S:mHEC2-GFP plant showed similar levels of phyB and PIF4 to that of the wild-type (Fig 5E and 5F). Moreover, PIF4-myc is less abundant in the hec1 hec2 double mutant background compared to wild type at 22°C and 28°C (Fig 5G). To test whether the reduced level of PIF4 in hec1 hec2 background is due to the 26S proteasome-mediated degradation, we treated seedlings with the proteasome inhibitor bortezomib before Western blot was performed. Results show that PIF4-myc level was stabilized in the hec1 hec2 mutant and became similar to that of wild-type (S11 Fig), suggesting that HEC-PIF4 heterodimerization inhibits the 26S proteasome-mediated degradation of PIF4. These data are consistent with HEC2-mediated stabilization of PIF1 [37]. Overall, these data suggest that HEC2 stabilizes both phyB and PIF4, but might inhibit PIF4 function by heterodimerization during thermomorphogenesis.

HECs inhibit PIF4 promoter binding

PIF4 binds to the promoters of growth genes and promotes their transcription [13,15]. Since HEC2-PIF4 interaction was crucial for inhibiting thermomorphogenesis (Fig 5), we hypothesized that heterodimerization of HEC2-PIF4 could suppress the binding of PIF4 to the target promoters. To test this hypothesis, we performed in vitro DNA binding of PIF4 in the absence and presence of HEC2. Result shows that PIF4 robustly binds to DNA even in the presence of GST control. However, addition of HEC2 along with PIF4 strongly inhibits PIF4 binding to DNA (S12 Fig). To substantiate these in vitro data, we generated 35S:PIF4-Myc in hec1 hec2 mutant and compared the promoter binding to that of the 35S:PIF4-Myc in Col-0 background. Chromatin Immunoprecipitation (ChIP) assays were performed using the 35S:PIF4-Myc in Col-0 and hec1 hec2 mutant backgrounds at both 22°C and 28°C. The results show that even though PIF4 is less abundant in the hec1 hec2 background compared to wild type (Figs 5G and S11), the enrichment of PIF4 on the direct target gene promoters such as YUC8 and IAA29 was strongly higher in the hec1 hec2 mutant compared to Col-0 background (Fig 5H). These data are consistent with the expression level of these genes (Fig 3E), as well as the thermo-induced hypocotyl elongation phenotype of the 35S:PIF4-Myc/hec1 hec2 compared to 35S:PIF4-Myc (Fig 2J and 2K).

Previously, PIF1, PIF3, PIF4 and PIF5 have been shown to directly regulate the expression of HECs in darkness [37]. To examine if PIFs also regulate the expression of HECs under high ambient temperature, we performed RT-qPCR for HEC1 and HEC2 in wild type, pif4 and pifQ grown at 22°C and 28°C. Results show that the thermo-induced expression of both HEC1 and HEC2 is strongly down-regulated in the pif4 and almost eliminated in the pifQ background (Fig 6A). Consistent with the expression level, PIF4 binding to the HEC1 and HEC2 promoters was also enhanced under high ambient temperature (Fig 6C). In contrast, the thermo-induced expression of PIF4 is strongly up-regulated in the hec1 hec2 background compared to the wild type (Figs 6B and S13). Strikingly, the binding of PIF4 on its own promoter was also strongly enhanced in the hec1 hec2 background compared to the wild type (Fig 6D). Thus, PIF4 autoregulates itself as well as the expression of HECs under high ambient temperature. Conversely, HECs also regulate the expression and the promoter occupancy of PIF4 to fine tune thermomorphogenesis.

Fig 6 — (A) PIFs activate the expression of *HEC1* and *HEC2*. RT-qPCR analysis of *HEC1* and *HEC2* gene expression using Col-0, *pif4* and *pifQ* mutant. (B) HECs inhibit the expression of *PIF4* under high ambient temperature. RT-qPCR analysis of *PIF4* using Col-0 and *hec1hec2* mutant. Relative gene expression levels were normalized using expression levels of *ACT7*. (C) PIF4 directly activates the expression of *HEC*s. ChIP-qPCR analysis of PIF4 binding to the *HEC1* and *HEC2* promoter region using 35S:PIF4-myc in Col-0 background. *ACT7* was used for the control. (D) PIF4 activates its own expression in a HEC-dependent manner. ChIP-qPCR analysis of *PIF4* promoter region using 35S:PIF4-myc in Col-0 and *hec1hec2* backgrounds. For both RT-qPCR and ChIP-qPCR samples, whole seedlings grown for 5 days at 22°C and then either kept at 22°C or transferred to 28°C for 4 hours. Three biological repeats were performed. The letters a-c indicate statistically significant differences based on one-way ANOVA analysis with Tukey’s HSD test. Simple genomic region scheme is described with PIF4 binding motif in vertical line. (D) Simplified model shows the role of an autoregulatory module controlling thermomorphogenesis. At high ambient temperature, PIF4 activates the expression of both *PIF4* and *HEC*s by transcriptional regulation. HECs heterodimerize with PIF4 and inhibit PIF4 function which regulates growth genes at transcriptional level.

Discussion

High ambient temperature due to global warming has a far-reaching impact on food security, ecological balance, and planet sustainability [1–3,47]. Fundamental understanding of how plant growth and development is regulated by high ambient temperature will play a vital role in developing crop plants resilient to climate change. In this study, we provide multiple evidence in support of a new role for the HEC family of bHLH transcription factors to fine tune thermomorphogenesis. First, the expression of HEC1 and HEC2 is upregulated and HEC2-GFP is post-translationally stabilized in response to high ambient temperature (Figs 1A, 1B, and S1). Second, hec1 hec2 double mutant display elongated hypocotyl and petiole length, and early flowering (Figs 1C, 1D, and S3), hallmark phenotypes of thermomorphogenesis. In contrast, HEC2-GFP overexpression line displays attenuated thermomorphogenesis. Third, HECs regulate the expression of a large number of genes in response to high ambient temperature (Fig 3). Fourth, hec1 hec2 enhances the exaggerated phenotype of PIF4 overexpression line under high ambient temperature (Fig 2J and 2K). These data firmly establish HECs as negative regulators of thermomorphogenesis.

Previously, multiple negative regulators have been described in thermomorphogenesis pathway [4]. These include Cry1, ELF3, GI-DELLA module, TOC1, and FCA [29,31–33,35,48]. All of these factors directly interact with the central hub transcription factor, PIF4 and inhibit its function. By contrast, being the same HLH family of transcription factors, HECs heterodimerize with PIF4 and other PIFs, and produce a non-functional PIF-HEC heterodimer that is unable to bind to DNA (Figs 5, 6, and S12) (37). In addition, HECs also stabilize PIF4 and destabilize the thermo sensor phyB (Fig 4A and 4B). Thus, HECs regulate thermomorphogenesis by controlling the activity of PIFs in a heterodimerization-dependent manner. This behavior is reflected in the global gene expression analyses where HECs and PIFs regulate the expression of a large number of genes oppositely (Figs 3 and S9). Consistently, hec1 hec2 pifQ hexuple mutant displays similar phenotype as pifQ, suggesting PIFs are epistatic to HECs in regulating thermomorphogenesis (Fig 2H and 2I).

Downstream of the thermo sensor phyB, PIF4 has been shown to play central role in regulating thermomorphogenesis [12]. PIF7 also plays important roles in two ways: first, along with PIF4, PIF7 contributes to regulating thermomorphogenesis [39]; and second, an RNA thermoswitch at the 5’untranslated region of PIF7 mRNA plays a sensory role in perceiving temperature and regulating the translational efficiency of PIF7 mRNA [11]. Our data show that not only PIF4 and PIF7, other PIFs also contribute to regulating thermomorphogenesis (Figs 2 and S4). The expression and post-translational stability of PIF1, PIF3 and PIF5 are also upregulated by high ambient temperature (S4 and S6 Figs). Overexpression of PIF1, PIF3 and PIF5 displays longer hypocotyl compared with wild type (S4A and S4B Fig), while the pif single, double, triple, and quadruple mutants display an additive role in regulating thermomorphogenesis (Fig 2A–2C). Moreover, pifQ phyB quintuple mutant displays an intermediate phenotype between phyB and pifQ (Fig 2F and 2G), suggesting that other factors including other PIFs might contribute to regulating thermomorphogenesis.

