
==== Front
J Adv Res
J Adv Res
Journal of Advanced Research
2090-1232
2090-1224
Elsevier

S2090-1232(23)00324-7
10.1016/j.jare.2023.11.001
Basic and Biological Science
Modulation of warm temperature-sensitive growth using a phytochrome B dark reversion variant, phyB[G515E], in Arabidopsis and rice
Jeon Jin a
Rahman Md Mizanor b
Yang Hee Wook a
Kim Jaewook a
Gam Ho-Jun c
Song Ji Young a
Jeong Seok Won a
Kim Jeong-Il d
Choi Myoung-Goo e
Shin Dong-Ho a
Choi Giltsu f
Shim Donghwan a
Jung Jae-Hoon g
Lee In-Jung c
Jeon Jong-Seong jjeon@khu.ac.kr
b⁎
Park Youn-Il yipark@cnu.ac.kr
a⁎
a Department of Biological Sciences, Chungnam National University, Daejeon 34134, Republic of Korea
b Graduate School of Green-Bio Science and Crop Biotech Institute, Kyung Hee University, Yongin 17104, Republic of Korea
c Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea
d Department of Molecular Biotechnology and Kumho Life Science Laboratory, Chonnam National University, Gwangju 61186, Republic of Korea
e National Institute of Crop Science, Rural Development Administration, Wanju 55365, Republic of Korea
f Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea
g Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea
⁎ Corresponding authors. jjeon@khu.ac.kryipark@cnu.ac.kr
04 11 2023
9 2024
04 11 2023
63 5772
23 6 2023
19 9 2023
2 11 2023
© 2024 The Authors. Published by Elsevier B.V. on behalf of Cairo University.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Graphical abstract

Highlights

• Phytochrome B variant phyB[G515E] with a slow dark reversion was generated.

• phyB[G515E] forms warm temperature-insensitive nuclear photobodies.

• PHYB[G515E] expression inhibited the phytochrome interacting factor4-auxin pathway.

• PHYB[G515E] transgenic Arabidopsis and rice showed warming-insensitive growth.

• phyB[G515E] is a powerful tool for engineering warm temperature-insensitive crops.

Introduction

Ambient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB–PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants.

Objectives

This study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress.

Methods

The thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species.

Results

At an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth.

Conclusion

Transient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4–auxin-mediated thermomorphogenic responses in plants.

Keywords

Arabidopsis
Heat shock-inducible promoters
Phytochrome B dark reversion variants
PIF4
Rice
Thermomorphogenic response
==== Body
pmcIntroduction

Warming-induced morphological changes observed in the aerial parts of plants include elongated hypocotyls, petioles, and stems, hyponastic growth, and thinned leaves. Such warm temperature-dependent changes, which constitute thermomorphogenesis, enhance plant adaptation to heat stress by increasing the cooling capacity of leaves [1], [2], [3]. In higher plants, red/far-red light sensing phytochromes transduce the light signals to regulate plant growth and development, which is mediated by downstream transcription factors known as phytochrome-interacting factors (PIFs). Arabidopsis thaliana genome encodes eight PIFs [4], among which PIF4 acts a central hub in the regulation of warm temperature-induced morphogenesis in plants [5]. Under heat stress conditions, for instance, direct binding of the evening complex (EC) to the PIF4 promoter is prevented, resulting in increased transcription of PIF4 [6]. This enhances the level of PIF4 protein, which directly activates the expression of cell growth-related genes involved in growth-promoting factors (ATHB2, PREs, and LNGs), auxin signaling (AUXIN RESPONSE FACTORs [ARFs]), and biosynthesis (YUCCA8 [YUC8], TAA1, and CYP79B2) and brassinosteroid biosynthesis [5].

PIF4 activity is negatively regulated by phytochrome B (phyB) at the post-translational level. In plants, phyB is synthesized as the red (R) light-absorbing Pr form, which can be phototransformed into the far-red (FR) light-absorbing Pfr form upon exposure to R light. The physiologically active Pfr form is converted into the inactive Pr form upon the absorption of FR light or upon incubation in darkness (known as dark reversion). Since dark reversion is accelerated under increased ambient temperature conditions both in the light and dark, this process is better described as thermal reversion [7]. Once activated by light, the nuclear-localized phyB directly interacts with PIF4, leading to its degradation via the 26S proteasome pathway. In view of thermal reversion, phyB also functions as a thermosensor that detects ambient temperature. The dual function of phyB as a light and temperature sensor has been experimentally validated, and PIF4–auxin pathway has been identified as its downstream cascade [8], [9], [10]. phyB is expressed in all plant tissues, but the epidermal phyB–PIF4 module is responsible for auxin-dependent, warming-induced hypocotyl growth [6], shade avoidance and hypocotyl gravitropism responses [11]. Thus, the leaf or cotyledon epidermal phyB–PIF4 module perceives the increase in temperature, which triggers de novo biosynthesis of auxin and its accumulation in the epidermal tissue. Once accumulated in source cells, auxin is transported to petioles and hypocotyl through the vascular bundle, leading to cell elongation in the target organs. Therefore, we speculate that thermomorphogenic responses in plants could be manipulated by genetically modulating PIF4 stability at the post-translational level.

PIF4 stability and hence its activity could be altered by expressing phyB variants with higher or lower dark reversion rate in a tissue-specific or warm temperature-dependent manner. Variants with lower dark reversion rate would favor the photoequilibrium of phyB toward the active Pfr form, destabilizing PIF4, and hence lowering auxin signaling. Because the N-terminal PHY domain is critical for Pfr stabilization [12], mutations in this domain led to changes in the dark stability of the Pfr form [7]. For example, phyB[R582A] and phyB[G564E] mutants exhibited increase in dark stability, whereas phyB[S584A/E/F] and phyB[A587T] mutants showed decreases in dark stability. Structural analysis of the phyB[G564E] variant revealed the importance of the PHY domain hairpin for Pfr stabilization; in this variant, mutation in the 563WGG565 motif weakened its interaction with the GAF domain in Pr, decreasing the dark reversion rate by approximately 450-fold [13]. By contrast, the phyB[S584A] and phyB[S584E] variants, which carry mutations in the 581PRXSF585 sequence (located opposite to 563WGG565 motif) within the two β-strands in the hairpin structure, exhibited highly increased dark reversion rates [13]. The enhanced reversion activity is likely to arise from the disruption of the contact between phyB[D307] in the GAF domain and phyB[S584] in the PHY domain hairpin in the Pfr state.

However, the importance of the PHY domain in Pfr stabilization is not restricted to the hairpin structure. Recently, Vierstra and colleagues reported a cryo-electron microscopy structure of full-length Arabidopsis phyB in the Pr state [14]. Accordingly, the phyB formed an asymmetric dimer assembly that differs from the symmetric dimers of prokaryotic phy families. Such complex structural organization of full-length phyB reflects a head-to-head association of His kinase-related domains (HKRDs), linkage between the GAF of the first protomer and the PAS2 of the second protomer, and interaction of the PAS2 modular loop with the PHY domain in its own protomer. The latter dimerization contacts seen in the Pr structure were inversely related to Pfr stabilization. Disruption of hydrophobic contact between the PHY domain and the modular loop (phyB[F780E] and phyB[I783P]) resulted in slower dark reversion of a truncated phyB construct [14], suggesting that hydrophobic contact disruption between the modular loop and the PHY domain of full-length phyB might be a promising means for improving temperature acclimation on our warming planet.

