Report. High Temperature-Mediated Adaptations in Plant Architecture Require the bhlh Transcription Factor PIF4

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1 Current iology 9, 8, March, 9 ª9 Elsevier Ltd ll rights reserved DOI./j.cub.9.. High Temperature-Mediated daptations in Plant rchitecture Require the bhlh Transcription Factor PIF Report Maria. Koini, Liz lvey, Trudie llen, Ceinwen. Tilley, Nicholas P. Harberd,, Garry C. Whitelam, and Keara. Franklin, Department of iology University of Leicester University Road Leicester LE 7RH UK John Innes Centre Colney Norwich NR 7UH UK Summary Exposure of rabidopsis plants to high temperature (8 C) results in a dramatic change in plant development. Responses to high temperature include rapid extension of plant axes, leaf hyponasty, and early flowering [, ]. These phenotypes parallel plant responses to the threat of vegetational shade and have been shown to involve the hormone auxin [, ]. In this work, we demonstrate that high temperature-induced architectural adaptations are mediated through the bhlh transcriptional regulator PHYTOCHROME INTERCTING FCTOR (PIF). Roles for PIF have previously been established in both light and gibberellin (G) signaling, through interactions with phytochromes and DELL proteins, respectively [ ]. Mutants deficient in PIF do not display elongation responses or leaf hyponasty upon transfer to high temperature. High temperature-mediated induction of the auxin-responsive gene I9 is also abolished in these plants. n early flowering response to high temperature is maintained in pif mutants, suggesting that architectural and flowering responses operate via separate signaling pathways. The role of PIF in temperature signaling does not, however, appear to operate through interaction with either phytochrome or DELL proteins, suggesting the existence of a novel regulatory mechanism. We conclude that PIF is an important component of plant high temperature signaling and integrates multiple environmental cues during plant development. Results High Temperature-Mediated Hypocotyl Elongation Is bolished in pif Mutants The phenotypic similarity of plant responses to high temperature ([, ], this study) and low red to far red ratio light (low R:FR) [, ] suggests the possible existence of shared regulatory mechanisms in the perception of these environmental stimuli. The latter is a component of vegetational shade and is perceived by the phytochrome family of plant photoreceptors [5]. Correspondence: kaf5@le.ac.uk Present address: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX R, UK Responses to low R:FR are regulated, in part, by stabilization of the transcriptional regulators PIF and PIF5 [5]. The role of PIF and PIF5 in mediating plant responses to elevated temperature was therefore investigated. The elongation of wild-type () and pif mutant hypocotyls was recorded after day treatment at both Cand8 C. Mutants deficient in PIF and PIF5 [] were analyzed alongside mutants deficient in PIF. The latter performs a significant role in seedling de-etiolation [7] but has not been demonstrated to perform a significant role in low R:FR signaling. Striking hypocotyl elongation responses were observed in, pif, and pif5 mutant seedlings at the higher temperature, although a mildly attenuated response was observed in pif5 mutants (Figure ). No elongation response was observed in pif seedlings transferred to 8 C, suggesting a fundamental role for PIF in regulating this process (Figure ). Similar data were obtained with a second allele, pif- (Figure S available online). Petiole Elongation and Leaf Hyponasty Responses to High Temperature re Severely Defective in pif Mutants The role of PIF in mediating petiole elongation responses to high temperature was investigated after transfer of -weekold rosettes from Cto8 C. Petiole length was recorded in leaves and 5 at both temperatures when plants at 8 C displayed a cm bolt. Wild-type, pif, and pif5 plants displayed clear increases in petiole length at the higher temperature, although a slightly attenuated response was observed in pif5 mutants. s with hypocotyl elongation, petiole elongation responses to high temperature were abolished in pif mutants (Figure ). similar response was observed in the pif- allele (Figure S). Upwards elevation of leaves (leaf hyponasty) is a common response to both low R:FR and high temperature in many plants [8]. This response was analyzed in and pif mutants transferred from Cto8 C for week. dramatic hyponasty response was observed in leaves and 5 of plants (Figures and ). This response was severely defective in pif mutants. small hyponasty response was observed in leaf of pif- mutants but no hyponasty responses were recorded in mutants containing the pif- allele (Figures and ; Figure S). Together, these data suggest a major role for PIF in mediating petiole elongation and leaf hyponasty responses to elevated temperature. n Early Flowering Response to High Temperature Is Retained in pif Mutants The potent induction of rabidopsis flowering by high temperature is well established and has been demonstrated to involve the transcriptional regulators FLOWERING LOCUS C and FLOWERING LOCUS M []. Mutants deficient in PIF displayed a similarly early flowering phenotype to plants upon transfer to 8 C (Figure C; Figure SC). Such data suggest that the role of PIF is confined to plant architectural adaptations to high temperature. The genetic separation of signaling pathways regulating elongation growth and early flowering responses to environmental stress is consistent with studies of low R:FR-treated plants [9, ].

2 Role of PIF in Plant High Temperature Responses 9 pif o C 8 o C Pe tol i e leng th ( mm) 8 Leaf Leaf + 8 Leaf 5 Leaf pif pif pif pif5 o C 8 o C pif5 Hy po c oylle t n g th ( mm) pif pif pif5 Figure. High Temperature-Mediated Hypocotyl Elongation in pif Mutants Photographs () and hypocotyl lengths () of and pif mutant seedlings grown in continuous irradiation at different temperatures. Plants were grown at C for days before transfer to 8 C for days. Control plants were maintained at C. Error bars represent SE. sterisks represent a significant difference from the C sample with Student s t test (p %.5). High Temperature-Mediated lterations in Plant rchitecture Can Operate Independently of Phytochromes and DELL Proteins Two molecular mechanisms have been documented to regulate PIF function. bundance of PIF has been shown to be modulated via physical interaction with the phytochromes. The interaction of PIF with active phy results in the phosphorylation and degradation of PIF, thereby suppressing elongation growth in high R:FR conditions [, 5]. y contrast, binding of phy to PIF has been observed to be relatively weak []. The activities of both PIF and PIF have been shown to be modulated through physical interaction with DELL proteins [, ]. The interaction of PIF with DELLs restrains elongation growth by preventing PIF binding to target pif mm Figure. High Temperature-Mediated Petiole Elongation in pif Mutants () Petiole lengths of and pif mutants grown in continuous irradiation at different temperatures. Plants were grown at C for weeks before transfer to 8 C. Control plants were maintained at C. Measurements were performed when plants grown at 8 C displayed a cm bolt. Error bars represent SE. sterisks represent a significant difference from the C sample with Student s t test (p %.5). () Photographs of and pif mutants at 5 weeks. promoters. Degradation of DELLs by the plant hormone G relieves growth restraint, in part through enhancing PIF activity []. It is well established that phytochrome photoequilibrium is temperature sensitive []. The possibility that high temperature may inactivate phytochrome activity was investigated through analysis of high temperature-mediated hypocotyl elongation in phytochrome-deficient mutants. Triple mutants, deficient in phytochromes, D, and E, displayed significant hypocotyl elongation after transfer to 8 C(Figure ). These phytochromes are the major phytochromes repressing elongation growth in high R:FR conditions []. The elongation observed in phyde mutants transferred to 8 C confirms that high temperature responses can operate independently of these phytochromes. The weak interaction of phy and PIF [] and limited activity of phyc in the absence of phy, D, and E [] suggests that high temperature-mediated

3 Current iology Vol 9 No Col pif mm Lea fa ngl e f rom s oil s u rfa c e (de grees ) Leaf Leaf + 8 Leaf 5 Leaf pif C Ro sette leav es a t bolting Figure. High Temperature-Mediated Leaf Hyponasty and Flowering Responses in pif Mutants ( and ) Photographs () and leaf angle measurements () of plants grown in continuous irradiation at different temperatures. Plants were grown at C for weeks before transfer to 8 C. Control plants were maintained at C. Measurements were recorded of the leaf angle from the soil surface after week of high temperature treatment. (C) Flowering time of pif mutants grown at high temperature. Flowering time was recorded as the number of rosette leaves when plants displayed a cm bolt. Error bars represent SE. sterisks represent a significant difference from the C sample with Student s t test (p %.5). elongation growth is not regulated through inactivation of the phytochromes. Low R:FR ratio-mediated gene expression responses were additionally retained at 8 C(Figure 5, next section), confirming significant phytochrome activity at elevated temperatures. The possibility that PIF/DELL interaction may be altered at 8 C was investigated through analysis of a DELL global mutant, deficient in all five DELL proteins. Mutant plants pif Figure. High Temperature-Mediated Hypocotyl Elongation in Phytochrome and DELL-Deficient Mutants and phyde mutants () and and DELL global deficient mutants () grown in the presence and absence of the G biosynthesis inhibitor Paclobutrazol (PC). Plants were grown at C for days before transfer to 8 C for days. Control plants were maintained at C. Error bars represent SE. sterisks represent a significant difference from the C sample with Student s t test (p %.5). displayed significant hypocotyl and petiole elongation upon transfer to high temperature (Figure ; Figure S). These data demonstrate that high temperature-mediated elongation growth can occur independently of DELL action, although a possible contribution of DELL proteins to responses cannot be completely eliminated. The infusion of soil with Paclobutrazol (PC), a G biosynthesis inhibitor, abolished high temperature-mediated hypocotyl elongation in plants, but not DELL global mutants (Figure ). These data suggest G biosynthesis to be permissive, rather than regulatory, for this response. The G-deficient ga- mutant has been shown to display a mildly attenuated hypocotyl elongation growth response to high temperature []. It is therefore likely that the paclobutrazol treatment used in these experiments is more effective in reducing G levels than is the ga- mutation. The absence of PC-mediated inhibition of elongation responses to high temperature in DELL global mutants suggests that the inhibition observed in seedlings results from unnaturally high DELL accumulation in these plants. It is likely that these accumulated DELLs act, in part, to prevent high temperature-mediated elongation growth through inhibition of PIF function. The possibility does, however, exist that unnaturally high DELL levels may inhibit

4 Role of PIF in Plant High Temperature Responses Rel ative transcr ipt pif pif + 8 Figure 5. I9 and PIF Transcript bundance after High Temperature and Low R:FR Treatments () I9 transcript abundance measured by qpcr in and pif mutants grown in continuous light for 7 days at C and transferred to a water bath at either Cor8 C for hr. Samples were harvested at,,,, 8, and hr. Mean data from two biological repeats are shown with range bars. () I9 expression in -week-old rosettes grown in continuous light at C, transferred to the same light conditions at different temperatures for hr, and treated with low R:FR at each temperature for a further hr. Error bars represent the SE from determinations. (C) PIF transcript abundance in plants measured as in (). Data from two biological repeats are shown ( and ). Rel ative trans crpt i C Relt ai ve trans crpt i High R:FR Low R:FR Time (h) High R:FR 8 8 Time (h) Low R:FR () + 8 () () + 8 () High R:FR 8 Low R:FR 8 high temperature-mediated elongation growth through additional PIF-independent mechanisms. High Temperature-Mediated Increases in Transcript bundance of the uxin-responsive Gene I9 re bolished pif Mutants The similarities between rabidopsis responses to high temperature [, ] and low R:FR [] were investigated at the molecular level through expression analysis of marker genes associated with low R:FR signaling. Plates of seedlings grown in continuous irradiation at C were transferred to water baths at either Cor8 C in identical light conditions. This treatment ensured uniform heating of plants and standardized gene expression kinetics between biological repeats. Plant temperature was monitored throughout the experiment with a thermal imaging camera to ensure that only plants displaying uniform heating were harvested. The transcript abundance of multiple genes reported to respond significantly and robustly to low R:FR treatment was analyzed throughout a hr time course ([, 5], data not shown). One gene (I9) was observed to repeatedly display increased transcript abundance after transfer to high temperature. This increase peaked at hr after transfer and decreased again by hr (Figure 5). No high temperature-mediated increase in I9 transcript abundance was observed in pif mutants (Figure 5). Similar data were obtained in adult rosettes, although differences between C and 8 C were not observed until the hr time point (Figure S). Rapid and sustained increases in I9 transcript abundance have been reported after low R:FR treatment of rabidopsis [, ]. The possible additivity of increased temperature and low R:FR signals on I9 levels was investigated through analysis of plants transferred to elevated temperatures and low R:FR. Plants were initially grown at a cool temperature ( C), acclimated to a higher temperature (either Cor8 C) for hr, and then subjected to a hr low R:FR treatment. This temperature shift alone resulted in considerably increased I9 transcript abundance at 8 C. smaller increase was observed at C(Figure 5). Responsivity to low R:FR was retained at all temperatures, providing further evidence that phytochrome function is not inactivated at higher temperatures. Transfer to High Temperature Transiently Elevates PIF Levels The increased PIF function observed at higher temperatures was investigated through analysis of PIF transcript abundance. Seedlings grown in continuous irradiation at C were transferred to water baths at either Cor8 C, as described above. Tissue was harvested at multiple time points over a hr time course. small transient increase in PIF transcript abundance was recorded after transfer to both water baths in two separate biological repeats and was greater at the higher temperature ( and, Figure 5C). The elevated transcript abundance observed after transfer to the C water bath likely represents the small temperature increase experienced by these plants (see Experimental Procedures). similar response to both treatments was recorded in adult rosettes (Figure S). Conclusions In this work, we have shown that PIF is essential for high temperature-mediated adaptations in plant architecture.

5 Current iology Vol 9 No 5 Given the functional redundancy between PIF family members in regulating light-mediated elongation growth [5, 8, 9], the dominance of PIF in this response is highly unusual. We have demonstrated that high temperature-mediated elongation growth can occur in the absence of phytochromes and DELL proteins, suggesting the existence of a novel molecular mechanism. Hypocotyl elongation responses to high temperature involve the plant hormone auxin []. We observed the auxin-responsive gene I9 to display increased transcript abundance upon transfer to high temperature, a response that was abolished in pif mutants. This gene has previously been shown to be a component of auxin-mediated elongation growth in shade-avoidance responses to low R:FR []. It is therefore possible that PIF functions as a key regulator of an auxin-mediated signaling pathway controlling architectural adaptations to high temperature. This pathway may additionally function as a component of shade avoidance, suggesting a prominent role for PIF as a node of crosstalk between light and temperature signaling [,, 5]. Light and cold signals have previously been shown to intersect through a bhlh protein, SPTUL, in the control of rabidopsis seed germination [7]. Together, these data suggest that bhlh proteins may function as integrators of multiple environmental signals. Mildly attenuated elongation responses to high temperature were observed in pif5 mutants. bhlh proteins can heterodimerize [8], so it is possible that an absence of PIF5 may perturb the activity of PIF signaling complexes. Transiently increased PIF transcript abundance was observed after transfer of plants to high temperature. nalysis of protein abundance under similar conditions would therefore be of interest. Low R:FR ratio-mediated elongation responses have been shown to operate, in part, through stabilization of PIF [5]. The possibility exists that high temperature acts to stabilize PIF protein in addition to transiently elevating PIF transcript levels. This would provide a mechanism for the role of PIF in longer- and shorter-term responses to high temperature, respectively. lternatively, high temperature may act to enhance PIF function to mediate the dramatic architectural responses observed. The identification of such a key regulator provides an important advance in understanding how plants respond to changes in ambient temperature. Furthermore, the future molecular dissection of PIF signaling pathways may provide valuable insight into the response of plants to temperature elevations associated with global climate change. Experimental Procedures Plant Material ll experiments were performed with the functionally null pif- and pif- alleles. oth are in the Columbia background and have been described previously [, 7]. The pif5 (Columbia), pif- (Columbia), and phyphydphye (Landsberg erecta, La-er) alleles are described elsewhere [5, 7,, ]. The global DELL mutant is homozygous for mutant alleles at the five rabidopsis DELL loci (gai-t, rga-t, rgl-, rgl-, and rgl-) and was constructed by crossing a gai-t rga-t rgl- rgl- homozygote [] with a rgl- homozygote (a T-DN insertion from the publicly available SIL collection [Columbia background]) that had been backcrossed six times successively onto the La-er background. line homozygous for the mutant alleles at all five loci were selected from the F on the basis of resistance to paclobutrazol (a G biosynthesis inhibitor) and further screened via PCR assay. Plant Growth Seeds were germinated on 5 cm Petri dishes containing a : mixture of soil and horticultural sand as described previously [5]. ll experiments were performed in continuous irradiation at constant temperature in matching growth cabinets (Fi-troton H, Sanyo Gallencamp). White light was provided by fluorescent tubes at a photon irradiance of mmol m s and a red to far-red ratio of.7. For low R:FR treatments, the same photon irradiance of Photosynthetically ctive Radiation (PR) was used with a R:FR of.. For mature plant analyses, uniformly sized seedlings were transferred to individual pots at 7 days. For experiments with paclobutrazol, seedlings were grown as above for 7 days and watered with mm Paclobutrazol in.% EtOH on days and 5. Control plants were watered similarly with.% EtOH. Physiological nalyses Measurements of hypocotyl and petiole length were recorded with IMGE J software ( Leaf hyponasty was recorded as the angle between individual leaves and the soil surface with a protractor. Flowering time was recorded as the number of rosette leaves when plants displayed a cm bolt. minimum of seedlings were used for hypocotyl measurement and plants for petiole, hyponasty, and flowering measurements. Each experiment was repeated multiple times with similar results. Gene Expression Gene expression was analyzed in 7-day-old seedlings and -week-old rosettes. Plants were transferred to a water bath at either Cor8 Cinthe light conditions described above. This treatment ensured rapid and uniform heating of plants. Plant temperature was monitored with a portable thermal imaging camera throughout the experiment ( co.uk/). Plants displaying uniform heating were harvested at,,, 8, and hr. Thermal imaging showed high temperature-treated seedlings to increase from Cto8 C within hr of water bath transfer. Control seedlings displayed a small increase in temperature from Cto C (data not shown). RN extraction and qpcr procedures have been described previously []. The primers used were ctinf (TCGTGCCCGGTGTTGTTCC), ctinr (CCGTCGTCCTTCCTGTTCC), I9F (TCCCTCTTGCC CGTT), I9R (TTGCCCCCTCCTCTT), PIFF (GCCGTGGGTG TTGGT), and PIFR (CCCCTGTGGTCCCG). Supplemental Data Supplemental Data include fourfigures and can be found with this article online at cknowledgments We thank Christian Fankhauser and Séverine Lorrain (University of Lausanne) for provision of pif- and pif5 lines and Peter Quail (PGEC, C) for provision of the pif- allele. This work was supported by a iotechnology and iological Sciences Research Council grant (P9) to G.C.W. and a Royal Society University Research Fellowship to K..F. Work in N.P.H. s lab was funded by the iotechnology and iological Sciences Research Council (Core Strategic Grant to the John Innes Centre and Response Modes Grants 8/997 and /D55/) and by a Gatsby Charitable Foundation studentship to L.. Received: December 5, 8 Revised: January, 9 ccepted: January 5, 9 Published online: February, 9 References. Gray, W.M., Östin,., Sandberg, G., Romano, C.P., and Estelle, M. (998). High temperature promotes auxin-mediated hypocotyl elongation in rabidopsis. Proc. Natl. cad. Sci. 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