Our data also show that PIF4 regulates its own expression by directly binding to its own promoter as recently described [49]. Thus, in response to high ambient temperature, PIF4 is stabilized and the stable PIF4 activates its own expression as well as other target growth genes (Figs 5H, 6B, 6D and S12) [4]. In addition, PIFs activate the expression of HECs by directly binding to the HEC promoters (Fig 6A and 6C) [37]. Under high ambient temperature, PIF4 plays a major role in this regulation in concert with other PIFs (Fig 6A). By contrast, HECs physically interact with PIF4 and inhibit the promoter occupancy to control its own transcription as well as other downstream target genes (Figs 5, 6B, 6D and S12). Consistently, the expression of PIF4 is strongly enhanced in hec1 hec2 background in response to high ambient temperature (Figs 6B and S13). In addition, HECs also interact with other PIFs (PIF1, PIF3 and PIF5) and inhibit their promoter occupancy as previously shown [37]. However, these PIFs do not autoregulate themselves, and HECs only inhibit their target gene expression [37]. The HEC function is analogous to the HFR1 function in inhibiting PIFs activity by heterodimerization [42,45,50]. Similar to HECs, the expression and stability of HFR1 are also up-regulated by high ambient temperature [43]. However, the regulation of HFR1 is blue-light-dependent, whereas HECs are regulated under white light conditions. Further studies are necessary to dissect which monochromatic light pathway controls the expression and stability of HECs.

In summary, HECs and PIFs are forming an autoregulatory composite negative feedback loop (Fig 6D), where PIFs transcriptionally activate the expression of HECs in response to high ambient temperature. HECs in turn physically heterodimerize with PIFs and stabilize them post-translationally. However, even though PIFs are stabilized by HECs, PIF activity is inhibited by HECs by direct heterodimerization. Because PIF4 controls its own expression, this PIF-HEC negative feedback loop fine tunes thermomorphogenesis in a temperature-dependent manner.

Materials and methods

Plant materials, growth conditions and phenotypic analyses

In this study, mutants and transgenic lines were generated from Col-0 ecotype of Arabidopsis thaliana. Seeds were surface-sterilized and plated on Murashige and Skoog (MS) medium without sucrose. Seeds were stratified for 3 days at 4°C in dark, and the plates were placed at 22°C for 2 days and then transferred to 22°C or 28°C for additional 4 days. Hypocotyl and petiole lengths were measured using ImageJ software (n>10) and statistically analyzed using one-way ANOVA analysis with Tukey’s HSD test. For flowering time measurement, plants were grown under long day conditions (16L:8D) and then the leaf number was counted at bolting for >10 plants.

Protein extraction and Western blot analyses

Total protein extracts were made from 50 seedlings for each sample using 50 μL urea extraction buffer [8 M urea, 0.35 M Tris-Cl pH 7.5, and 1× protease inhibitor cocktail]. Subsequently, 6X SDS loading buffer was added to the samples and boiled for 5 min. Supernatant from the samples was loaded into SDS-PAGE gels after centrifugation at 16,000 g for 15 min. PVDF membrane (EMD Millipore, Burlington, MA) were used for transfer, and Western blots were detected using anti-myc (Cell Signaling Technologies, Danvers, MA), anti-GFP (Abiocode, Agoura Hills, CA), anti-phyB, anti-PIF4 (AgriSera AB, Vännäs, Sweden) or anti-RPT5 (Abiocode, Agoura Hills, CA) antibodies. Coomassie blue staining and/or anti-RPT5 were used for the loading control.

RNA extraction, cDNA synthesis, and qRT-PCR

RNA extraction was performed as previously described [28]. Briefly, 6-day-old white light-grown seedlings were used with three independent biological replicates (n = 3). Seeds were kept in 22°C under continuous white light for 6 days. After 6 days, seedlings were either kept at 22°C or transferred to 28°C for additional 24 hours under continuous white light. Plant RNA purification kit (Sigma-Aldrich Co., St. Louis, MO) were used for total RNA extraction according to the manufacturer’s protocols. For cDNA synthesis, 1 mg of total RNA was used for reverse transcription with M-MLV Reverse Transcriptase (Thermofischer Scientific Inc., Waltham, MA). SYBR Green PCR master mix (Thermofischer Scientific Inc., Waltham, MA) and gene-specific oligonucleotides were used to conduct qPCR analyses using primers shown in S1 Table. Finally, relative transcription level was calculated using 2^ΔCt using ACT7 normalization.

RNA-seq analyses

3’Tag-Seq method was used in this study for RNA-seq analysis [51]. FastQC was used to examine raw read quality (www.bioinformatics.babraham.ac.uk/projects/fastqc/). The raw reads were aligned to the Arabidopsis genome with Bowtie2 [52] and TopHat [53]. The annotation of the Arabidopsis genome was from TAIR10 (www.arabidopsis.org/). Read count data were obtained using HTseq [54] (htseq.readthedocs.io/en/master/). Differentially expressed genes in WT/hec1 hec2/pifQ were identified using the EdgeR [55]. Cutoff and adjusted P value (FDR) for the differential gene expression were defined ≥2-fold and ≤0.05 respectively. Venn diagrams were generated using the website (http://bioinformatics.psb.ugent.be/webtools/Venn/) and Heatmap was generated using Morpheus (https://software.broadinstitute.org/morpheus/). For the heatmap analysis, we used the hierarchical clustering with one minus cosine similarity metric combined with average linkage method. Also, GO enrichment analyses were performed using (http://geneontology.org). GO bar graphs were generated based on the result of the significant enriched terms with the lowest P value and FDR (≤0.05) in GO terms. Raw data and processed data for RNAseq in Col-0, hec1 hec2 and pifQ can be accessed from the Gene Expression Omnibus database under accession number GSE158992.

Yeast two-hybrid analyses

Cloning of HEC2 and mHEC2 to pGADT7 and PIF4 into pGBT9 was described previously [37]. Different combinations of plasmids were introduced into yeast strain Y187 and selected on -Leu, -Trp minimal synthetic medium. β-galactosidase assay was performed according to the manufacturer’s protocol (Matchmaker Two-Hybrid System; Takara Bio, Mountain View, CA, https://www.takarabio.com).

In vivo co-immunoprecipitation (co-IP) assays

For in vivo co-immunoprecipitation (co-IP) assays, 35S:HEC2-GFP, 35S:mHEC2-GFP and 35S:PIF4-myc were crossed for each combination and double transgenic plants expressing both proteins were selected. Immunoprecipitation was conducted using 50 seedlings for each sample with Dynabeads protein A and anti-myc (Abcam, Cambridge, MA). Western blots using anti-myc (Cell Signaling Technologies, Danvers, MA) or anti-GFP antibody (Abcam, Cambridge, MA) were used to detect the proteins.

In Vitro DNA pull-down assay using biotinylated DNA

The DNA pull-down assay was conducted as previously described with some modifications [56]. The biotin-labeled primer pair amplifying the PIF4 G-box region is listed in S1 Table as previously described [49]. One microgram of biotin-labeled DNA from PIF4 G-box region was used for binding assay with 100 ng of GST, GST-PIF4, and GST-HEC2 as previously expressed and purified from E. coli [28,38]. After 2hr of incubation for protein dimerization followed by DNA binding, streptavidin agarose beads (Catalog # S1420S; New England Biolabs, Ipswich, MA) were added and incubated for additional 30 min at 4°C. Beads were thoroughly washed and boiled in SDS loading buffer. Western blots using anti-GST HRP (Catalog # RPN1236; GE healthcare, Pittsburgh, PA) was used to detect the proteins.

Chromatin Immunoprecipitation (ChIP) assays

ChIP assays were performed as previously described [57]. Six-day-old seedlings of 35S:PIF4-myc in Col-0 or hec1hec2 background were transferred to 22°C or 28°C for 24 hr in continuous white light and harvested. Sonication of chromatin pellet was performed using Branson digital sonifier (Emerson, St. Louis, MO). ChIP grade anti-Myc antibody (9B10, Abcam, Cambridge, MA) coupled to dynabeads were used for immunoprecipitation. Finally, PCR purification kit (Qiagen, Valencia, CA) was used for DNA purification. Samples without IP were used as input DNA. Enrichment (% of input) was calculated from each sample relative to their corresponding input.

Supporting information

S1 Table. Primers used in this study.

(PDF)

Click here for additional data file.^{(116.6KB, pdf)}

S1 Fig. The expression of HEC1 and HEC2 is upregulated at high ambient temperature.

RNA-seq data show transcription level of HEC1 and HEC2 in WT comparing normal and high ambient temperature. Numbers in the bar graph indicate p-value. Sequence data were obtained from publicly available GEO web site under accession number GSE142354.

(TIFF)

Click here for additional data file.^{(4.1MB, tiff)}

S2 Fig. Relative transcript level of HEC2-GFP.