Photobodies are nuclear compartments whose assembly and disassembly are triggered by photoactive Pfr and inactive Pr forms of phyB, respectively. The number and size of photobodies are highly dynamic, varying either dark-to-light transition or steady-state light intensities. Pfr-stabilizing conditions, such as high light intensity, stimulate the formation of photobodies (0.7–2 μm in diameter). Under low light or shade, phyB photobodies are relocalized to small foci in the nucleoplasm (0.1–0.7 μm in diameter) [15]. Transitions between small foci and photobodies also occur during thermomorphogenic responses in Arabidopsis [16], [17]. Considering the pivotal role of the phyB–PIF4–auxin signaling pathway in plants, the colocalization of PIF4 with phyB-containing photobodies was recently reported [18].

Rice is a major cereal crop cultivated across the diverse environments of Asia, Africa, America, Oceania, and Europe, and a major source of energy, carbohydrate, protein, vitamins, and nutrients for over 50 % of the global population [19], [20]. Regardless of whether the region has a temperate or tropical climate, critical temperatures for rice seed germination range from 10 to 45 °C (optimum, 20–35 °C), and for seedling emergence and establishment range from 12 to 35 °C (optimum, 25–30 °C) [21]. Temperatures outside the optimum limits cause stress, which hamper normal rice seed germination and seedling growth, thus reducing grain yield. Heat stress negatively affects plant height, root length, leaf number, biomass, and chlorophyll concentration in rice [22]. Reactive oxygen species accumulation, chloroplast degradation, cell membrane instability, spikelet sterility, and impaired grain quality are the other detrimental impacts of heat stress in rice [23], [24], [25], [26], [27]. Therefore, studying the early vegetative growth response of rice genotypes at high temperature could be an effective approach for the development of high-yielding heat-tolerant rice cultivars.

In present study, we aimed to manipulate warm temperature-induced elongation of plants at the post-translational level of PIF4 in Arabidopsis using phyB variants with slower dark reversion rates. Consistent with recent finding that the substitution of phyB F780, the counter part of G515, to Glu (phyB[F780E]) resulted in hydrophobic disruption and slower dark reversion rate [14], the Gly515 to Glu substitution (phyB[G515E]) led to slower dark reversion and hence was used to manipulate PIF4 stability. In addition, phyB[G564E] [12], [13], [28], [29] and phyB[S584F] [12], [13] variants were chosen as they exhibited slower and faster dark reversion properties, respectively. Additionally, phyB[G767R], which fails to localize to the nucleus [30], [31], was included as a negative control. These phyB variants as well as wild-type phyB were expressed either constitutively or in a tissue-specific or warm temperature-dependent manner. To monitor the impact of only phyB variants, and not of endogenous phyB, on Arabidopsis growth under warm temperature, the phyB variants were overexpressed in the phyB-9 mutant background. To generate Arabidopsis lines that exhibit warm temperature insensitivity primarily upon the rise in ambient temperature, the phyB variants were transiently expressed in Arabidopsis Columbia-0 (Col-0) ecotype using heat shock-inducible promoters. Arabidopsis phyB[G515E] or phyB[G564E] was also overexpressed in rice to test whether warming-induced seedling growth could be manipulated by heterogenous Arabidopsis phyB variants. Our results demonstrated that the warm temperature-specific expression and constitutive expression of phyB variants with lower dark reversion rates increased the warm temperature insensitivity of the respective Arabidopsis and rice plants. Thus, phyB variants with slower dark reversion could be used as a genetic tool for producing global warming-insensitive plants of important agricultural crops such as rice.

Materials and methods

Plant growth conditions and phenotype analysis

Arabidopsis thaliana and Nicotiana benthamiana plants were grown in a growth room at 22 °C or 28 °C under a 16-h light/8-h dark photoperiod and 120 µmol m−2s−1 light intensity. Arabidopsis seeds were sterilized with 70 % ethanol for 30 s and 2 % (v/v) sodium hypochlorite for 5 min, and then rinsed five times in sterile water. The sterilized seeds were grown on agar plates containing half-strength Murashige and Skoog (1/2 MS) medium (pH 5.7) and 0.8 % agar. Hypocotyl lengths were measured using seedlings grown vertically on 1/2 MS medium at 22 °C or 28 °C for 5 days. To conduct phenotypic analysis at vegetative growth stage, plants were grown in soil at 22 °C or 28 °C for 17 days. Hypocotyls, petioles, and leaves were photographed, and hypocotyl and petiole lengths and leaf areas were measured using the ImageJ software (https://rsb.info.nih.gov/ij).

Wild-type (WT) Japonica rice (Oryza sativa) cultivar Dongjin (DJ) and its transgenic counterparts expressing the Arabidopsis PHYB and its amino acid substitution variants, PHYB[G515E] and PHYB[G564E], were used in this study. Rice plants were grown in a greenhouse at 28 °C/25 °C day/night temperature under 14-h light/10-h dark cycle, 70–80 % relative humidity and 120 µmol m−2s−1 light intensity or in a paddy field for living modified organisms at Kyung Hee University (Yongin, Korea) under natural environmental conditions during summer 2022. To check warming-induced plant growth phenotype, WT and transgenic rice seedlings were grown at 25 °C and 34 °C under 14-h light/10-h dark cycle and 70–80 % relative humidity, and the seedlings were sampled after 5 days for taking length measurements and extracting RNA and endogenous IAAs.

Dark reversion analysis

The QuickChangeTM site-directed mutagenesis kit (Agilent Technologies; CA, USA) was used to generate Arabidopsis phyB variants, phyB[G515E] and phyB[S584F]. Full-length phyB recombinant apoproteins, with a 10-amino acid streptavidin affinity-tag (SAWRHPQFGG) at the C-terminus, were expressed using the Pichia pastoris protein expression system (ThermoFisher Scientific; MA, USA) and purified by streptavidin affinity chromatography (IBA), as described previously [32]. Phycocyanobilin (PCB) was added as a chromophore (final concentration = 20 μM) before purification of the holoproteins under dim green light. The purified Pr form of phyB was exposed to R light to generate the Pfr form, which was confirmed using Varian Cary 3E UV–VIS Spectrophotometer (Cary, Varian Inc, Canada). Photochemical difference spectra (ΔA) were obtained by subtracting the absorption spectrum of the Pfr form from that of the Pr form. The rates of dark reversion were estimated by calculating the reduction percentages in the Pfr absorption spectrum within 30 min at room temperature.

Plasmid construction and plant transformation

Plants of Arabidopsis Col-0, phyB-9 mutant in Col-0 background, and rice cultivar DJ were used in this study. To generate transgenic plants overexpressing Arabidopsis PHYB and its single amino acid substitution variants, PHYB[G515E], -[G564E], -[S584F], and -[G767R], the full-length PHYB coding region was amplified by PCR from Arabidopsis cDNA using Phusion DNA polymerase (ThermoFisher Scientific; MA, USA) and site-direct mutant primers (Supplementary Table S1). To induce constitutive expression, PHYB and variants were cloned into pJJ461 and pBI121 vectors [33], [34] containing the Cauliflower mosaic virus 35S (CaMV35Sp) or Cassava vein mosaic virus (CsVMVp) promoter with an adaptor sequence encoding the green fluorescent protein (GFP) tag to yield CaMV35Sp::PHYB-GFP, CsVMVp::PHYB[G515E]-GFP, CsVMVp::PHYB[G564E]-GFP, CsVMVp::PHYB[S584F]-GFP, and CaMV35Sp::PHYB[G767R]-GFP DNA constructs.