RT-qPCR was performed to detect HEC2-GFP transcript level. Samples were from 35S:HEC2-GFP whole seedling grown for 5 days in 22°C and transferred to 22°C or 28°C for 4 hours. Three biological replicates were used in this study. Relative gene expression levels were normalized using expression levels of ACT7. n.s. stands for not significant according to Student’s t-test (P<0.05).

(TIFF)

Click here for additional data file.^{(5.1MB, tiff)}

S3 Fig. HECs regulate petiole elongation and flowering time at high ambient temperature.

(A) Box plot shows the petiole lengths of genotypes indicated. Seedlings were grown for two days in continuous white light at 22°C and then either kept at 22°C or transferred to 28°C for additional 5 days. More than 10 seedlings were measured. The letters a-c indicate statistically significant differences based on one-way ANOVA analysis with Tukey’s HSD test. Tukey’s box plot was used with median as a center value. (B) Box plot shows the leaf number for bolting under long day conditions (16L:8D). Seedlings were grown for two days in continuous white light at 22°C and then either kept at 22°C or transferred to 28°C until bolting. More than 10 seedlings were measured. The letters a-b indicate statistically significant differences based on one-way ANOVA analysis with Tukey’s HSD test. Tukey’s box plot was used with median as a center value.

(TIFF)

Click here for additional data file.^{(5.9MB, tiff)}

S4 Fig. PIF family members contribute to thermomorphogenesis.

(A) Photograph shows seedling phenotypes of PIFs-overexpression lines and phyB-9 at normal and high ambient temperature. Seedlings were grown for two days in continuous white light at 22°C and then either kept at 22°C or transferred to 28°C for additional four days before being photographed. The scale bar represents 5 mm. (B) Box plot shows the hypocotyl lengths of seedlings described in (A). More than 10 seedlings were measured for each experiment and was repeated 3 times. The letters a-c indicate statistically significant differences between means of hypocotyl lengths (P<0.05) based on one-way ANOVA analysis with Tukey’s HSD test. Tukey’s box plot was used with median as a center value. (C, E) Western blots show the level of PIFs (C) and phyB (E) in Wild-type and various PIF overexpression lines. Seedlings were grown for 5 days at 22°C and either kept at 22°C or transferred to 28°C for 4 hours. Coomassie staining or anti-RPT5 was used as a control. Red number indicates the quantitation value from anti-phyB or anti-myc detection divided by the control. (D and F) Dot plots show the relative amount of PIF4 (D, n = 3) or phyB (F, n = 3). Asterisks indicate statistically significant difference using Student’s t-test; *p < 0.05 and **p < 0.01.

(TIFF)

Click here for additional data file.^{(10.9MB, tiff)}

S5 Fig. Transcript levels of PHYB and PIFs in PIFs-overexpression lines.

RT-qPCR was performed to detect tagged-PIFs and PHYB transcript levels. Samples were from WT, 35S:TAP-PIF1, PIF3-myc, PIF4-myc and PIF5-myc seedlings grown for 5 days at 22°C and transferred to 22°C or 28°C for 4 hours. Three biological replicates were used in this study. Relative gene expression levels were normalized using the expression level of ACT7. n.s. stands for not significant according to Student’s t-test (P<0.05).

(TIFF)

Click here for additional data file.^{(14.9MB, tiff)}

S6 Fig. The expression of four major PIFs is upregulated at high ambient temperature.

RT-qPCR was performed to detect the transcript levels of four major PIFs (PIF1, PIF3, PIF4 and PIF5). Samples were from WT seedling grown for 5 days at 22°C and transferred to 22°C or 28°C for 4 hours. Three biological replicates were used in this study. Relative gene expression levels were normalized using the expression level of ACT7. Asterisks indicate statistically significant difference using Student’s t-test; *p < 0.05 and **p < 0.01.

(TIFF)

Click here for additional data file.^{(2.3MB, tiff)}

S7 Fig. PIFs are epistatic to HECs in regulating petiole length and flowering time.

(TIFF)

Click here for additional data file.^{(6.4MB, tiff)}

S8 Fig. Gene Ontology (GO) analysis of the temperature-dependent 735 genes.

Gene Ontology (GO) analysis of temperature-dependent 735 genes that are common among three genotypes. Six-day-old white light-grown seedlings were transferred to 22°C or 28°C for additional 24 hours and total RNA was extracted from three biological replicates for RNA-seq analyses.

(TIFF)

Click here for additional data file.^{(6.7MB, tiff)}

S9 Fig. The expression patterns of HEC- and PIF-dependent genes are opposite.

(A-B) Hierarchical clustering displaying 844 (638+206) HEC-dependent DEGs (A) and 1067 (638+429) PIF-dependent DEGs (B) shows distinct pattern in hec1hec2 and pifQ mutant at high ambient temperature.

(TIFF)

Click here for additional data file.^{(7.5MB, tiff)}

S10 Fig. Mutated HEC does not affect flowering time at high ambient temperature.

(A) Box plot shows the leaf number for bolting (16L:8D). More than 10 seedlings were measured. n.s. stands for not significant based on one-way ANOVA analysis with Tukey’s HSD test. Tukey’s box plot was used with median as a center value.

(TIFF)

Click here for additional data file.^{(3.5MB, tiff)}

S11 Fig. HEC-PIF4 heterodimerization inhibits 26S proteasome-mediated degradation of PIF4.

(A) Western blot shows the level of PIF4-myc from whole seedlings of 35S:PIF4-myc in either hec1hec2 or wild-type backgrounds. Seedlings were grown for 5 days at 22°C and either kept at 22°C or transferred to 28°C for 4 hours. B stands for bortezomib treatment. Coomassie staining was used as a control. Red number indicates the quantitation value from anti-myc divided by the control.

(TIFF)

Click here for additional data file.^{(4.4MB, tiff)}

S12 Fig. HEC2 inhibits PIF4 binding to DNA in vitro.

(Upper panel) Immunoblot shows the amount of GST only (as a control), GST-PIF4, and GST-HEC2 and their combinations as input. All GST-fusion proteins were expressed and purified from E. coli and detected using anti-GST antibody. (Lower panel) Immunoblot shows the amount of GST-PIF4 bound to the DNA. Biotin labeled PIF4 G-box promoter region was precipitated using streptavidin beads after pre-binding with combinations of proteins as indicated by + and/or -.

(TIFF)

Click here for additional data file.^{(7.7MB, tiff)}

S13 Fig. The expression levels of PIFs in WT and hec1hec2 at high ambient temperature.

RNA-seq data show transcription level of PIF1, PIF3, PIF4, PIF5, and PIF7 in WT and hec1hec2 background comparing normal and high ambient temperature. h12 indicates hec1 hec2 mutant. Numbers in the bar graph indicate p-value.

(TIFF)

Click here for additional data file.^{(4.4MB, tiff)}

Acknowledgments

We thank Dr. Inyup Paik for helpful discussion throughout this project, members of the Huq laboratory for critical reading of the manuscript, and Dr. Peter Quail for sharing anti-phyB antibody and phyB pifQ seeds. The authors acknowledge the Texas Advanced Computing Center (TACC) at The University of Texas at Austin for providing High Performance Computing, visualization, and database resources that have contributed to the research results reported in this paper.

Data Availability

RNA sequencing data are available from the Gene Expression Omnibus database (accession number GSE158992). All other relevant data are within the paper and its supporting information file.

Funding Statement

This work was supported by grants from the National Science Foundation (MCB-2014408) and National Institute of Health (NIH) (GM-114297) to E.H., and Integrative Biology (IB) Research Fellowship grant from the University of Texas at Austin to S.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Grimm NB, Chapin III FS, Bierwagen B, Gonzalez P, Groffman PM, Luo Y, et al. The impacts of climate change on ecosystem structure and function. Frontiers in Ecology and the Environment. 2013;11(9):474–82. [Google Scholar]
2.Challinor AJ, Watson J, Lobell DB, Howden SM, Smith DR, Chhetri N. A meta-analysis of crop yield under climate change and adaptation. Nature Climate Change. 2014;4(4):287–91. [Google Scholar]
3.Burdon JJ, Zhan J. Climate change and disease in plant communities. PLOS Biology. 2020;18(11):e3000949. doi: 10.1371/journal.pbio.3000949 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Vu LD, Xu X, Gevaert K, De Smet I. Developmental Plasticity at High Temperature. Plant Physiology. 2019;181(2):399–411. doi: 10.1104/pp.19.00652 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Casal JJ, Balasubramanian S. Thermomorphogenesis. Annual Review of Plant Biology. 2019;70(1):321–46. doi: 10.1146/annurev-arplant-050718-095919 [DOI] [PubMed] [Google Scholar]
6.Lin J, Xu Y, Zhu Z. Emerging Plant Thermosensors: From RNA to Protein. Trends in Plant Science. 2020;25(12):1187–9. doi: 10.1016/j.tplants.2020.08.007 [DOI] [PubMed] [Google Scholar]
7.Hayes S, Schachtschabel J, Mishkind M, Munnik T, Arisz SA. Hot Topic: Thermosensing in Plants. Plant, Cell & Environment. 2021;n/a(n/a). [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Legris M, Klose C, Burgie ES, Costigliolo C, Neme M, Hiltbrunner A, et al. Phytochrome B integrates light and temperature signals in Arabidopsis. Science. 2016;354:897–900. doi: 10.1126/science.aaf5656 [DOI] [PubMed] [Google Scholar]
9.Jung J-H, Domijan M, Klose C, Biswas S, Ezer D, Gao M, et al. Phytochromes function as thermosensors in Arabidopsis. Science. 2016;354:886–9. doi: 10.1126/science.aaf6005 [DOI] [PubMed] [Google Scholar]
10.Jung J-H, Barbosa AD, Hutin S, Kumita JR, Gao M, Derwort D, et al. A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis. Nature. 2020;585(7824):256–60. doi: 10.1038/s41586-020-2644-7 [DOI] [PubMed] [Google Scholar]
11.Chung BYW, Balcerowicz M, Di Antonio M, Jaeger KE, Geng F, Franaszek K, et al. An RNA thermoswitch regulates daytime growth in Arabidopsis. Nature Plants. 2020;6(5):522–32. doi: 10.1038/s41477-020-0633-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, et al. High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4. Curr Biol. 2009;19(5):408–13. doi: 10.1016/j.cub.2009.01.046 [DOI] [PubMed] [Google Scholar]
13.Franklin KA, Lee SH, Patel D, Kumar SV, Spartz AK, Gu C, et al. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) regulates auxin biosynthesis at high temperature. Proceedings of the National Academy of Sciences. 2011;108(50):20231–5. doi: 10.1073/pnas.1110682108 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Oh E, Zhu J-Y, Wang Z-Y. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nature Cell Biology. 2012:802–9. doi: 10.1038/ncb2545 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Sun J, Qi L, Li Y, Chu J, Li C. PIF4–Mediated Activation of YUCCA8 Expression Integrates Temperature into the Auxin Pathway in Regulating Arabidopsis Hypocotyl Growth. PLOS Genetics. 2012;8(3):e1002594. doi: 10.1371/journal.pgen.1002594 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Stavang JA, Gallego-Bartolomé J, Gómez MD, Yoshida S, Asami T, Olsen JE, et al. Hormonal regulation of temperature-induced growth in Arabidopsis. The Plant Journal. 2009;60(4):589–601. doi: 10.1111/j.1365-313X.2009.03983.x [DOI] [PubMed] [Google Scholar]
17.Qiu Y, Li M, Kim RJ-A, Moore CM, Chen M. Daytime temperature is sensed by phytochrome B in Arabidopsis through a transcriptional activator HEMERA. Nature Communications. 2019;10(1):140. doi: 10.1038/s41467-018-08059-z [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Ding L, Wang S, Song ZT, Jiang Y, Han JJ, Lu SJ, et al. Two B-Box Domain Proteins, BBX18 and BBX23, Interact with ELF3 and Regulate Thermomorphogenesis in Arabidopsis. Cell Rep. 2018;25(7):1718–28 e4. doi: 10.1016/j.celrep.2018.10.060 [DOI] [PubMed] [Google Scholar]
19.Han X, Yu H, Yuan R, Yang Y, An F, Qin G. Arabidopsis Transcription Factor TCP5 Controls Plant Thermomorphogenesis by Positively Regulating PIF4 Activity. iScience. 2019;15:611–22. doi: 10.1016/j.isci.2019.04.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Zhou Y, Xun Q, Zhang D, Lv M, Ou Y, Li J. TCP Transcription Factors Associate with PHYTOCHROME INTERACTING FACTOR 4 and CRYPTOCHROME 1 to Regulate Thermomorphogenesis in Arabidopsis thaliana. iScience. 2019;15:600–10. doi: 10.1016/j.isci.2019.04.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Yan Y, Li C, Dong X, Li H, Zhang D, Zhou Y, et al. MYB30 Is a Key Negative Regulator of Arabidopsis Photomorphogenic Development That Promotes PIF4 and PIF5 Protein Accumulation in the Light. The Plant Cell. 2020;32(7):2196–215. doi: 10.1105/tpc.19.00645 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Dong X, Yan Y, Jiang B, Shi Y, Jia Y, Cheng J, et al. The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures. The EMBO Journal. 2020;39(13):e103630. doi: 10.15252/embj.2019103630 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Sun Q, Wang S, Xu G, Kang X, Zhang M, Ni M. SHB1 and CCA1 interaction desensitizes light responses and enhances thermomorphogenesis. Nature Communications. 2019;10(1):3110. doi: 10.1038/s41467-019-11071-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Delker C, Sonntag L, James Geo V, Janitza P, Ibañez C, Ziermann H, et al. The DET1-COP1-HY5 Pathway Constitutes a Multipurpose Signaling Module Regulating Plant Photomorphogenesis and Thermomorphogenesis. Cell Reports. 2014;9(6):1983–9. doi: 10.1016/j.celrep.2014.11.043 [DOI] [PubMed] [Google Scholar]
25.Gangappa SN, Kumar SV. DET1 and HY5 Control PIF4-Mediated Thermosensory Elongation Growth through Distinct Mechanisms. Cell Reports. 2017;18(2):344–51. doi: 10.1016/j.celrep.2016.12.046 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Park YJ, Lee HJ, Ha JH, Kim JY, Park CM. COP1 conveys warm temperature information to hypocotyl thermomorphogenesis. New Phytol. 2017;215(1):269–80. doi: 10.1111/nph.14581 [DOI] [PubMed] [Google Scholar]
27.Jang K, Gil Lee H, Jung S-J, Paek N-C, Joon Seo P. The E3 Ubiquitin Ligase COP1 Regulates Thermosensory Flowering by Triggering GI Degradation in Arabidopsis. Scientific Reports. 2015;5(1):12071. doi: 10.1038/srep12071 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Lee S, Paik I, Huq E. SPAs promote thermomorphogenesis by regulating the phyB-PIF4 module in Arabidopsis. Development. 2020;147(19):dev189233. doi: 10.1242/dev.189233 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Ma D, Li X, Guo Y, Chu J, Fang S, Yan C, et al. Cryptochrome 1 interacts with PIF4 to regulate high temperature-mediated hypocotyl elongation in response to blue light. Proc Natl Acad Sci U S A. 2016;113(1):224–9. doi: 10.1073/pnas.1511437113 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Hayes S, Sharma A, Fraser DP, Trevisan M, Cragg-Barber CK, Tavridou E, et al. UV-B Perceived by the UVR8 Photoreceptor Inhibits Plant Thermomorphogenesis. Current Biology. 2017;27(1):120–7. doi: 10.1016/j.cub.2016.11.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Lee H-J, Jung J-H, Cortés Llorca L, Kim S-G, Lee S, Baldwin IT, et al. FCA mediates thermal adaptation of stem growth by attenuating auxin action in Arabidopsis. Nature Communications. 2014;5(1):5473. doi: 10.1038/ncomms6473 [DOI] [PubMed] [Google Scholar]
32.Zhu JY, Oh E, Wang T, Wang ZY. TOC1-PIF4 interaction mediates the circadian gating of thermoresponsive growth in Arabidopsis. Nat Commun. 2016;7:13692. doi: 10.1038/ncomms13692 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Box Mathew S, Huang BE, Domijan M, Jaeger Katja E, Khattak Asif K, Yoo Seong J, et al. ELF3 Controls Thermoresponsive Growth in Arabidopsis. Current Biology. 2015;25(2):194–9. doi: 10.1016/j.cub.2014.10.076 [DOI] [PubMed] [Google Scholar]
34.Nieto C, Lopez-Salmeron V, Daviere JM, Prat S. ELF3-PIF4 interaction regulates plant growth independently of the Evening Complex. Curr Biol. 2015;25(2):187–93. doi: 10.1016/j.cub.2014.10.070 [DOI] [PubMed] [Google Scholar]
35.Park Y-J, Kim JY, Lee J-H, Lee B-D, Paek N-C, Park C-M. GIGANTEA Shapes the Photoperiodic Rhythms of Thermomorphogenic Growth in Arabidopsis. Molecular Plant. 2020;13(3):459–70. doi: 10.1016/j.molp.2020.01.003 [DOI] [PubMed] [Google Scholar]
36.Gremski K, Ditta G, Yanofsky MF. The HECATE genes regulate female reproductive tract development in Arabidopsis thaliana. Development. 2007;134:3593–601. doi: 10.1242/dev.011510 [DOI] [PubMed] [Google Scholar]
37.Zhu L, Xin R, Bu Q, Shen H, Dang J, Huq E. A negative feedback loop between PHYTOCHROME INTERACTING FACTORs and HECATE proteins fine tunes photomorphogenesis in Arabidopsis. Plant Cell. 2016;28(4):855–74. doi: 10.1105/tpc.16.00122 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Kathare PK, Xu X, Nguyen A, Huq E. A COP1-PIF-HEC regulatory module fine-tunes photomorphogenesis in Arabidopsis. The Plant Journal. 2020;104(1):113–23. doi: 10.1111/tpj.14908 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Fiorucci A-S, Galvão VC, Ince YÇ, Boccaccini A, Goyal A, Allenbach Petrolati L, et al. PHYTOCHROME INTERACTING FACTOR 7 is important for early responses to elevated temperature in Arabidopsis seedlings. New Phytologist. 2020;226:50–8. doi: 10.1111/nph.16316 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Leivar P, Quail PH. PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci. 2011;16:19–28. doi: 10.1016/j.tplants.2010.08.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Tavridou E, Schmid-Siegert E, Fankhauser C, Ulm R. UVR8-mediated inhibition of shade avoidance involves HFR1 stabilization in Arabidopsis. PLOS Genetics. 2020;16(5):e1008797. doi: 10.1371/journal.pgen.1008797 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Xu X, Kathare PK, Pham VN, Bu Q, Nguyen A, Huq E. Reciprocal proteasome-mediated degradation of PIFs and HFR1 underlies photomorphogenic development in Arabidopsis. Development. 2017;144(10):1831–40. doi: 10.1242/dev.146936 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Foreman J, Johansson H, Hornitschek P, Josse EM, Fankhauser C, Halliday KJ. Light receptor action is critical for maintaining plant biomass at warm ambient temperatures. Plant J. 2011;65(3):441–52. doi: 10.1111/j.1365-313X.2010.04434.x [DOI] [PubMed] [Google Scholar]
44.Luo Q, Lian H-L, He S-B, Li L, Jia K-P, Yang H-Q. COP1 and phyB physically interact with PIL1 to regulate its stability and photomorphogenic development in Arabidopsis. The Plant Cell Online. 2014;26(6):2441–56. doi: 10.1105/tpc.113.121657 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Hornitschek P, Lorrain S, Zoete V, Michielin O, Fankhauser C. Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers. Embo J. 2009;28:3893–902. doi: 10.1038/emboj.2009.306 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Paulišić S, Qin W, Arora Verasztó H, Then C, Alary B, Nogue F, et al. Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation. The EMBO Journal. 2020;n/a(n/a):e104273. doi: 10.15252/embj.2019104273 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Lippmann R, Babben S, Menger A, Delker C, Quint M. Development of Wild and Cultivated Plants under Global Warming Conditions. Current Biology. 2019;29(24):R1326–R38. doi: 10.1016/j.cub.2019.10.016 [DOI] [PubMed] [Google Scholar]
48.Nieto C, López-Salmerón V, Davière J-M, Prat S. ELF3-PIF4 Interaction Regulates Plant Growth Independently of the Evening Complex. Current Biology. 2014;25:187–93. doi: 10.1016/j.cub.2014.10.070 [DOI] [PubMed] [Google Scholar]
49.Zhai H, Xiong L, Li H, Lyu X, Yang G, Zhao T, et al. Cryptochrome 1 Inhibits Shoot Branching by Repressing the Self-Activated Transciption Loop of PIF4 in Arabidopsis. Plant Communications. 2020;1(3):100042. doi: 10.1016/j.xplc.2020.100042 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Shi H, Zhong S, Mo X, Liu N, Nezames CD, Deng XW. HFR1 Sequesters PIF1 to Govern the Transcriptional Network Underlying Light-Initiated Seed Germination in Arabidopsis. The Plant Cell. 2013;25(10):3770–84. doi: 10.1105/tpc.113.117424 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Lohman BK, Weber JN, Bolnick DI. Evaluation of TagSeq, a reliable low-cost alternative for RNAseq. Mol Ecol Resour. 2016;16(6):1315–21. doi: 10.1111/1755-0998.12529 [DOI] [PubMed] [Google Scholar]
52.Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012;9:357. doi: 10.1038/nmeth.1923 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature protocols. 2012;7(3):562–78. doi: 10.1038/nprot.2012.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015;31(2):166–9. doi: 10.1093/bioinformatics/btu638 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139–40. doi: 10.1093/bioinformatics/btp616 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Heo JB, Sung S. Vernalization-Mediated Epigenetic Silencing by a Long Intronic Noncoding RNA. Science. 2011;331(6013):76–9. doi: 10.1126/science.1197349 [DOI] [PubMed] [Google Scholar]
57.Paik I, Chen F, Ngoc Pham V, Zhu L, Kim J-I, Huq E. A phyB-PIF1-SPA1 kinase regulatory complex promotes photomorphogenesis in Arabidopsis. Nature Communications. 2019;10(1):4216. doi: 10.1038/s41467-019-12110-y [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS Genet. doi: 10.1371/journal.pgen.1009595.r001