To generate transgenic Arabidopsis plants expressing PHYB[G515E] under heat stress or in a tissue-specific manner, the heat stress-responsive promoters of Heat Shock Protein 21 (HSP21), HSP15.7, and Heat shock factor A1 (HsfA1) genes and tissue-specific promoters such as CER6 (epidermis), SCR (endodermis), and SULTR1 (vascular phloem) were amplified by PCR from Arabidopsis genomic DNA using sequence-specific primers (Supplementary Table S1). These promoter fusion constructs were cloned into a promoter-deleted pJJ461 vector with an adaptor sequence encoding GFP to yield HSP21p::PHYB[G515E]-GFP, HSP15.7p::PHYB[G515E]-GFP, HSFA1Ep::PHYB[G515E]-GFP, CER6p::PHYB[G515E]-GFP, SCRp::PHYB[515E]-GFP, and SULTR1p::PHYB[G515E]-GFP DNA constructs. All vectors were then transformed into Col-0 or phyB-9 mutant plants by Agrobacterium-mediated transformation using the floral-dip method [35], and homozygous transgenic lines were isolated (Supplementary Figs. S2, S3 and S5).

To generate transgenic plants expressing Arabidopsis PIF4 gene in genotypes expressing PHYB variants, the full-length PIF4 coding region was amplified by PCR from Arabidopsis cDNA, and cloned into pDONRTM221 (Invitrogen; MA, USA) using the Gateway® BP ClonaseTM II enzyme mix (Invitrogen; MA, USA) to yield pDONR221:PIF4. This PIF4 sequence was inserted into pGWB417 (kanamycin) (Addgene plasmid ID: 74811) and pGWB517 (hygromycin) (Addgene plasmid ID: 74859) vectors using the Gateway® LR ClonaseTM II enzyme mix (Invitrogen; MA, USA), yielding the CaMV35Sp::PIF4-4xMYC construct. The CaMV35Sp::PIF4-4xMYC construct was introduced into Col-0, phyB-9 mutant, and transgenic plants expressing phyB or its site-specific variants using the Agrobacterium-mediated transformation method, and T3 homozygous transgenic plants were obtained.

Arabidopsis plants expressing the synthetic auxin-responsive promoter Direct Repeat 5 (DR5)-driven β-glucuronidase (GUS) reporter gene (DR5p::GUS) [36] were crossed with the phyB-9 mutant or transgenic plants expressing PHYB or site-specific variants to generate phyB-9 × DR5p::GUS, CaMV35Sp::PHYB-GFP × DR5p::GUS, CsVMVp::PHYB[G515E]-GFP × DR5p::GUS, CsVMVp::PHYB[S584F]-GFP × DR5p::GUS, and CaMV35Sp::PHYB[G767R]-GFP × DR5p::GUS transgenic plants. Transformants were initially screened on 1/2 MS medium containing 250 mg/l cefotaxime and either 20 mg/l hygromycin or 50 mg/l kanamycin, and then selected on plates containing 20 mg/l hygromycin or 50 mg/l kanamycin during seedling growth (Supplementary Figs. S2, S3, and S5). All constructs were confirmed via DNA sequencing prior to plant transformation.

To generate transgenic rice lines expressing Arabidopsis PHYB, PHYB[G515E], and PHYB[G564E], the full-length coding regions (minus the stop codon) were amplified by PCR using pfu DNA polymerase (BioFACTTM), and then cloned in between the maize Ubiquitin-1 promoter (ZmUbi) and synthetic GFP (sGFP) reporter genes of the pGA3427 binary vector [37]. Subsequently, the correct constructs were transformed into Agrobacterium tumefaciens strain LBA4404, which was then used for rice transformation using the Agrobacterium-mediated co-cultivation method [38]. Transgenic rice plants were regenerated from the transformed calli by selection on media containing 50 mg/l hygromycin. Primer sequences used for vector construction are summarized in Supplementary Table S1.

Transient expression in Arabidopsis and N. benthamiana leaves

PIF4 was transiently expressed in 10-day-old transgenic Arabidopsis plants expressing PHYB and its variants using the Agrobacterium-mediated transient expression method. Briefly, A. tumefaciens strain GV3101 harboring 35Sp::PIF4-mCherry vector was suspended in the activation solution (10 mM MgCl2, 10 mM MES, 150 μM acetosyringone) for 4 h, and then infiltrated into seedlings placed in a Petri dish by applying vacuum for 2 min twice at a 30-s interval. The infiltrated seedlings were incubated in a growth room for 2 days and used for confocal microscopy. To transiently express Arabidopsis PIF4 and PHYB in N. benthamiana, the CaMV35Sp::PIF4-mCherry construct and CaMV35Sp::PHYB-GFP, CsVMVp::PHYB[G515E]-GFP, CsVMVp::PHYB[S584F]-GFP or CaMV35Sp::PHYB[G767R]-GFP construct were mobilized into A. tumefaciens strain GV3101 using the freeze–thaw method [39]. Recombinant A. tumefaciens cells were delivered into the abaxial surface of leaves of 3–4-week-old N. benthamiana plants using a blunt tipped plastic syringe and gentle pressure [40]. To perform co-transformations, recombinant bacteria carrying different plasmids were mixed at an equimolar ratio prior to infiltration. The infiltrated plants were incubated in a growth room for 2–3 days and used for confocal imaging.

RNA isolation and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from 10-day-old Arabidopsis seedlings using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich; MO, USA), treated with DNaseI (Takara; Kyoto, Japan), and purified using the NucleoSpin RNA Clean-up Kit (Macherey-Nagel; Düren, Germany). Then, 500 ng of total RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad; CA, USA), and qRT-PCR was conducted using PrimeScriptTM RT master mix (Takara; Kyoto, Japan) on the CFX96TM Real-Time PCR detection system (Bio-Rad; CA, USA), according to the manufacturer’s instructions. All qRT-PCR assays were performed in triplicate and subjected to statistical analysis. Data analysis were performed as described previously [41], [42]. ACTIN7 was used as the internal control gene.

Total RNA of rice was extracted from mature leaves and 0.5-cm shoot bases of DJ (WT) and transgenic plants using RNAiso plus reagent (Takara, Tokyo, Japan). Then, 2 μg of total RNA was reverse-transcribed using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). The resultant cDNA was amplified by qRT-PCR using Prime Q-Master Mix 2X (GentiBio; MA, USA) in Qiagen 154 Rotor-Gene Q real-time PCR cycler (Hilden, Germany). The relative transcript level and fold-change in gene expression were analyzed using the 2−ΔCt and 2−ΔΔCt methods [43], respectively. Rice Ubiquitin5 (LOC_Os01g22490) was used as the internal control [44]. Primers sequences used to perform qRT-PCR are listed in Supplementary Table S1.

Protein extraction and immunoblot analysis

Plant tissues (200 mg) were ground using TissueLyser II (Qiagen; Hilden, Germany), and then mixed with 200 µL of extraction buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, 20 mM dithiothreitol, 80 µM MG132 [Sigma-Aldrich; MO, USA], 1 × EDTA-free protease inhibitor cocktail [Roche; Basel, Switzerland]). After centrifugation, protein concentration was determined using the Bio-Rad protein assay kit (CA, USA), with bovine serum albumin as the standard. Total protein extract prepared from the different samples was loaded on an 8 % sodium dodecyl sulfate (SDS)-polyacrylamide gel (50 µg total protein per lane), separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), and electroblotted on to polyvinylidene difluoride (PVDF) membranes (Millipore; MA, USA). The membranes were subsequently incubated with anti-GFP, -MYC, or -ACTIN antibody (Takara Bio, USA; 1:2,000 dilution), washed, and then incubated with horse radish peroxidase-conjugated goat anti-rabbit IgG (secondary antibody; 1:10,000 dilution). The resultant protein bands were visualized by treating the membrane with Clarity MaxTM Western ECL Substrate (Bio-Rad; CA, USA), according to the manufacturer’s instructions. Band intensities were quantified using ImageJ.