Decision Letter 0

Claudia Köhler, Philip Anthony Wigge

1 Mar 2021

Dear Dr Huq,

Thank you very much for submitting your Research Article entitled 'An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. All reviewers are in support of this manuscript and highlight its significant interest to the field. In light of the level of interest in the manuscript, we would be willing to consider a resubmission that addresses the points raised by the reviewers. Reviewer 2 in particular has made a detailed suggestion about the logical flow of the manuscript, which would make the study easier to follow and clarify the underlying biology. We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Philip Anthony Wigge, Ph.D

Guest Editor

PLOS Genetics

Claudia Köhler

Section Editor: Plant Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The manuscript by Lee et al. reports the regulatory role of HEC1 and HEC2 in thermomorphogenesis. The authors first demonstrate the involvement of HEC1/2 in thermomorphogenesis by showing that high ambient temperature induced HEC1/2 gene expression level and HEC2 protein stability, and that hec1/2 mutants and HEC2-ox lines displayed opposite thermomorphogenic phenotypes. They then show that all four PIFs (PIF1/3/4/5) contribute to promote thermomorphogenesis, and that pifQ is epistatic to hec1/2 by genetic analysis. Moreover, the authors show that HECs could interact and form heterodimers with PIF4, thus HECs could inhibit PIF binding to their target gene promoters. Consequently, HECs and PIFs oppositely control the expression of many high temperature-regulated genes, thus forming a negative feedback loop that fine-tunes plant growth under high ambient temperature.

Generally, this manuscript contains data that are both interesting and important, and would contribute to a better understanding of high ambient temperature-induced architecture changes of sessile plants. Here are my suggestions to improve this manuscript.

1) Abstract, “PIF4-HEC forms an autoregulatory composite negative feedback loop”: please rephrase.

2) Introduction, “Furthermore, several factors have been shown to control either the PIF4 abundance and/or activity to regulate thermomorphogenesis”: it was recently shown that the cold response regulator CBF1 promotes PIF4 abundance at ambient temperatures, and that MYB30 promotes PIF4 protein re-accumulation under prolonged light irradiation (EMBO J, 39:e103630; Plant Cell, 32:2196-2215). These recent reports are relevant to this statement.

3) It is important to show that all four PIFs contribute to promote thermomorphogenesis. In support of this claim, I would prefer to show the data of pif mutants instead of PIF-OE lines in the main figure, if only one could be included as a main figure. In this sense, I would suggest the authors to exchange Figure 2 with Figure S4.

4) Since the authors show that all four PIFs contribute to promote thermomorphogenesis, I would suggest the authors to make this story bigger, i.e., HECs not only interacts with and regulates the activity of PIF4, but also do the same with other PIFs. In this sense, the authors could detect the protein levels of PIF1/3/5 in Figure 1E, and could examine the expression levels of PIF1/3/5 in Figure 6B.

5) Figure 1E shows that both PIF4 and phyB protein levels are decreased in hec1 hec2 mutants at 28 degree. How could HEC1/2 promote PIF4 and phyB stability simultaneously at 28 degree? The authors may do some tests, at least make some discussion.

6) The authors may do some assays, such EMSAs, to provide direct evidence showing that HEC could inhibit PIF4 binding to its target gene promoter.

7) The model in Figure 6D should be improved to better illustrate the findings. The HEC inhibition of PIF4 is confusing because HECs promote PIF4 protein stability at high temperature.

Reviewer #2: The manuscript by Lee et al. shows that helix-loop-helix transcriptional regulators HECATE 1 (HEC1) and HEC2 inhibit thermomorphogenesis by physically interacting with PIF4 transcription factor and reducing its binding to PIF4 target genes. The carefully designed experiments are statistically solid and provide the necessary support to the conclusions. The work provides insight into our understanding of the mechanisms involved in thermomorphogenesis in plants.

My major concern is that the logic behind the general structure of the text is no easy to follow. I do not refer here to the wording itself but to the order in which the information is presented. Perhaps the authors are describing their findings in the order in which they did the experiments. It took me a while to find the main message. Therefore, I propose substantial cut and paste and some rearrangement of the figures as follows:

The first issue is that HECs increase their abundance in response to warmth and repress thermomorphogenesis. Therefore, Fig. 1 should contain parts A-D, leaving E and F for a later stage.

The second point of the manuscript is that the action of HECs requires PIFs. I know that the observation that all PIFs contribute to thermomorphogenesis is interesting in itself but it should not complicate the message. Therefore, I would merge Figs 2 and 3A-B into Fig. 2. Then, it makes sense if the authors are using the pifQ mutant background to demonstrate that the action of HECs requires PIFQ, to provide the information that all the PIFQ members contribute to thermomorphogenesis. By passing, since the authors have produced hec1 hec2 pifQ, they are likely to have hec1 hec2 pif4 in the segregating population. Since the rest of the paper deals with PIF4 it would be interesting to include the latter mutant (although I would not consider that a must do).

I would use current Fig. 5A-E as the third display (Fig. 3) because the convergence of the hec1 hec2 and pifQ transcriptomes further supports the conclusion reached by the genetic experiments in Fig. 2.

The fourth point is that although HECs affect PIF4 abundance these effects do not fully account for the requirement of PIFs for HECs action. Therefore, I would fuse PIF4 data from Fig. 1F-G plus Fig. 3C-D (PIFOX data) into Fig. 4.

The fifth point is that PIF4-HEC2 heterodimerization is crucial for HECs function in thermomorphogenesis and HECs reduce PIF4 binding to its targets. Therefore, I would use Fig. 4 plus Fig. 5G-H in Fig. 5.

The sixth point is the autoregulatory negative feedback loop (Fig. 6 as it is).

The subtitle “phyB and PIFs are oppositely regulated at high ambient temperature” deviates from the main stream and is confusing. It could be placed at the end, after the sequence described above.

Minor concerns

There are several known positive and negative regulators of thermomorphogenesis. Do expression patterns or other features of the HEC system tell us when (e.g. time of day, growth conditions) or where (organs) HECs could be important to reduce thermomorphogenesis?

Introduction: “Analyses of the four major PHYTOCHROME INTERACTING FACTORs (PIF1, PIF3, PIF4 and PIF5) mutants”. The “four major” would exclude PIF7 from the major PIFs, which is actually misleading.

Introduction: “while PIFs are acting positively downstream of phyB in attenuating thermormoprhogenesis”. I do not think “attenuating” is correct here.

Introduction: “An intriguing finding in our study is that PIF4 appears to regulate its own expression by directly binding to its own promoter”. Please, check carefully, I think that this had already been shown in the literature.

Figure S4. PIFs additively rgeulate thermomorphogenesis. It should be “regulate”

Reviewer #3: This manuscript describes the functional involvement of HEC1 and HEC2 in thermo responses. Their transcription and accumulation are enhanced by a higher ambient temperature. Overexpression of HEC2 attenuates thermo-induced hypocotyl elongation but enhances PIF4 stability or accumulation. hec1 hec2 double mutant shows enhanced thermo responses, and PIF4 is epistatic to HECs. HEC2 physically interacts with PIF4. Their interaction may inhibit 26S proteosome-mediated degradation of PIF4 but on the other hand may insulate PIF4 from binding to it target sites. PIF4 (or other PIFs) directly up-regulate the expression of HECs and HECs in turn inhibit the expression of PIF4. All discoveries are summarized in Figure 6D.

Major

1) In the fourth paragraph of the introduction, you discussed various ways that regulate the stability or activity of PIF4, and one particular case is mentioned for TOC1. There is a publication reflecting the involvement by the CCA1/LHY clock components that directly bind to the PIF4 promoter and activate its expression and likely accumulation (Sun et al., 2019, NC).

2) For Figures 5H and 6C, it would be helpful to audients by showing the promoter regions for ChIP-PCR analysis in a simple diagram.

Minor

1) In Figure S1 legend, add the reference number for your citation of Lee et al.

2) In Figure 2 legend, revise the description for C through F from WT, 35S:HEC2-GFP and hec12 mutant to wild type and various PIF overexpression lines.

3) In line 181, cite Figure 3A.

4) In Figure 3 legend, revise line 4 from as described in (B) to as described in (A).

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: None

Reviewer #2: Yes

Reviewer #3: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS Genet. 2021 Jun 1;17(6):e1009595. doi: 10.1371/journal.pgen.1009595.r002

Author response to Decision Letter 0

29 Apr 2021

Attachment

Submitted filename: Response to reviewers comments.docx

Click here for additional data file.^{(26.5KB, docx)}

PLoS Genet. doi: 10.1371/journal.pgen.1009595.r003

Decision Letter 1

Claudia Köhler, Philip Anthony Wigge

11 May 2021

Dear Dr Huq,

We are pleased to inform you that your manuscript entitled "An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Philip Anthony Wigge, Ph.D

Guest Editor

PLOS Genetics

Claudia Köhler

Section Editor: Plant Genetics

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly:

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-21-00104R1

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.

PLoS Genet. doi: 10.1371/journal.pgen.1009595.r004

Acceptance letter

Claudia Köhler, Philip Anthony Wigge

24 May 2021

PGENETICS-D-21-00104R1

An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis

Dear Dr Huq,

We are pleased to inform you that your manuscript entitled "An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Katalin Szabo

PLOS Genetics

On behalf of:

The PLOS Genetics Team

Carlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdom

plosgenetics@plos.org | +44 (0) 1223-442823

plosgenetics.org | Twitter: @PLOSGenetics

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Primers used in this study.

(PDF)

Click here for additional data file.^{(116.6KB, pdf)}

S1 Fig. The expression of HEC1 and HEC2 is upregulated at high ambient temperature.

(TIFF)

Click here for additional data file.^{(4.1MB, tiff)}

S2 Fig. Relative transcript level of HEC2-GFP.

(TIFF)

Click here for additional data file.^{(5.1MB, tiff)}

S3 Fig. HECs regulate petiole elongation and flowering time at high ambient temperature.

(TIFF)

Click here for additional data file.^{(5.9MB, tiff)}

S4 Fig. PIF family members contribute to thermomorphogenesis.

(TIFF)

Click here for additional data file.^{(10.9MB, tiff)}

S5 Fig. Transcript levels of PHYB and PIFs in PIFs-overexpression lines.

(TIFF)

Click here for additional data file.^{(14.9MB, tiff)}

S6 Fig. The expression of four major PIFs is upregulated at high ambient temperature.

(TIFF)

Click here for additional data file.^{(2.3MB, tiff)}

S7 Fig. PIFs are epistatic to HECs in regulating petiole length and flowering time.

(TIFF)

Click here for additional data file.^{(6.4MB, tiff)}

S8 Fig. Gene Ontology (GO) analysis of the temperature-dependent 735 genes.

(TIFF)

Click here for additional data file.^{(6.7MB, tiff)}

S9 Fig. The expression patterns of HEC- and PIF-dependent genes are opposite.

(TIFF)

Click here for additional data file.^{(7.5MB, tiff)}

S10 Fig. Mutated HEC does not affect flowering time at high ambient temperature.

(TIFF)

Click here for additional data file.^{(3.5MB, tiff)}

S11 Fig. HEC-PIF4 heterodimerization inhibits 26S proteasome-mediated degradation of PIF4.

(TIFF)

Click here for additional data file.^{(4.4MB, tiff)}

S12 Fig. HEC2 inhibits PIF4 binding to DNA in vitro.

(TIFF)

Click here for additional data file.^{(7.7MB, tiff)}

S13 Fig. The expression levels of PIFs in WT and hec1hec2 at high ambient temperature.