Histochemical GUS staining and activity assays

Seven-day-old DR5p::GUS transgenic plants expressing in phyB-9, CaMV35Sp::PHYB-GFP, CsVMVp::PHYB[G515E]-GFP, CsVMVp::PHYB[S584F]-GFP, and CaMV35Sp::PHYB[G767R]-GFP backgrounds were dark incubated at 37 °C for 24 h in a solution containing 100 mM sodium phosphate (pH 7.0), 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6], 10 mM EDTA, 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (Duchefa; Haarlem, Netherlands), and 20 % (v/v) methanol. The stained tissues were washed and dehydrated using an ethanol series and then mounted in 90 % (v/v) glycerol. Samples were examined using the Olympus SZ microscope (Tokyo, Japan) at 8 × magnification and photographed with a Canon PowerShot G9 Camera (Tokyo, Japan). GUS activities were measured using 1 mM 4-methylumberlliferyl-β-D-glucuronide (MUG) in GUS extraction buffer, as described previously [45]. After terminating the reaction with 0.2 M Na2CO3, the GUS reaction product 4-methylumbelliferone (MU) was measured with the LS-55 spectrofluorometer (Perkin-Elmer; MA, USA) at excitation and emission wavelengths of 365 and 455 nm, respectively.

Confocal imaging

Agroinfiltrated Arabidopsis and N. benthamiana leaves were subjected to fluorescence imaging using a confocal microscope (LSM800 with Airyscan; Carl Zeiss, Jena, Germany) equipped with a 40 × water-immersion objective lens. To perform steady-state fluorescence imaging, tissues were fixed with 1 % paraformaldehyde in phosphate-buffered saline (PBS; pH 7.0) for 20 min to prevent excitation light-induced rapid alteration of fluorescence protein localization during live cell imaging [46]. After staining the nuclei with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich; MO, USA) for 10 min, samples were mounted on a slide glass with distilled water.

To observe the subcellular localization of WT Arabidopsis phyB and its G515E and G564E variants in rice, the leaf sheaths of each transgenic rice line were examined. Dehusked seeds of the transgenic lines were sterilized and sown on 1/2 MS medium containing 50 mg/l hygromycin. Seeds of each transgenic line were grown under 14-h light/10-h dark cycle at 25 °C. Leaf sheaths of 7-day-old seedlings were collected after a minimum of 3 h incubation in the light, and GFP signals were detected using a confocal microscope (LSM510 META; Carl Zeiss, Jena, Germany). The signals of GFP, mCherry, and DAPI were detected at emission wavelengths ranging from 505 to 530, 600–620, and 400–485 nm, respectively.

Chlorophyll quantification

Leaves (50 mg) of 2-month-old rice plants were cut into small pieces, transferred into 1.5-ml tubes containing glass beads, and frozen in liquid nitrogen. The samples were ground using TissueLyser II (Qiagen; Hilden, Germany). Then, 1 mL of 80 % (v/v) acetone was added to each tube and vortexed. The mixtures were centrifuged at 12,000 rpm for 10 min. Supernatants were separated and diluted up to a final volume of 10 mL by adding 80 % (v/v) acetone. The absorbance of the diluted supernatants was measured using a UV–Vis Spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan), and the chlorophyll content was calculated using equations described previously [47]. Soil-plant analysis development (SPAD) chlorophyll meter (SPAD-502Plus, Konica Minolta Sensing Inc., Tokyo, Japan) was also used to measure chlorophyll concentration in leaves. An average of six readings on different parts of the same leaf was taken to obtain a more representative value of the chlorophyll content in that leaf.

IAA quantification

For the quantification of endogenous IAA, 5-day-old rice seedling samples were placed in 2 mL E-tubes and pulverized using a grinder in liquid nitrogen. The ground samples were extracted with 1.5 mL of 80 % methanol (MeOH) containing ascorbic acid (200 mg/L). The crude extract was then centrifuged (10,000g, 10 min, 4 °C), and the resulting supernatant was collected in a round-bottom flask. To this pooled supernatant, indole-2,4,5,6,7-d5-3-acetic acid (Cayman Chemical, USA) was added as an internal standard. The extract was then evaporated under reduced pressure using a rotary evaporator at 38 °C, and the remaining residue was dissolved in 0.1 M phosphate buffer. The aqueous layer was partitioned using 5 mL of ethyl acetate, and the aqueous layer was recovered while adjusting its pH to 2.7. The pH-adjusted aqueous layer was once again partitioned with ethyl acetate, and the organic solvent layer was recovered. The organic solvent layer was then dried under reduced pressure and rinsed with 20 % MeOH. Further purification was conducted using a Sep-Pak C18 cartridge, with modifications based on the method [48]. The samples purified through the C18 cartridge were dried under reduced pressure, dissolved in 100 % MeOH, collected in 1 mL reaction vials, and dried under N2 gas. Methyl ester derivatization was achieved by reacting with 100 µL of diazomethane for 40 min, followed by analysis using GC/MS.

The ethics statement

All institutional and national guidelines for the care and use of plants were followed.

Results

PHY domain mutant phyB[G515E] exhibits slow dark reversion

Most previous studies that aimed to manipulate the dark reversion activity of phyB focused on the residues surrounding the bilin-binding pocket of GAF and PHY domains (Fig. 1) [7]. During the analysis of phyB mutants in the PHY domain, the G515 residue of phyB was chosen for testing whether the disruption of hydrophobic contact between the modular loop and the PHY domain in full-length phyB would reduce dark reversion activity when replaced with Glu. Similar to the phyB[F780E] variant [14], phyB[G515E] assembled with phycocyanobilin as a chromophore showed a significant reduction in dark reversion. The absorption spectra of the Pr and Pfr forms of phyB[G515E] (649 and 713 nm, respectively) similar to those of phyB (650 and 712 nm, respectively) (Fig. 2a). However, phyB[G515E] exhibited a slower dark reversion rate than phyB (Fig. 2b). In contrast to phyB[G515E], the phyB[S584F] variant, which is mutated in the dark reversion-affecting 581PRXSF585 sequence [12], [13], exhibited a strikingly increased dark reversion rate (Fig. 2b). Based on these results, we used phyB mutants with slow (phyB[G515E]) or rapid (phyB[S584F]) dark reversion rates in subsequent experiments. Moreover, in this study, we included the phyB[G564E] variant, which was previously reported for its slower dark reversion, and a missense mutant (phyB[G767R]) impaired in the localization of phyB into the nucleus, showing no phyB activity [31]. Next, we used phyB and its variants fused with GFP instead of streptavidin to further characterize their physiological functions, as GFP-tagged full-length phys are biologically active [49], [50]. This allowed us to monitor dynamic nature of photobodies in response to changes in light quality and quantity, as well as temperatures [15], [16], [17], [18].Fig. 1 Residues enhancing or reducing phyB dark reversion. Schematic of the phyB module (center) showing its structures and domains. PCM, photosensory core module; NTE, N-terminal extension; PAS, Per/Arnt/Sim domain; KA, knotted architecture; GAF, cGMP phosphodiesterase/adenylylcyclase/FhlAdomain; PΦB, phytochromobilin; PHY, phytochrome domain; FT, flexible tongue; Mod, modular loop; DHp, dimerization histidine phosphotransfer domain; CA, catalytic ATP-binding domain; HKRD, histidine kinase-related domain. Substituted residues led to enhanced (red) or reduced (blue) dark reversion. Light-insensitive mutation is indicated in green. Residues examined in this study are indicated in orange. Full-length wild-type (WT) phyB structure (PDB: 7RZW) was used for indicating the reported residues. PAS1 structure was predicted using Alphafold2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 Phyb[G515E] variant exhibits slow dark reversion rate. (A) differential absorbance spectra of phyb proteins. the differential spectra (ΔA) of wild-type (phyB) and two variants (G515E and S584F) were obtained by subtracting the Pfr absorption spectra from the Pr absorption spectra. (B) Dark reversion of phyB proteins. The percentages of decreases in the Pfr absorption spectra were measured during dark incubation at the indicated time points. (C) Subcellular localization of phyB-GFP and G515E-GFP in cotyledon epidermal cells of seedlings. Maximum-intensity-projection fluorescence microscopy images of cotyledon epidermal cells are shown. Scale bars = 5 μm. (D) Fluence rate-dependent hypocotyl growth inhibition. Scale bars = 5 mm. In C and D, Col-0 (circles), phyB-9 mutant (triangles), CaMV35Sp::PHYB-GFP;phyB-9 (PHYB, asterisks) and CsVMVp::PHYB[G515E]-GFP;phyB-9 (G515E, squares) seedlings were grown for 5 days under darkness (D) or continuous white light illumination at varying intensities (L; 0.02, 0.2, 2, and 27 μmol m−2 s−1). For G515E plants, three transgenic lines (#3–2, #16–3 and #27–1) with varying protein levels (Supplementary Fig. S1) were presented. In D, data represent mean ± SD (n = 4).

The assembly of photobodies is promoted by the light-induced conversion of Pr to Pfr; however, assembled photobodies are disassembled by inactive Pr [15], [16], [17], [18]. This implies that number of phyB photobodies is negatively correlated with the dark or thermal reversion rate of Pfr. To test this view, we examined the subcellular localization of WT phyB and phyB[G515E] fused with enhanced GFP (GFP) in cotyledon epidermal cells of seedlings using confocal microscopy (Fig. 2c). In transgenic plants overexpressing GFP-fused phyB, the GFP signal was diffused in the cytoplasm in the dark but formed nuclear-localized photobodies under light intensities greater than 2 μmol m−2 s−1. However, in plants overexpressing GFP-fused phyB[G515E], photobodies were formed at a much lower light intensity (0.02 μmol m−2 s−1) and remained unchanged in size and number upon the increase in light intensity.

To correlate photobody formation with hypocotyl growth, we measured fluence rate-dependent hypocotyl growth in lines containing different forms of phyB such as Col-0, phyB-9, and CaMV35Sp::PHYB-GFP (PHYB) and CsVMVp::PHYB[G515E]-GFP (G515E) in phyB-9 backgrounds (Fig. 2d and Supplementary Fig. S1). These plants showed similar hypocotyl length and morphology under dark conditions; however, their sensitivity towards continuous white light was different. The phyB-9 mutant seedling showed a decreased sensitivity towards the fluence rate of white light compared with Col-0, whereas the G515E transgenic plants showed increased sensitivity. Transgenic plants containing PHYB and CsVMVp::PHYB[G564E]-GFP (G564E) were similar to that of plants containing G515E seedlings, while the response of transgenic plants containing phyB variant with rapid dark conversion rate CsVMVp::PHYB[S584F]-GFP (S584F) and light insensitive CaMV35Sp::PHYB[G767R]-GFP (G767R) was similar to that of the phyB-9 mutant (Supplementary Fig. S2). Overall, light inhibition of hypocotyl elongation was observed when photobodies were clearly formed in the nucleus.

Epidermis-specific and constitutive overexpression of PHYB[G515E] in phyB-9 leads to heat insensitivity

In Arabidopsis seedlings, epidermis-localized phyB senses warm temperature and activates the PIF4–auxin pathway [6]. Thus, we reasoned that epidermis-specific expression of phyB variants with slow dark reversion rates using the CER6 promoter (CER6p) would inhibit warming-induced cell growth. CER6 encodes ketoacyl-CoA synthase 6, which is necessary for wax biosynthesis. Its expression is limited to the epidermis, and hence its promoter is widely used to drive epidermis-specific expression [51]. For comparison, transgenic lines were generated to express phyB variants specifically in the endodermis, leaf bundle sheath cells, or vascular bundle using promoters SCRp and SULTR1p (Supplementary Fig. S3). While SCARECROW (SCR), along with SHORT-ROOT (SHR), is a key regulator of radial patterning in the Arabidopsis root [52], [53], it is also specifically expressed in the leaf bundle sheath cells and plays a redundant role in bundle sheath cell-fate specification [54]. SULFATE TRANSPORTER 1;3 (SULTR1;3) is exclusively expressed in phloem cells [55]. The levels of phyB[G515E] protein in the three transgenic strains (CER6p, SCRp, and SULTR1p), as determined by actin levels, were not similar. The expression level in SCRp lines was significantly lower than in the others (Supplementary Fig. S3d). When we consider the volume proportion of tissue types (mesophyll: epidermal: vascular) in mature Arabidopsis leaves, which are approximately 82.4: 16.2: 1.3 [56], and compare the cell numbers in the whole leaf, epidermis and bundle-sheath [56], [57], [58], [59], [60], we find that phyB[G515E] protein levels in CER6p transgenics are comparable to those in SCRp, but about 15-fold lower than those in SULTR1p transgenics (Supplementary Fig. S3e).

As a result, light-induced hypocotyl growth in phyB-9 mutants was fully or partially rescued by the expression of PHYB[G515E] driven by CER6p and SULTR1p (Supplementary Fig. S3f, g). However, CER6p transgenic lines only exhibited inhibition of temperature-dependent hypocotyl growth, whereas SCRp and SULTR1p transgenic lines showed temperature sensitivity similar to that of Col-0 plants (Fig. 3a–d). This supports the notion that epidermal cell-localized phyB acts as a thermosensor [6].Fig. 3 Epidermis-specific promoter- and constitutive promoter-driven PHYB[G515E] represses warm temperature-induced elongation of hypocotyls. (A and B) Maximum-intensity-projection confocal images of the roots (A) and cotyledon epidermal cells (B). Scale bars = 50 μm (A) and 5 μm (B). In B, G515E-GFP expressed by CER6p is localized to the DAPI-stained nucleus. (C - F) Images (C, E) and hypocotyl length (D, F). Scale bars = 5 mm. Col-0, phyB-9 mutant, CER6p::PHYB[G515E]-GFP;phyB-9 (CER6p), SCRp::PHYB[G515E]-GFP;phyB-9 (SCRp), SULTR1p::PHYB[G515E]-GFP;phyB-9 (SULTR1p), CaMV35Sp::PHYB-GFP;phyB-9 (PHYB), CsVMVp::PHYB[G515E]-GFP;phyB-9 (G515E), CsVMVp::PHYB[G564E]-GFP;phyB-9 (G564E), CsVMVp::PHYB[S584F]-GFP;phyB-9 (S584F), and CaMV35Sp::PHYB[G767R]-GFP;phyB-9 (G767R) transgenic plants were used. Plants were grown at 22 °C and 28 °C for 5 days (A and B) or at 22 °C for 2 days, and then either kept at 22 °C or transferred to 28 °C for additional 3 days (C-F) under continuous white light conditions (90 μmol m−2 s−1). In D and F, different letters indicate significant differences (one-way ANOVA; p < 0.05).

Next, we reasoned that lines containing dark stable phyB variants, G515E and G564E, maintain higher amounts of Pfr in the nucleus than that of phyB, resulting in less sensitive growth in response to elevated temperature. By contrast, a line containing a mutant in the C-terminal PAS domain of phyB, G767R [30], [31], was expected to show a temperature-independent growth response, similar to that of phyB-9, as most of phyB would be inactive in the cytoplasm. Indeed, transgenic lines constitutively expressing PHYB variants, G515E and G564E, accumulating phyB proteins to high, but similar levels (Supplementary Fig. S1) exhibited 0.3–0.8-fold reduction in hypocotyl length compared with the phyB-9 mutant, while other variant lines, S584F and G767R, failed to rescue the phyB-9 mutant phenotype under elevated growth temperature (Fig. 3e, f). They showed comparable growth at both room and elevated temperatures. These data strongly imply that the phyB[G515E] variant would affect photobody dynamics and the PIF4–auxin pathway during warming stress.

Warm temperature-dependent disintegration of photobodies is impaired in the G515E line

Warm temperature-induced conversion of active Pfr into inactive Pr is closely related to photobody dynamics as photobody is disassembled by the dark conversion of Pfr to Pr (Fig. 2) [15], [16], [17], [18], implying that number of phyB photobodies is negatively correlated with the dark reversion rate of Pfr. If this correlation is true, the number or size of photobodies in G515E seedlings would remain unchanged upon exposure to elevated temperature, because of the slow dark reversion of phyB[G515E]. Consistent with previous reports [8], [17], phyB-GFP formed photobodies in the nucleus, and the number of nuclear photobodies decreased from 6 to 3 when plants were grown at a warmer temperature (28 °C) (Fig. 4a, b). By comparison, in G515E seedlings, although photobodies were formed in a similar manner as those in PHYB, the photobodies were insensitive to the higher temperature and therefore did not decrease in number. The loss-of-function phyB mutant lines, S584F and G767R, failed to form nuclear photobodies. Instead, GFP fluorescence in these lines was predominantly retained in the cytosol and remained unchanged in response to the elevated temperature. Thus, the warm temperature insensitivity of photobody dynamics in G515E plants implies that hypocotyl growth stimulation at elevated temperature is associated with the heat-induced reduction in photobody number, and that phyB-mediated inhibition of PIF4 takes place within photobodies.Fig. 4 phyB[G515E] impairs photobody disintegration and PIF4–auxin pathway during thermomorphogenesis. (A and B) Subcellular localization of phyB-GFP, phyB[G515E]-GFP, phyB[S584F]-GFP, and phyB[G767R]-GFP (A) and the number of photobodies (B) of the cotyledon epidermal cells. Scale bars = 5 μm. In A, phyB-GFP and G515E-GFP are colocalized with DAPI-stained nucleus, while S584F-GFP and G767R-GFP are mostly localized to the cytoplasm. (C) phyB and PIF4 levels in different phyB variant-expressing transgenic plants. The phyB-GFP and PIF4-Myc proteins were detected using anti-GFP and anti-MYC antibodies, respectively. ACTIN was used as a loading control. (D – F) GUS staining assay (D) and quantification of GUS activity (E), and transcript levels of auxin biosynthesis genes, IAA19, IAA29, and YUC8 (F) in seedlings. In D, plants were incubated with 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) for 20 h. (G) Correlation of PIF4 levels, hypocotyl length and GUS activity. PIF4 level were quantified from the images shown in Fig. 4c. Col-0, phyB-9 mutants, CaMV35Sp::PHYB-GFP;phyB-9 (PHYB), CsVMVp::PHYB[G515E]-GFP;phyB-9 (G515E), CsVMVp::PHYB[S584F]-GFP;phyB-9 (S584F), and CaMV35Sp::PHYB[G767R]-GFP;phyB-9 (G767R) transgenic seedlings with (C and D) or without (A) the PIF4-MYC (C) and DR5p::GUS (D) co-expression were used. Plants were grown at 22 °C and 28 °C for 5 days (A, C, and D) or at 22 °C for 2 days, and then either kept at 22 °C or transferred to 28 °C for 6 h (F) under continuous white light conditions (90 μmol m−2 s−1). In B and E, different letters indicate significant differences (one-way ANOVA; p < 0.05). In F, data represent mean ± SD (n = 3 ∼ 5; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, NS = non-significant).

Nuclear PIF4-associated phyB photobodies are sensitive to warm temperature

The localization of PIF4 to photobodies is dependent on the presence of active phyB. Upon light illumination, PIF4 is incorporated into phyB-containing photobodies [18]. However, upon dark or FR light treatment, which converts active phyB into the inactive state, PIF4 is dispersed in the nucleoplasm [61]. Indeed, PIF4 showed two different localization patterns: one in which PIF4 localized to phyB-containing photobodies, and another in which PIF4 was diffused freely throughout the nucleoplasm. Dark-incubated N. benthamiana leaves transiently transformed with the Arabidopsis PIF4-cherry construct as well as light-treated N. benthamiana leaves co-transfected with G767R failed to form apparent photobodies (Supplementary Fig. S4a). However, light-induced PIF4 localization to the nuclear photobodies was observed in PHYB and G515E lines (Supplementary Fig. S4a). Number of PIF4-localized photobody was lowered in PHYB line upon ambient temperature increases, which was not observed in G515E and PIF4 co-expressing line (Supplementary Fig. S4b, c).

PIF4–auxin module is lowered by G515E introduction

To monitor whether the level of PIF4 is sensitive to the dark reversion rate of phyB and hence the number of photobodies, 4 × Myc-tagged PIF4 was constitutively expressed in Col-0, phyB-9 mutant, and PHYB, G515E, and G767R transgenic lines. Western blotting clearly revealed that the level of PIF4 was considerably lower in PHYB and G515E transgenic lines but higher in the phyB-9 mutant and G767R transgenic plants, compared with Col-0, at both room temperature and elevated temperature (Fig. 4c). In addition to posttranslational regulation of PIF4 protein levels, transcriptional regulation also contributes to the increased PIF4 level [5], [6]. Thus, constitutive expression of phyB[G515E] seems override such transcriptional regulation, implying the importance of post-translational regulatory pathway for manipulating PIF4-mediated thermomorphogenesis.

During thermomorphogenesis, auxin biosynthesis is the immediate target of PIF4 [8], [9]. Therefore, the lowering of PIF4 level in G515E lines strongly implies that phyB[G515E] downregulated auxin biosynthesis. In the present study, the DR5::GUS reporter system [62] was used to monitor auxin responses and distribution as auxin levels at specific sites is quantitatively correlated with the GUS expression level driven by the synthetic promoter DR5 [63]. Consistently, the relative auxin responses in response to warming temperature, estimated by GUS staining (Fig. 4d) and activity quantification (Fig. 4e), remained unchanged in phyB-9 mutant but significantly enhanced in Col-0. Such enhancement in GUS staining was significantly inhibited in PHYB, G515E, S584F, and G767R expressing transgenic plants.

Among the auxin biosynthesis genes regulated by PIF4, transcript levels of IAA19, IAA29 and YUC8 were enhanced by warming, which were inhibited by the expression of G515E, but not by S584F and G767R (Fig. 4f). Accordingly, the hypocotyls of PHYB and G515E lines, which had lower PIF4 levels, were shorter than those of the G767R transgenic line that had enhanced PIF4 level (Fig. 4g). Thus, reduced auxin response due to decreased PIF4 levels (R2 = 0.85) appears to be responsible for the warm-temperature insensitive growth of G515E. Overall, hypocotyl lengths were inversely correlated with nuclear phyB levels but positively correlated with PIF4 levels at both normal and elevated temperatures (R2 = 0.79). Although the moderate correlation between auxin responses and hypocotyl growth (R2 = 0.71, Fig. 4g) needs further clarification, these data strongly support the view that phyB acts as a thermosensor, which could be manipulated by altering the dark reversion rate of phyB.

Heat shock-inducible promoters driven expression of PHYB[G515E] modulates Arabidopsis thermomorphogenesis

We speculated that the phyB–PIF4–auxin pathway could be manipulated at elevated temperatures by expressing nuclear phyB variants, such as phyB[G515E], under the control of high temperature-inducible promoters. In this study, HSP21, HSP157, and HsfA1 were selected as strong, intermediate, and weak heat stress-inducible promoters, respectively [64]. Thus, transgenic Arabidopsis lines expressing PHYB[G515E] under the control of HSP21, HSP157, and HsfA1 promoters were generated in Col-0 background (Supplementary Fig. S5a-e). Transgenic lines expressing PHYB[G515E] under the control of the three heat shock-inducible promoters were insensitive to warm temperature, as indicated by their hypocotyl and leaf petiole growth (Supplementary Fig. S5f-i). Plants carrying the HSP21p::PHYB[G515E]-GFP construct were the most insensitive to temperature change, although their growth was inhibited even at normal temperature. By contrast, the HSFA1p::PHYB[G515E]-GFP plants were the most sensitive to warm temperature, although they showed no growth retardation.

Constitutive overexpression of Arabidopsis PHYB[G515E] in rice leads to warm temperature insensitivity

To test whether Arabidopsis phyB could modulate rice thermomorphogenesis, Arabidopsis phyB and slow-reverting variants (phyB[G515E] and phyB[G564E]) were constitutively expressed in WT rice variety DJ plants (Supplementary Fig. S6a). Phenotypic analysis was carried out using T3-generation transgenic rice plants grown in a greenhouse under natural environmental conditions during summer. Like in Arabidopsis, nuclear localization of Arabidopsis phyB and its slow-reverting variants was detected in ZmUbip::AtPHYB-GFP (PHYBr), ZmUbip:: AtPHYB[G515E]-GFP (G515Er), and ZmUbip::AtPHYB[G564E]-GFP (G564Er) transgenic rice plants (Supplementary Fig. S6b). The height of the mature PHYBr plants was almost the same as that of WT plants; however, mature G515Er and G564Er plants were significantly shorter than mature WT plants (Fig. 5a, b). In addition, compared with the leaves of DJ and PHYBr plants, those of G515Er and G564Er plants were greener (Supplementary Fig. S6c), because of the higher total chlorophyll content (Supplementary Fig. S6d). Consistently, SPAD values, which represent the ratio of chlorophyll absorbance measured at 650 nm to leaf thickness measured at 940 nm [65], were higher in G515Er and G564Er leaves than in WT leaves, whereas the PHYBr leaves showed no significant difference in SPAD values relative to the WT (Supplementary Fig. S6e).Fig. 5 Constitutive expression of Arabidopsis PHYB and its slow dark reversion variants PHYB[G515E] and PHYB[G564E] represses warm temperature-induced growth in rice. Representative mature plants (A), plant height (B), seedling phenotypes (C), shoot lengths (D), transcript levels of auxin biosynthesis genes, OsYUC1, OsYUC2, OsYUC3, OsYUC4, OsYUC5, and OsYUC6 (E), and endogenous auxin levels (F) of WT DJ and transgenic lines (ZmUbip::AtPHYB-GFP (PHYBr), ZmUbip::AtPHYB[G515E]-GFP (G515Er), and ZmUbip::AtPHYB[G564E]-GFP) (G564Er). In A, plants were grown in a paddy field during summer. Scale bars = 15 cm. In C, D, E, and F plants were grown under 14-h light/10-h dark photoperiod at 25 °C and 34 °C for 5 days. Scale bars = 2 cm. In F, the endogenous auxin contents in rice seedling shoots ranged from 12 to 39 ng/g FW. In D, different letters indicate significant differences (one-way ANOVA; p < 0.05). In E, OsUbi5 was used as the internal control. In E and F, data represent mean ± SD (n = 3 or 7; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, NS = non-significant).

The warming-induced growth phenotypes of DJ and transgenic rice seedlings were evaluated by growing them separately at 25 °C and 34 °C for 5 days and measuring shoot length. The shoots of WT and AtPHYB-overexpressing seedlings were significantly longer at 34 °C than at 25 °C. However, this warm temperature-induced growth was strongly reduced in G515Er and G564Er transgenic rice (Fig. 5c, d).

This warm temperature-insensitive growth observed in transgenic rice lines expressing of warm-insensitive Arabidopsis phyB variants in this study could be attributed to the inhibition of auxin-mediated cell elongation, similar to what is observed in Arabidopsis. To test this possibility, we measured the transcript levels of genes involved in auxin biosynthesis and the levels of shoot endogenous auxins. Among the six OsYUC genes encoding rate-limiting enzymes in the endogenous auxin biosynthesis pathway, the expression levels of OsYUC1 and OsYUC6 increased by approximately two-fold in DJ and PHYBr plants but remained unchanged in G515Er and G564Er seedlings at warm temperature (Fig. 5e). In line with enhanced transcript levels of auxin biosynthesis genes, the warming-induced increase in endogenous free auxin contents was observed in wild type and PHYBr, but not in G515Er and G564Er transgenic rice lines (Fig. 5f). This suggests that OsYUC1 and OsYUC6 play important roles in the accumulation of auxin induced by warm temperature, which, in turn, leads to growth enhancement in rice.

Discussion

Warming-induced hypocotyl growth in Arabidopsis is mediated by auxin, a mobile signal that travels down from cotyledons [66] where thermosensing and signaling are mediated by the epidermis-localized phyB–PIF4 module [6]. Thus, from a practical point of view, to generate warm temperature-sensitive or -insensitive plants, genetic manipulation of the thermoresponsive phyB–PIF4–auxin pathway is required at the post-translational level, considering the complex transcriptional regulation of PIF4 [1], [6], [67]. Among the various post-translational regulators of PIF4 activity, phyB was chosen as a genetic target as its gain-of-function and loss-of-function mutants affecting light signaling, photobody formation, chromophore binding, and dark reversion are well characterized [7]. In the present study, phyB variants with slower or faster dark reversion rates than the phyB exhibited warm temperature-insensitive hypocotyl growth via lowering or enhancing PIF4–auxin pathway, respectively (Fig. 6). Thus, our results suggest that molecular genetic modulation of the phyB–PIF4–auxin pathway involved in warm temperature perception, signaling, and response in terms of hypocotyl and leaf petiole growth provides a promising approach for engineering thermomorphogenic responses such as accelerating flowering time, which is dependent on PIF4 and PIF5 [68].Fig. 6 Schematic illustrating for the transformation of warm sensitivity using phyB dark variants. Under given light intensity and temperature conditions, a dynamic equilibrium between inactive cytosolic Pr dimer (shown in red) and active nuclear photobody-localized Pfr dimers (in crimson red) downregulates auxin biosynthesis. When the temperature increases without change in light intensity, the level of nuclear Pfr decreases due to its dark reversion to the Pr form. This, in turn, leads to warming-induced auxin biosynthesis and subsequent cell elongation/growth. Such warm temperature-sensitive plants could be transformed into warm temperature-insensitive plants by expressing phyB variants, phyB[G515E] and phyB[G564E], with slower dark reversion rates (indicated in blue). This expression can be either constitutive or induced by heat shock. Consequently, in plants expressing PHYB[G515E] and PHYB[G564E], the level of nuclear Pfr remains higher than that of WT phyB, thus weakening the PIF4–auxin pathway. Thick and thin arrows indicate high and low conversion rates, respectively.

The present study also illustrates a feasible approach for manipulating the elevated temperature signaling pathway using heat stress-inducible promoters. Compared with Col-0 plants, the plants expressing PHYB under the control of heat shock-inducible promoters were less sensitive to elevated temperature (Fig. 6). However, alternative warm temperature-inducible promoters are needed, since the leaky expression of PHYB[G515E] and PHYB[G564E] by heat shock-inducible promoters used in the current study stunted or enhanced growth, respectively, even at normal growth temperature. In addition, phyB dark variants could potentially be used to improve plant growth and development under various abiotic stress conditions. In fact, phyB is known to regulate acclimation to other abiotic stresses including salinity, drought, cold, high light intensity, and heavy metal toxicity by altering the expression levels of genes involved in the regulation of leaf transpiration, plant antioxidant capacity, and protective pigment content [69].

One of the unexpected findings in the present study was that the number of photobodies containing phyB and PIF4 was inversely correlated with PIF4 and consequently with hypocotyl growth enhancement (Supplementary Fig. S7). Thus, we propose that photobody-bound PIF4 is inactive, presumably phosphorylated and targeted for degradation, while the free form of PIF4 is active and regulates the transcription of target genes, such as auxin biosynthesis genes in this case. Another interesting finding was the ambient temperature-induced lowering in photobody number, which seemed to be caused by the reversion of active Pfr to inactive Pr as the phyB[G515E] variant resists the warming effect. Otherwise, nuclear phyB[G515E] might suppress the direct involvement of photobody-regulating proteins such as HEMERA (HMR), REGULATOR OF CHLOROPLAST BIOGENESIS (RCB), and NUCLEAR CONTROL OF PEP ACTIVITY (NCP) [70]. Both HMR and RCB interact directly with phyB and are required for thermomorphogenesis via PIF4 stabilization [71]. Further investigation is needed to determine whether protein–protein interaction between phyB variants and HMR/RCB vary in response to growth temperatures, along with PIF4 as a full-time player. Purification of the protein components of intact photobodies from plants grown at normal and elevated growth temperatures, and their characterization, might be a good starting point for gaining further insights.

Responses of rice seedlings to warm environment are important as seedling growth is a decisive stage for ensuring proper plant development and, therefore, rice grain yield and global food security. Rice seedling growth is almost linearly correlated with the increasing growth temperature up to the critical temperature of 35 °C [21]. Similar to Arabidopsis, inhibition of warm temperature-induced rice seedling growth was achieved by the heterologous expression of Arabidopsis PHYB variants, PHYB[G515E] and PHYB[G564E]. The inhibition of warm temperature-induced increases in the transcript levels of auxin biosynthesis genes OsYUC1 and OsYUC6, as well as free auxin contents, by nuclear-localized Arabidopsis phyB[G515E] and phyB[G564E], strongly implicates the presence of phyB-mediated temperature perception and the PIF–auxin module in rice, similar to that in Arabidopsis. Consistently, rice phyB localizes to the cytoplasm in the dark and to the nucleus under continuous light [72]. Furthermore, rice phyB knockout mutant plants exhibit early senescence [73], whereas mature leaves of Arabidopsis PHYB[G515E] and PHYB[G564E] transgenic plants showed delayed senescence (Supplementary Fig. S6). Differences in the heat sensitivity of rice cultivars BIM and N22 [73] might be caused by the genetic modulation of phyB-dependent thermosensing and signaling.

Three rice phytochrome members, OsphyA, OsphyB, and OsphyC are involved in the regulation of seedling de-etiolation, plant architecture, and heading time [74]. Among the 14 rice phytochrome-interacting factor-like proteins (PILs), six OsPILs (OsPIL11 to OsPIL16) share high protein sequence similarity with Arabidopsis PIFs (AtPIF3, AtPIF4, and AtPIF5) [75], [76]. OsphyB interacts with OsPIL14, OsPIL15, and OsPIL16, contributing to various physiological and stress responses, such as cold resistance [77], [78], [79]. Moreover, the overexpression of OsPIL15 has been shown to inhibit auxin signaling and is speculated to be involved in photomorphogenesis in rice [80]. However, the involvement of Osphy-OsPIL-auxin module in rice thermomorphogenesis has not been reported yet. Our current study strongly suggests that rice may also possess similar temperature-sensing and signaling-cascade mechanisms to those found in A. thaliana, which warrants further investigation.

Conclusion

Overall, disrupting the hydrophobic contact between the PHY domain and modular loop of phyB appears to be an effective strategy for engineering phyB variants with slower dark reversion from Pfr to Pr. The phyB[G515E] variant strongly suppressed PIF4-mediated auxin biosynthesis in epidermal cells, resulting in reduced hypocotyl elongation during Arabidopsis thermomorphogenesis. Our study also revealed that nuclear PIF4 localization to phyB photobodies is sensitive to changes in temperature. Additionally, engineered Arabidopsis phyB variants proved effective in inhibiting warm temperature-induced shoot elongation in rice. Consequently, the introduction of phyB variants represents an effective approach for developing global warming-insensitive plants, which can contribute to ensure food security for current and future generations.

CRediT authorship contribution statement

Jin Jeon: Methodology, Investigation, Visualization, Writing – original draft. Md Mizanor Rahman: Methodology, Investigation. Hee Wook Yang: Methodology, Investigation, Visualization, Writing – original draft. Jaewook Kim: Methodology, Investigation. Ho-Jun Gam: Methodology, Investigation. Ji Young Song: Methodology, Investigation. Seok Won Jeong: Methodology, Investigation. Jeong-Il Kim: Methodology, Investigation, Writing – review & editing. Myoung-Goo Choi: Methodology, Investigation. Dong-Ho Shin: . Giltsu Choi: Conceptualization, Writing – review & editing. Donghwan Shim: Methodology, Investigation. Jae-Hoon Jung: Conceptualization, Writing – review & editing. In-Jung Lee: Conceptualization, Writing – review & editing. Jong Seong Jeon: Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Visualization, Funding acquisition, Project administration, Writing – original draft. Youn-Il Park: Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Visualization, Funding acquisition, Project administration, Writing – original draft.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary data 1

Acknowledgements

We thank J. Clark Lagarias for helpful discussions and for critically reading the manuscript. This research was supported by Korea Forestry Promotion Institute (2021368B10-2123-BD0 to S.D.H. and P.Y.-I.), KRIBB Research Initiative Program (KGM1002311 to P.Y.-I.), NIAS Research Program for Agricultural Science & Technology Development (RS-2023-00223126 to P.Y.-I.), and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2023R1A2C1003142 to J.J.-S.), and in part by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (grant no. 2021R1A2C1012562 to K.J.-I.).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2023.11.001.
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