(TIFF)

Click here for additional data file.^{(4.4MB, tiff)}

Attachment

Submitted filename: Response to reviewers comments.docx

Click here for additional data file.^{(26.5KB, docx)}

Data Availability Statement

RNA sequencing data are available from the Gene Expression Omnibus database (accession number GSE158992). All other relevant data are within the paper and its supporting information file.

[pgen.1009595.ref001] 1.Grimm NB, Chapin III FS, Bierwagen B, Gonzalez P, Groffman PM, Luo Y, et al. The impacts of climate change on ecosystem structure and function. Frontiers in Ecology and the Environment. 2013;11(9):474–82. [Google Scholar]

[pgen.1009595.ref002] 2.Challinor AJ, Watson J, Lobell DB, Howden SM, Smith DR, Chhetri N. A meta-analysis of crop yield under climate change and adaptation. Nature Climate Change. 2014;4(4):287–91. [Google Scholar]

[pgen.1009595.ref003] 3.Burdon JJ, Zhan J. Climate change and disease in plant communities. PLOS Biology. 2020;18(11):e3000949. doi: 10.1371/journal.pbio.3000949 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref004] 4.Vu LD, Xu X, Gevaert K, De Smet I. Developmental Plasticity at High Temperature. Plant Physiology. 2019;181(2):399–411. doi: 10.1104/pp.19.00652 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref005] 5.Casal JJ, Balasubramanian S. Thermomorphogenesis. Annual Review of Plant Biology. 2019;70(1):321–46. doi: 10.1146/annurev-arplant-050718-095919 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref006] 6.Lin J, Xu Y, Zhu Z. Emerging Plant Thermosensors: From RNA to Protein. Trends in Plant Science. 2020;25(12):1187–9. doi: 10.1016/j.tplants.2020.08.007 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref007] 7.Hayes S, Schachtschabel J, Mishkind M, Munnik T, Arisz SA. Hot Topic: Thermosensing in Plants. Plant, Cell & Environment. 2021;n/a(n/a). [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref008] 8.Legris M, Klose C, Burgie ES, Costigliolo C, Neme M, Hiltbrunner A, et al. Phytochrome B integrates light and temperature signals in Arabidopsis. Science. 2016;354:897–900. doi: 10.1126/science.aaf5656 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref009] 9.Jung J-H, Domijan M, Klose C, Biswas S, Ezer D, Gao M, et al. Phytochromes function as thermosensors in Arabidopsis. Science. 2016;354:886–9. doi: 10.1126/science.aaf6005 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref010] 10.Jung J-H, Barbosa AD, Hutin S, Kumita JR, Gao M, Derwort D, et al. A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis. Nature. 2020;585(7824):256–60. doi: 10.1038/s41586-020-2644-7 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref011] 11.Chung BYW, Balcerowicz M, Di Antonio M, Jaeger KE, Geng F, Franaszek K, et al. An RNA thermoswitch regulates daytime growth in Arabidopsis. Nature Plants. 2020;6(5):522–32. doi: 10.1038/s41477-020-0633-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref012] 12.Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, et al. High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4. Curr Biol. 2009;19(5):408–13. doi: 10.1016/j.cub.2009.01.046 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref013] 13.Franklin KA, Lee SH, Patel D, Kumar SV, Spartz AK, Gu C, et al. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) regulates auxin biosynthesis at high temperature. Proceedings of the National Academy of Sciences. 2011;108(50):20231–5. doi: 10.1073/pnas.1110682108 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref014] 14.Oh E, Zhu J-Y, Wang Z-Y. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nature Cell Biology. 2012:802–9. doi: 10.1038/ncb2545 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref015] 15.Sun J, Qi L, Li Y, Chu J, Li C. PIF4–Mediated Activation of YUCCA8 Expression Integrates Temperature into the Auxin Pathway in Regulating Arabidopsis Hypocotyl Growth. PLOS Genetics. 2012;8(3):e1002594. doi: 10.1371/journal.pgen.1002594 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref016] 16.Stavang JA, Gallego-Bartolomé J, Gómez MD, Yoshida S, Asami T, Olsen JE, et al. Hormonal regulation of temperature-induced growth in Arabidopsis. The Plant Journal. 2009;60(4):589–601. doi: 10.1111/j.1365-313X.2009.03983.x [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref017] 17.Qiu Y, Li M, Kim RJ-A, Moore CM, Chen M. Daytime temperature is sensed by phytochrome B in Arabidopsis through a transcriptional activator HEMERA. Nature Communications. 2019;10(1):140. doi: 10.1038/s41467-018-08059-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref018] 18.Ding L, Wang S, Song ZT, Jiang Y, Han JJ, Lu SJ, et al. Two B-Box Domain Proteins, BBX18 and BBX23, Interact with ELF3 and Regulate Thermomorphogenesis in Arabidopsis. Cell Rep. 2018;25(7):1718–28 e4. doi: 10.1016/j.celrep.2018.10.060 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref019] 19.Han X, Yu H, Yuan R, Yang Y, An F, Qin G. Arabidopsis Transcription Factor TCP5 Controls Plant Thermomorphogenesis by Positively Regulating PIF4 Activity. iScience. 2019;15:611–22. doi: 10.1016/j.isci.2019.04.005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref020] 20.Zhou Y, Xun Q, Zhang D, Lv M, Ou Y, Li J. TCP Transcription Factors Associate with PHYTOCHROME INTERACTING FACTOR 4 and CRYPTOCHROME 1 to Regulate Thermomorphogenesis in Arabidopsis thaliana. iScience. 2019;15:600–10. doi: 10.1016/j.isci.2019.04.002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref021] 21.Yan Y, Li C, Dong X, Li H, Zhang D, Zhou Y, et al. MYB30 Is a Key Negative Regulator of Arabidopsis Photomorphogenic Development That Promotes PIF4 and PIF5 Protein Accumulation in the Light. The Plant Cell. 2020;32(7):2196–215. doi: 10.1105/tpc.19.00645 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref022] 22.Dong X, Yan Y, Jiang B, Shi Y, Jia Y, Cheng J, et al. The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures. The EMBO Journal. 2020;39(13):e103630. doi: 10.15252/embj.2019103630 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref023] 23.Sun Q, Wang S, Xu G, Kang X, Zhang M, Ni M. SHB1 and CCA1 interaction desensitizes light responses and enhances thermomorphogenesis. Nature Communications. 2019;10(1):3110. doi: 10.1038/s41467-019-11071-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref024] 24.Delker C, Sonntag L, James Geo V, Janitza P, Ibañez C, Ziermann H, et al. The DET1-COP1-HY5 Pathway Constitutes a Multipurpose Signaling Module Regulating Plant Photomorphogenesis and Thermomorphogenesis. Cell Reports. 2014;9(6):1983–9. doi: 10.1016/j.celrep.2014.11.043 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref025] 25.Gangappa SN, Kumar SV. DET1 and HY5 Control PIF4-Mediated Thermosensory Elongation Growth through Distinct Mechanisms. Cell Reports. 2017;18(2):344–51. doi: 10.1016/j.celrep.2016.12.046 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref026] 26.Park YJ, Lee HJ, Ha JH, Kim JY, Park CM. COP1 conveys warm temperature information to hypocotyl thermomorphogenesis. New Phytol. 2017;215(1):269–80. doi: 10.1111/nph.14581 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref027] 27.Jang K, Gil Lee H, Jung S-J, Paek N-C, Joon Seo P. The E3 Ubiquitin Ligase COP1 Regulates Thermosensory Flowering by Triggering GI Degradation in Arabidopsis. Scientific Reports. 2015;5(1):12071. doi: 10.1038/srep12071 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref028] 28.Lee S, Paik I, Huq E. SPAs promote thermomorphogenesis by regulating the phyB-PIF4 module in Arabidopsis. Development. 2020;147(19):dev189233. doi: 10.1242/dev.189233 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref029] 29.Ma D, Li X, Guo Y, Chu J, Fang S, Yan C, et al. Cryptochrome 1 interacts with PIF4 to regulate high temperature-mediated hypocotyl elongation in response to blue light. Proc Natl Acad Sci U S A. 2016;113(1):224–9. doi: 10.1073/pnas.1511437113 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref030] 30.Hayes S, Sharma A, Fraser DP, Trevisan M, Cragg-Barber CK, Tavridou E, et al. UV-B Perceived by the UVR8 Photoreceptor Inhibits Plant Thermomorphogenesis. Current Biology. 2017;27(1):120–7. doi: 10.1016/j.cub.2016.11.004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref031] 31.Lee H-J, Jung J-H, Cortés Llorca L, Kim S-G, Lee S, Baldwin IT, et al. FCA mediates thermal adaptation of stem growth by attenuating auxin action in Arabidopsis. Nature Communications. 2014;5(1):5473. doi: 10.1038/ncomms6473 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref032] 32.Zhu JY, Oh E, Wang T, Wang ZY. TOC1-PIF4 interaction mediates the circadian gating of thermoresponsive growth in Arabidopsis. Nat Commun. 2016;7:13692. doi: 10.1038/ncomms13692 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref033] 33.Box Mathew S, Huang BE, Domijan M, Jaeger Katja E, Khattak Asif K, Yoo Seong J, et al. ELF3 Controls Thermoresponsive Growth in Arabidopsis. Current Biology. 2015;25(2):194–9. doi: 10.1016/j.cub.2014.10.076 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref034] 34.Nieto C, Lopez-Salmeron V, Daviere JM, Prat S. ELF3-PIF4 interaction regulates plant growth independently of the Evening Complex. Curr Biol. 2015;25(2):187–93. doi: 10.1016/j.cub.2014.10.070 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref035] 35.Park Y-J, Kim JY, Lee J-H, Lee B-D, Paek N-C, Park C-M. GIGANTEA Shapes the Photoperiodic Rhythms of Thermomorphogenic Growth in Arabidopsis. Molecular Plant. 2020;13(3):459–70. doi: 10.1016/j.molp.2020.01.003 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref036] 36.Gremski K, Ditta G, Yanofsky MF. The HECATE genes regulate female reproductive tract development in Arabidopsis thaliana. Development. 2007;134:3593–601. doi: 10.1242/dev.011510 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref037] 37.Zhu L, Xin R, Bu Q, Shen H, Dang J, Huq E. A negative feedback loop between PHYTOCHROME INTERACTING FACTORs and HECATE proteins fine tunes photomorphogenesis in Arabidopsis. Plant Cell. 2016;28(4):855–74. doi: 10.1105/tpc.16.00122 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref038] 38.Kathare PK, Xu X, Nguyen A, Huq E. A COP1-PIF-HEC regulatory module fine-tunes photomorphogenesis in Arabidopsis. The Plant Journal. 2020;104(1):113–23. doi: 10.1111/tpj.14908 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref039] 39.Fiorucci A-S, Galvão VC, Ince YÇ, Boccaccini A, Goyal A, Allenbach Petrolati L, et al. PHYTOCHROME INTERACTING FACTOR 7 is important for early responses to elevated temperature in Arabidopsis seedlings. New Phytologist. 2020;226:50–8. doi: 10.1111/nph.16316 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref040] 40.Leivar P, Quail PH. PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci. 2011;16:19–28. doi: 10.1016/j.tplants.2010.08.003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref041] 41.Tavridou E, Schmid-Siegert E, Fankhauser C, Ulm R. UVR8-mediated inhibition of shade avoidance involves HFR1 stabilization in Arabidopsis. PLOS Genetics. 2020;16(5):e1008797. doi: 10.1371/journal.pgen.1008797 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref042] 42.Xu X, Kathare PK, Pham VN, Bu Q, Nguyen A, Huq E. Reciprocal proteasome-mediated degradation of PIFs and HFR1 underlies photomorphogenic development in Arabidopsis. Development. 2017;144(10):1831–40. doi: 10.1242/dev.146936 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref043] 43.Foreman J, Johansson H, Hornitschek P, Josse EM, Fankhauser C, Halliday KJ. Light receptor action is critical for maintaining plant biomass at warm ambient temperatures. Plant J. 2011;65(3):441–52. doi: 10.1111/j.1365-313X.2010.04434.x [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref044] 44.Luo Q, Lian H-L, He S-B, Li L, Jia K-P, Yang H-Q. COP1 and phyB physically interact with PIL1 to regulate its stability and photomorphogenic development in Arabidopsis. The Plant Cell Online. 2014;26(6):2441–56. doi: 10.1105/tpc.113.121657 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref045] 45.Hornitschek P, Lorrain S, Zoete V, Michielin O, Fankhauser C. Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers. Embo J. 2009;28:3893–902. doi: 10.1038/emboj.2009.306 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref046] 46.Paulišić S, Qin W, Arora Verasztó H, Then C, Alary B, Nogue F, et al. Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation. The EMBO Journal. 2020;n/a(n/a):e104273. doi: 10.15252/embj.2019104273 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref047] 47.Lippmann R, Babben S, Menger A, Delker C, Quint M. Development of Wild and Cultivated Plants under Global Warming Conditions. Current Biology. 2019;29(24):R1326–R38. doi: 10.1016/j.cub.2019.10.016 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref048] 48.Nieto C, López-Salmerón V, Davière J-M, Prat S. ELF3-PIF4 Interaction Regulates Plant Growth Independently of the Evening Complex. Current Biology. 2014;25:187–93. doi: 10.1016/j.cub.2014.10.070 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref049] 49.Zhai H, Xiong L, Li H, Lyu X, Yang G, Zhao T, et al. Cryptochrome 1 Inhibits Shoot Branching by Repressing the Self-Activated Transciption Loop of PIF4 in Arabidopsis. Plant Communications. 2020;1(3):100042. doi: 10.1016/j.xplc.2020.100042 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref050] 50.Shi H, Zhong S, Mo X, Liu N, Nezames CD, Deng XW. HFR1 Sequesters PIF1 to Govern the Transcriptional Network Underlying Light-Initiated Seed Germination in Arabidopsis. The Plant Cell. 2013;25(10):3770–84. doi: 10.1105/tpc.113.117424 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref051] 51.Lohman BK, Weber JN, Bolnick DI. Evaluation of TagSeq, a reliable low-cost alternative for RNAseq. Mol Ecol Resour. 2016;16(6):1315–21. doi: 10.1111/1755-0998.12529 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref052] 52.Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012;9:357. doi: 10.1038/nmeth.1923 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref053] 53.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature protocols. 2012;7(3):562–78. doi: 10.1038/nprot.2012.016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref054] 54.Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015;31(2):166–9. doi: 10.1093/bioinformatics/btu638 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref055] 55.Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139–40. doi: 10.1093/bioinformatics/btp616 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1009595.ref056] 56.Heo JB, Sung S. Vernalization-Mediated Epigenetic Silencing by a Long Intronic Noncoding RNA. Science. 2011;331(6013):76–9. doi: 10.1126/science.1197349 [DOI] [PubMed] [Google Scholar]

[pgen.1009595.ref057] 57.Paik I, Chen F, Ngoc Pham V, Zhu L, Kim J-I, Huq E. A phyB-PIF1-SPA1 kinase regulatory complex promotes photomorphogenesis in Arabidopsis. Nature Communications. 2019;10(1):4216. doi: 10.1038/s41467-019-12110-y [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis

Sanghwa Lee

Ling Zhu

Enamul Huq

Roles

Abstract

Author summary

Introduction

Results

HECATEs (HECs) inhibit thermomorphogenesis

Fig 1. HECs inhibit thermomorphogenesis.

PIF family members contribute to thermomorphogenesis

Fig 2. Genetic relationship of PIFs and HECs in thermomorphogenesis.

Genetic relationships between HECs and PIFs

HECs alter global gene expression at high ambient temperature

Fig 3. HECs regulate global gene expression at high ambient temperature.

HECs alter phyB-PIF4 level at high ambient temperature

Fig 4. HECs alter phyB-PIF4 level at high ambient temperature.

HEC2 regulates thermomorphogenesis in part by heterodimerizing with PIF4

Fig 5. PIF4-HEC2 heterodimerization is crucial for HEC2 function in thermomorphogenesis.

HECs inhibit PIF4 promoter binding

Fig 6. An autoregulatory negative feedback module controls thermomorphogenesis.

Discussion

Materials and methods

Plant materials, growth conditions and phenotypic analyses

Protein extraction and Western blot analyses

RNA extraction, cDNA synthesis, and qRT-PCR

RNA-seq analyses

Yeast two-hybrid analyses

In vivo co-immunoprecipitation (co-IP) assays

In Vitro DNA pull-down assay using biotinylated DNA

Chromatin Immunoprecipitation (ChIP) assays

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Claudia Köhler

Philip Anthony Wigge

Roles

Author response to Decision Letter 0

Decision Letter 1

Claudia Köhler

Philip Anthony Wigge

Roles

Acceptance letter

Claudia Köhler

Philip Anthony Wigge

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases