Mol. Cells 2020; 43(7): 645-661
Published online June 22, 2020
https://doi.org/10.14348/molcells.2020.0117
© The Korean Society for Molecular and Cellular Biology
Correspondence to : gchoi@kaist.edu
Leaf senescence is a developmental process by which a plant actively remobilizes nutrients from aged and photosynthetically inefficient leaves to young growing ones by disassembling organelles and degrading macromolecules. Senescence is accelerated by age and environmental stresses such as prolonged darkness. Phytochrome B (phyB) inhibits leaf senescence by inhibiting phytochrome-interacting factor 4 (PIF4) and PIF5 in prolonged darkness. However, it remains unknown whether phyB mediates the temperature signal that regulates leaf senescence. We found the light-activated form of phyB (Pfr) remains active at least four days after a transfer to darkness at 20°C but is inactivated more rapidly at 28°C. This faster inactivation of Pfr further increases PIF4 protein levels at the higher ambient temperature. In addition, PIF4 mRNA levels rise faster after the transfer to darkness at high ambient temperature via a mechanism that depends on ELF3 but not phyB. Increased PIF4 protein then binds to the ORE1 promoter and activates its expression together with ABA and ethylene signaling, accelerating leaf senescence at high ambient temperature. Our results support a role for the phy-PIF signaling module in integrating not only light signaling but also temperature signaling in the regulation of leaf senescence.
Keywords Arabidopsis, PIF4, phytochrome, senescence, temperature
Plants display various adaptive responses to survive at different temperatures. For example, as an adaptation to low temperatures under weak blue light, plant chloroplasts move from periclinal cell surfaces to anticlinal cell surfaces (Kodama et al., 2008). This presumably avoids photodamage induced by light exposure that exceeds the plant’s photosynthetic capacity at low temperatures. Plant thermomorphogenesis is another example of plant adaptation to high ambient temperature. This process is characterized by the elongation of hypocotyls and stems, and the production of hyponastic leaves with elongated petioles (Casal and Balasubramanian, 2019; Gray et al., 1998; Koini et al., 2009; Lippmann et al., 2019; Quint et al., 2016). Hypocotyl elongation is thought to be an adaptation that avoids heat absorbed by the soil in the seedling stage, while hyponastic leaves with elongated petioles help dissipate heat more efficiently by opening a rosette structure in the vegetative stage. High ambient temperature also reduces leaf size and thickness, reduces leaf stomatal density, and hastens flowering (Blázquez et al., 2003; Crawford et al., 2012; Halliday et al., 2003; Kumar et al., 2012). It is particularly important to understand these adaptive changes in growth pattern and flowering time because they reduce overall plant productivity. As the globe continues to warm, this reduced productivity may exacerbate global food shortages (Bita and Gerats, 2013).
To adapt to changing temperatures, plants must first be able to sense temperature. Any biological molecule, in principle, could serve as a thermosensor because temperature affects all molecular kinetics. Indeed, different classes of biological molecule including DNA, RNA, protein, and membrane have been identified as thermosensors for different temperature responses (Casal and Balasubramanian, 2019; Chung et al., 2020; Vu et al., 2019). Plant photoreceptors have also been suggested as potential thermosensors. Phytochrome B (phyB), which shifts from its biologically active Pfr form to its inactive Pr form upon the exposure to high ambient temperature and far-red light, acts as a thermosensor for plant thermomorphogenesis (Jung et al., 2016; Legris et al., 2016). Phototropin, which is also deactivated thermally, acts as a thermosensor for cold temperature-induced chloroplast movement in
Phytochrome-interacting bHLH transcription factor 4 (PIF4) is one of the key factors in
High ambient temperature can also increase PIF4 activity partly by elevating PIF4 protein stability. PIF4 protein is degraded by light in part via its interaction with phyB and BLADE ON PETIOLE 1 and 2 (BOP1/2), which are BTB-domain containing substrate adaptor proteins in the CUL3 E3 ligase complex (Zhang et al., 2017). In addition to phyB and BOPs, UVR8 destabilizes PIF4 protein in UV-B-treated plants grown at high ambient temperature (Hayes et al., 2017), whereas DET1 and COP1/SPAs stabilize PIF4 protein regardless of temperature (Gangappa and Kumar, 2017). In a third and final mechanism, high ambient temperature regulates PIF4 protein activity via the activity of other proteins. LONG HYPOCOTYL IN FAR-RED 1 (HFR1), an atypical HLH protein that inhibits PIF4 DNA binding by forming heterodimers with PIF4, is stabilized by high ambient temperature, mitigating high temperature responses by PIF4 (Foreman et al., 2011; Hayes et al., 2017; Hornitschek et al., 2009; Ma et al., 2016). FCA, an RNA binding protein, is recruited more strongly to the
Leaf senescence is the final stage of leaf development characterized by the degradation of chlorophyll molecules and internal organs followed by the remobilization of nutrients to younger organs in plants. Leaf senescence is accelerated by endogenous factors such as leaf age and by environmental stresses such as prolonged darkness, drought, salt stress, and pathogen attack (Lim et al., 2007). Many plant hormones have shown to regulate leaf senescence either promotively (ABA, brassinosteroids, ethylene, jasmonic acid, salicylic acid) or repressively (auxin, cytokinin, gibberellic acids) in conjunction with intricate networks of various transcription factors including bZIP, NAC, and WRKY transcription factor families. Ethylene is a plant hormones that promotes leaf senescence. Ethylene binds to and inhibits the ethylene receptor-kinase complex, ETRs-CTR1, resulting in the activation of EIN2. Activated EIN2 stabilizes EIN3 and related EIL transcription factors (Dubois et al., 2018). This promotes leaf senescence either by activating the expression of senescence promoting other transcription factors such as ORE1 and AtNAP or by repressing miR164, which targets ORE1 (Li et al., 2013). ABA is another plant hormone that promotes leaf senescence. The core ABA signaling comprise the SnRK2s that activate group A bZIP transcription factors including ABI5, the group A PP2Cs that inhibit SnRK2s, and PYR/PYLs that bind to ABA. ABA binding to PYR/PYLs promotes the binding of PYR/PYLs to PP2Cs, inhibiting their activities. The inhibition of PP2Cs liberates SnRK2s allowing the phosphorylation and activation of the group A bZIP transcription factors that promote leaf senescence. These group A bZIPs do this by activating ORE1 and chlorophyll degradation genes (Cutler et al., 2010; Sakuraba et al., 2014). In leaf senescence induced by prolonged darkness, PIF4 and its homologs, PIF3 and PIF5, promote leaf senescence by directly activating the expression of senescence-associated downstream target genes such as
High ambient temperature may also regulate senescence. Among animals, the lifespan of
In this study, we asked whether high ambient temperature promotes leaf senescence in
For general growth and seed harvest,
To measure chlorophyll levels in senescent leaves, chlorophylls were extracted from 10 detached cotyledons or from cotyledons cut from seedlings in 100% ethanol for 1 day at 4°C in the dark. Then, the extracted chlorophyll levels were determined using a spectrophotometer (Lichtenthaler, 1987). All handling of the plates was done under a green safety light. Chlorophyll levels were normalized by the fresh weight of the cotyledons.
To inactivate Pfr before the transfer to darkness, the seedlings or detached cotyledons were irradiated with far-red light (2.5 µmol m-2 s-1) for 10 min or for the indicated times.
To measure the expression of
Total RNA was isolated from 80 seedlings using the MiniBEST Plant RNA Extract Kit (Takara, Japan). Total RNA (2 µg) was used to synthesize cDNAs using oligo (dT) 18 primers and the M-MLV reverse transcriptase (Promega, USA). Expression of specific mRNAs was determined via quantitative real-time polymerase chain reaction (PCR) with specific primer sets (Supplementary Table S1) using the CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). The expression of specific mRNAs was normalized by the expression levels of
Seven-day-old light-grown seedlings were transferred to darkness for 1 day either at 20°C or 28°C and sampled for chromatin immunoprecipitation (ChIP) assays. ChIP assays were performed as described (Oh et al., 2007). Briefly, seedlings were cross-linked with a crosslinking buffer (0.4 M Sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, 2.7% formaldehyde, 0.8% MG-132, 1 mM PMSF) for 20 min under vacuum, frozen with liquid nitrogen, and ground with a mortar and pestle. The ground samples were resuspended in nuclei isolation buffer (0.25 M Sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 0.1 mM CaCl2, 9% Triton X-100, 1 mM PMSF) and debris was removed with a miracloth. Nuclei were isolated by centrifugation (9,800
Seedlings were sampled and frozen immediately with liquid nitrogen in a dark room under a green safety light. Frozen seedlings were ground and resuspended in UREA protein extraction buffer (100 mM NaH2PO4 pH 7.8, 10 mM Tris-HCl pH 8.0, 1 mM PMSF, 8 M urea with the Calbiochem Protease Inhibitor Cocktail) and the debris was removed by centrifugation (9,800
Four-day-old light-grown
Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this paper are as follows: PHYB (AT2G18790); PIF4 (AT2G43010); PIF5 (AT3G59060); PIL1 (AT2G46970); HFR1 (AT1G02340); EIN2 (AT5G03280); EIN3 (AT3G20770); ABI5 (AT2G36270); ABA2 (AT1G52340); ORE1 (AT5G39610); NYE1 (AT4G22920); UBQ10 (AT4G05320); LRB1 (AT2G46260); LRB2 (AT3G61600); COP1 (AT2G32950); ELF3 (AT2G25930); and PP2AA3 (AT1G13320).
Leaf senescence is accelerated both by internal aging programs and environmental stresses such as darkness, drought, high salinity, and pathogen attacks (Lim et al., 2007). High ambient temperature, because it affects so many plant processes (e.g., seed germination, stem elongation, and flowering) may be another critical environmental factor that accelerates leaf senescence.
We examined leaf senescence by transferring light-grown seedlings or detached leaves to a dark condition at either 20°C or 28°C. Leaf senescence is marked by a gradual change of leaf color from green to yellow—due to reduced chlorophyll—and increased expression of senescence marker genes such as
Since PIF4 and PIF5 promote senescence at ambient temperature, we asked whether PIF4 and PIF5 also accelerate leaf senescence at high ambient temperatures. We found the
PIF4 accelerates senescence both by activating the expression of
We next asked how high ambient temperature regulates PIF4 by measuring
Since phyB suppresses senescence by inhibiting PIF4 (Sakuraba et al., 2014), we asked which step of
We found
Since COP1 and ELF3 regulate
Since leaf senescence occurs relatively slowly after the transfer to darkness, we were curious to see whether Pfr generated before the transfer to darkness remains active to suppress senescence after the transfer to darkness. We therefore investigated the role of preexisting Pfr by inactivating Pfr with a pre-FRp.
Wild type seedlings senesce faster at 20°C after exposure to a pre-FRp (Fig. 6A). Previous studies have shown PIF4/5 promote senescence downstream of phyB (Sakuraba et al., 2014; Song et al., 2014). Consistent with these results, not only did we find
Alternatively, it is possible pre-FRp exposure does not accelerate senescence at 28°C, not because high ambient temperature accelerates the thermal reversion of Pfr to Pr to levels below an activity threshold, but because Pfr cannot suppress senescence at high ambient temperature. If this is the case, pre-FRp exposure should not accelerate senescence even in seedlings with high levels of Pfr. We therefore examined senescence in
The suppression of senescence by pre-existing Pfr raises the question of how long Pfr remains active after the transfer to darkness. Since the number and size of phyB photobodies—distinct fluorescent dots in the nucleus—is correlated with phyB activity (Chen et al., 2003; Hahm et al., 2020), we examined how long phyB photobodies last after the transfer to darkness. As reported before, PhyB-GFP forms bright photobodies in the light (Fig. 7A). In the epidermis, the nuclear fluorescent signals grow more diffuse with fewer and weaker photobodies already at 12 h after the transfer to darkness. At 28°C, the effect is stronger, with more diffuse nuclear fluorescent signal and even fewer photobodies (Fig. 7B). One day after the transfer to darkness, nuclear fluorescent signals are even further reduced with virtually no discernible photobodies at either 20°C or 28°C. These results indicate phyB-GFP photobodies disappear quickly after the transfer to darkness at both 20°C than at 28°C, but lasting slightly longer at 20°C.
Although photobodies disappear quickly, diffuse GFP signal is still present in the nucleus 24 h after the transfer to darkness at both 20°C and 28°C (Fig. 7A). This suggests that a small portion of phyB may still be active. To more sensitively gauge the presence of active Pfr, we exposed seedlings to an FRp in the middle of the dark period (mid-FRp) at various times after the transfer to darkness and measured the expression of PIF target gene mRNAs (Fig. 7C). If Pfr is present at the time of the mid-FRp, then the mid-FRp should convert the remaining Pfr to Pr, releasing PIFs to activate the expression of PIF target gene mRNAs. We performed these experiments with
PIF4 promotes leaf senescence in a prolonged dark condition, whereas phyB suppresses senescence by inhibiting PIF4 (Sakuraba et al., 2014; Song et al., 2014). In this study, we show high ambient temperature accelerates leaf senescence in the dark by increasing
Our data indicate some Pfr remains active relatively long after the transfer to darkness. We demonstrated this by measuring the induction of PIF4 target gene expression by exposure to a mid-FRp even 4 days after the transfer to darkness at 20°C (Fig. 7D). Direct measurements of different absorption spectra in vitro, in yeast, and in plants indicate that recombinant phytochromes undergo an initial rapid thermal reversion of Pfr-Pr heterodimers followed by a slower thermal reversion of Pfr-Pfr homodimers (Ádám et al., 2011; Eichenberg et al., 1999; 2000; Jung et al., 2016; Klose et al., 2015; Kunkel et al., 1995; Legris et al., 2016; Remberg et al., 1998; Sweere et al., 2001). In plants, the Pfr form of phyB also undergoes thermal reversion with a half-life of about 60 min at 25°C, but some Pfr remains even after 4 h in darkness. In fluorescent image analyses of phyB-GFP, although phyB-GFP photobodies disappear within 16 to 18 h in darkness, there is residual diffuse nuclear phyB-GFP signal even 24 h after the transfer to darkness (Huang et al., 2016; Van Buskirk et al., 2014). This suggests a portion of the phyB-GFP remains in the Pfr form even after 24 h of darkness. As we further probed biologically active Pfr by measuring the expression of FRp-inducible PIF target genes, we confirmed that Pfr remains active even 4 days after the transfer to darkness at ambient temperature (Fig. 7D). This relatively long survival of Pfr after the transfer to darkness is also evidenced by the destabilization of PIF4 protein 1 day after the transfer of
Our data show phyB acts not only as a photoreceptor but also as a thermosensor that regulates leaf senescence in prolonged darkness. PhyB’s ability to sense temperature is likely due to a temperature-dependent increase in the rate of thermal reversion of the Pfr form to the Pr form. This was manifested in the reduced size of phyB-GFP photobodies and in direct absorbance measurements of Pfr level at high ambient temperature in light-grown seedlings (Jung et al., 2016; Legris et al., 2016). We found, however, that phyB photobodies disassemble quickly after the transfer to darkness, making it difficult in our experimental conditions to determine whether Pfr lasts long enough to suppress senescence after the transfer to darkness (Fig. 7A). To detect the presence of biologically active Pfr, we measured the induction of PIF4 target gene mRNAs by an FRp given in the middle of the period of darkness (mid-FRp) (Fig. 7C). Such a mid-FRp should revert all the Pfr present at the time of irradiation to Pr, releasing PIF4 to activate the expression of its target genes. We found a mid-FRp increases PIF4 target gene mRNAs even if given 4 days after the transfer to darkness at 20°C. At 28°C, however, a mid-FRp given 3-4 days after the transfer to darkness does not affect PIF4 target gene mRNAs (Fig. 7D). These results indicate high ambient temperature accelerates the inactivation of Pfr, making phytochromes less active and therefore less able to suppress senescence.
The phytochromes, however, are not the only thermosensors regulating leaf senescence via PIF4 as high ambient temperature increases
High ambient temperature increases PIF4 protein levels both phy-dependently and phy-independently. Since
Currently, no other thermosensors are known to regulate senescence via PIF4. Previous studies have shown that other plant photoreceptors, including phototropin (phot), cryptochrome (cry: cry1 and cry2), and UVR8, also undergo thermal reversion from an active state to an inactive state, making them good thermosensor candidates (Findlay and Jenkins, 2016; Fujii et al., 2017; Herbel et al., 2013). However, phot, cry, and UVR8 are unlikely to be the thermosensor that regulates senescence via PIF4 in prolonged darkness. First, phot has a short half-life (~30 s at 22°C), making it difficult to function in a prolonged darkness (Christie, 2007). Phot also does not regulate
ELF3 and COP1 could be closely associated with a thermosensor regulating high temperature-induced
This work was supported by the National Research Foundation of Korea (NRF-2018R1A3B1052617).
C.K., P.O.L., Y.I.P., and G.C. designed the experiments. C.K., S.J.K., J.J., E.P., and E.O. performed the experiments. C.K., E.O., P.O.L., and G.C. wrote the manuscript. All authors discussed the results and reviewed the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2020; 43(7): 645-661
Published online July 31, 2020 https://doi.org/10.14348/molcells.2020.0117
Copyright © The Korean Society for Molecular and Cellular Biology.
Chanhee Kim1 , Sun Ji Kim2, Jinkil Jeong3
, Eunae Park1
, Eunkyoo Oh4
, Youn-Il Park5
, Pyung Ok Lim6
, and Giltsu Choi1,*
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea, 2Center for Plant Aging Research, Institute for Basic Science, Daegu 42988, Korea, 3Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA, 4Division of Life Sciences, Korea University, Seoul 02841, Korea, 5Department of Biological Sciences and Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 34134, Korea, 6Department of New Biology, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu 42988, Korea
Correspondence to:gchoi@kaist.edu
Leaf senescence is a developmental process by which a plant actively remobilizes nutrients from aged and photosynthetically inefficient leaves to young growing ones by disassembling organelles and degrading macromolecules. Senescence is accelerated by age and environmental stresses such as prolonged darkness. Phytochrome B (phyB) inhibits leaf senescence by inhibiting phytochrome-interacting factor 4 (PIF4) and PIF5 in prolonged darkness. However, it remains unknown whether phyB mediates the temperature signal that regulates leaf senescence. We found the light-activated form of phyB (Pfr) remains active at least four days after a transfer to darkness at 20°C but is inactivated more rapidly at 28°C. This faster inactivation of Pfr further increases PIF4 protein levels at the higher ambient temperature. In addition, PIF4 mRNA levels rise faster after the transfer to darkness at high ambient temperature via a mechanism that depends on ELF3 but not phyB. Increased PIF4 protein then binds to the ORE1 promoter and activates its expression together with ABA and ethylene signaling, accelerating leaf senescence at high ambient temperature. Our results support a role for the phy-PIF signaling module in integrating not only light signaling but also temperature signaling in the regulation of leaf senescence.
Keywords: Arabidopsis, PIF4, phytochrome, senescence, temperature
Plants display various adaptive responses to survive at different temperatures. For example, as an adaptation to low temperatures under weak blue light, plant chloroplasts move from periclinal cell surfaces to anticlinal cell surfaces (Kodama et al., 2008). This presumably avoids photodamage induced by light exposure that exceeds the plant’s photosynthetic capacity at low temperatures. Plant thermomorphogenesis is another example of plant adaptation to high ambient temperature. This process is characterized by the elongation of hypocotyls and stems, and the production of hyponastic leaves with elongated petioles (Casal and Balasubramanian, 2019; Gray et al., 1998; Koini et al., 2009; Lippmann et al., 2019; Quint et al., 2016). Hypocotyl elongation is thought to be an adaptation that avoids heat absorbed by the soil in the seedling stage, while hyponastic leaves with elongated petioles help dissipate heat more efficiently by opening a rosette structure in the vegetative stage. High ambient temperature also reduces leaf size and thickness, reduces leaf stomatal density, and hastens flowering (Blázquez et al., 2003; Crawford et al., 2012; Halliday et al., 2003; Kumar et al., 2012). It is particularly important to understand these adaptive changes in growth pattern and flowering time because they reduce overall plant productivity. As the globe continues to warm, this reduced productivity may exacerbate global food shortages (Bita and Gerats, 2013).
To adapt to changing temperatures, plants must first be able to sense temperature. Any biological molecule, in principle, could serve as a thermosensor because temperature affects all molecular kinetics. Indeed, different classes of biological molecule including DNA, RNA, protein, and membrane have been identified as thermosensors for different temperature responses (Casal and Balasubramanian, 2019; Chung et al., 2020; Vu et al., 2019). Plant photoreceptors have also been suggested as potential thermosensors. Phytochrome B (phyB), which shifts from its biologically active Pfr form to its inactive Pr form upon the exposure to high ambient temperature and far-red light, acts as a thermosensor for plant thermomorphogenesis (Jung et al., 2016; Legris et al., 2016). Phototropin, which is also deactivated thermally, acts as a thermosensor for cold temperature-induced chloroplast movement in
Phytochrome-interacting bHLH transcription factor 4 (PIF4) is one of the key factors in
High ambient temperature can also increase PIF4 activity partly by elevating PIF4 protein stability. PIF4 protein is degraded by light in part via its interaction with phyB and BLADE ON PETIOLE 1 and 2 (BOP1/2), which are BTB-domain containing substrate adaptor proteins in the CUL3 E3 ligase complex (Zhang et al., 2017). In addition to phyB and BOPs, UVR8 destabilizes PIF4 protein in UV-B-treated plants grown at high ambient temperature (Hayes et al., 2017), whereas DET1 and COP1/SPAs stabilize PIF4 protein regardless of temperature (Gangappa and Kumar, 2017). In a third and final mechanism, high ambient temperature regulates PIF4 protein activity via the activity of other proteins. LONG HYPOCOTYL IN FAR-RED 1 (HFR1), an atypical HLH protein that inhibits PIF4 DNA binding by forming heterodimers with PIF4, is stabilized by high ambient temperature, mitigating high temperature responses by PIF4 (Foreman et al., 2011; Hayes et al., 2017; Hornitschek et al., 2009; Ma et al., 2016). FCA, an RNA binding protein, is recruited more strongly to the
Leaf senescence is the final stage of leaf development characterized by the degradation of chlorophyll molecules and internal organs followed by the remobilization of nutrients to younger organs in plants. Leaf senescence is accelerated by endogenous factors such as leaf age and by environmental stresses such as prolonged darkness, drought, salt stress, and pathogen attack (Lim et al., 2007). Many plant hormones have shown to regulate leaf senescence either promotively (ABA, brassinosteroids, ethylene, jasmonic acid, salicylic acid) or repressively (auxin, cytokinin, gibberellic acids) in conjunction with intricate networks of various transcription factors including bZIP, NAC, and WRKY transcription factor families. Ethylene is a plant hormones that promotes leaf senescence. Ethylene binds to and inhibits the ethylene receptor-kinase complex, ETRs-CTR1, resulting in the activation of EIN2. Activated EIN2 stabilizes EIN3 and related EIL transcription factors (Dubois et al., 2018). This promotes leaf senescence either by activating the expression of senescence promoting other transcription factors such as ORE1 and AtNAP or by repressing miR164, which targets ORE1 (Li et al., 2013). ABA is another plant hormone that promotes leaf senescence. The core ABA signaling comprise the SnRK2s that activate group A bZIP transcription factors including ABI5, the group A PP2Cs that inhibit SnRK2s, and PYR/PYLs that bind to ABA. ABA binding to PYR/PYLs promotes the binding of PYR/PYLs to PP2Cs, inhibiting their activities. The inhibition of PP2Cs liberates SnRK2s allowing the phosphorylation and activation of the group A bZIP transcription factors that promote leaf senescence. These group A bZIPs do this by activating ORE1 and chlorophyll degradation genes (Cutler et al., 2010; Sakuraba et al., 2014). In leaf senescence induced by prolonged darkness, PIF4 and its homologs, PIF3 and PIF5, promote leaf senescence by directly activating the expression of senescence-associated downstream target genes such as
High ambient temperature may also regulate senescence. Among animals, the lifespan of
In this study, we asked whether high ambient temperature promotes leaf senescence in
For general growth and seed harvest,
To measure chlorophyll levels in senescent leaves, chlorophylls were extracted from 10 detached cotyledons or from cotyledons cut from seedlings in 100% ethanol for 1 day at 4°C in the dark. Then, the extracted chlorophyll levels were determined using a spectrophotometer (Lichtenthaler, 1987). All handling of the plates was done under a green safety light. Chlorophyll levels were normalized by the fresh weight of the cotyledons.
To inactivate Pfr before the transfer to darkness, the seedlings or detached cotyledons were irradiated with far-red light (2.5 µmol m-2 s-1) for 10 min or for the indicated times.
To measure the expression of
Total RNA was isolated from 80 seedlings using the MiniBEST Plant RNA Extract Kit (Takara, Japan). Total RNA (2 µg) was used to synthesize cDNAs using oligo (dT) 18 primers and the M-MLV reverse transcriptase (Promega, USA). Expression of specific mRNAs was determined via quantitative real-time polymerase chain reaction (PCR) with specific primer sets (Supplementary Table S1) using the CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). The expression of specific mRNAs was normalized by the expression levels of
Seven-day-old light-grown seedlings were transferred to darkness for 1 day either at 20°C or 28°C and sampled for chromatin immunoprecipitation (ChIP) assays. ChIP assays were performed as described (Oh et al., 2007). Briefly, seedlings were cross-linked with a crosslinking buffer (0.4 M Sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, 2.7% formaldehyde, 0.8% MG-132, 1 mM PMSF) for 20 min under vacuum, frozen with liquid nitrogen, and ground with a mortar and pestle. The ground samples were resuspended in nuclei isolation buffer (0.25 M Sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 0.1 mM CaCl2, 9% Triton X-100, 1 mM PMSF) and debris was removed with a miracloth. Nuclei were isolated by centrifugation (9,800
Seedlings were sampled and frozen immediately with liquid nitrogen in a dark room under a green safety light. Frozen seedlings were ground and resuspended in UREA protein extraction buffer (100 mM NaH2PO4 pH 7.8, 10 mM Tris-HCl pH 8.0, 1 mM PMSF, 8 M urea with the Calbiochem Protease Inhibitor Cocktail) and the debris was removed by centrifugation (9,800
Four-day-old light-grown
Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this paper are as follows: PHYB (AT2G18790); PIF4 (AT2G43010); PIF5 (AT3G59060); PIL1 (AT2G46970); HFR1 (AT1G02340); EIN2 (AT5G03280); EIN3 (AT3G20770); ABI5 (AT2G36270); ABA2 (AT1G52340); ORE1 (AT5G39610); NYE1 (AT4G22920); UBQ10 (AT4G05320); LRB1 (AT2G46260); LRB2 (AT3G61600); COP1 (AT2G32950); ELF3 (AT2G25930); and PP2AA3 (AT1G13320).
Leaf senescence is accelerated both by internal aging programs and environmental stresses such as darkness, drought, high salinity, and pathogen attacks (Lim et al., 2007). High ambient temperature, because it affects so many plant processes (e.g., seed germination, stem elongation, and flowering) may be another critical environmental factor that accelerates leaf senescence.
We examined leaf senescence by transferring light-grown seedlings or detached leaves to a dark condition at either 20°C or 28°C. Leaf senescence is marked by a gradual change of leaf color from green to yellow—due to reduced chlorophyll—and increased expression of senescence marker genes such as
Since PIF4 and PIF5 promote senescence at ambient temperature, we asked whether PIF4 and PIF5 also accelerate leaf senescence at high ambient temperatures. We found the
PIF4 accelerates senescence both by activating the expression of
We next asked how high ambient temperature regulates PIF4 by measuring
Since phyB suppresses senescence by inhibiting PIF4 (Sakuraba et al., 2014), we asked which step of
We found
Since COP1 and ELF3 regulate
Since leaf senescence occurs relatively slowly after the transfer to darkness, we were curious to see whether Pfr generated before the transfer to darkness remains active to suppress senescence after the transfer to darkness. We therefore investigated the role of preexisting Pfr by inactivating Pfr with a pre-FRp.
Wild type seedlings senesce faster at 20°C after exposure to a pre-FRp (Fig. 6A). Previous studies have shown PIF4/5 promote senescence downstream of phyB (Sakuraba et al., 2014; Song et al., 2014). Consistent with these results, not only did we find
Alternatively, it is possible pre-FRp exposure does not accelerate senescence at 28°C, not because high ambient temperature accelerates the thermal reversion of Pfr to Pr to levels below an activity threshold, but because Pfr cannot suppress senescence at high ambient temperature. If this is the case, pre-FRp exposure should not accelerate senescence even in seedlings with high levels of Pfr. We therefore examined senescence in
The suppression of senescence by pre-existing Pfr raises the question of how long Pfr remains active after the transfer to darkness. Since the number and size of phyB photobodies—distinct fluorescent dots in the nucleus—is correlated with phyB activity (Chen et al., 2003; Hahm et al., 2020), we examined how long phyB photobodies last after the transfer to darkness. As reported before, PhyB-GFP forms bright photobodies in the light (Fig. 7A). In the epidermis, the nuclear fluorescent signals grow more diffuse with fewer and weaker photobodies already at 12 h after the transfer to darkness. At 28°C, the effect is stronger, with more diffuse nuclear fluorescent signal and even fewer photobodies (Fig. 7B). One day after the transfer to darkness, nuclear fluorescent signals are even further reduced with virtually no discernible photobodies at either 20°C or 28°C. These results indicate phyB-GFP photobodies disappear quickly after the transfer to darkness at both 20°C than at 28°C, but lasting slightly longer at 20°C.
Although photobodies disappear quickly, diffuse GFP signal is still present in the nucleus 24 h after the transfer to darkness at both 20°C and 28°C (Fig. 7A). This suggests that a small portion of phyB may still be active. To more sensitively gauge the presence of active Pfr, we exposed seedlings to an FRp in the middle of the dark period (mid-FRp) at various times after the transfer to darkness and measured the expression of PIF target gene mRNAs (Fig. 7C). If Pfr is present at the time of the mid-FRp, then the mid-FRp should convert the remaining Pfr to Pr, releasing PIFs to activate the expression of PIF target gene mRNAs. We performed these experiments with
PIF4 promotes leaf senescence in a prolonged dark condition, whereas phyB suppresses senescence by inhibiting PIF4 (Sakuraba et al., 2014; Song et al., 2014). In this study, we show high ambient temperature accelerates leaf senescence in the dark by increasing
Our data indicate some Pfr remains active relatively long after the transfer to darkness. We demonstrated this by measuring the induction of PIF4 target gene expression by exposure to a mid-FRp even 4 days after the transfer to darkness at 20°C (Fig. 7D). Direct measurements of different absorption spectra in vitro, in yeast, and in plants indicate that recombinant phytochromes undergo an initial rapid thermal reversion of Pfr-Pr heterodimers followed by a slower thermal reversion of Pfr-Pfr homodimers (Ádám et al., 2011; Eichenberg et al., 1999; 2000; Jung et al., 2016; Klose et al., 2015; Kunkel et al., 1995; Legris et al., 2016; Remberg et al., 1998; Sweere et al., 2001). In plants, the Pfr form of phyB also undergoes thermal reversion with a half-life of about 60 min at 25°C, but some Pfr remains even after 4 h in darkness. In fluorescent image analyses of phyB-GFP, although phyB-GFP photobodies disappear within 16 to 18 h in darkness, there is residual diffuse nuclear phyB-GFP signal even 24 h after the transfer to darkness (Huang et al., 2016; Van Buskirk et al., 2014). This suggests a portion of the phyB-GFP remains in the Pfr form even after 24 h of darkness. As we further probed biologically active Pfr by measuring the expression of FRp-inducible PIF target genes, we confirmed that Pfr remains active even 4 days after the transfer to darkness at ambient temperature (Fig. 7D). This relatively long survival of Pfr after the transfer to darkness is also evidenced by the destabilization of PIF4 protein 1 day after the transfer of
Our data show phyB acts not only as a photoreceptor but also as a thermosensor that regulates leaf senescence in prolonged darkness. PhyB’s ability to sense temperature is likely due to a temperature-dependent increase in the rate of thermal reversion of the Pfr form to the Pr form. This was manifested in the reduced size of phyB-GFP photobodies and in direct absorbance measurements of Pfr level at high ambient temperature in light-grown seedlings (Jung et al., 2016; Legris et al., 2016). We found, however, that phyB photobodies disassemble quickly after the transfer to darkness, making it difficult in our experimental conditions to determine whether Pfr lasts long enough to suppress senescence after the transfer to darkness (Fig. 7A). To detect the presence of biologically active Pfr, we measured the induction of PIF4 target gene mRNAs by an FRp given in the middle of the period of darkness (mid-FRp) (Fig. 7C). Such a mid-FRp should revert all the Pfr present at the time of irradiation to Pr, releasing PIF4 to activate the expression of its target genes. We found a mid-FRp increases PIF4 target gene mRNAs even if given 4 days after the transfer to darkness at 20°C. At 28°C, however, a mid-FRp given 3-4 days after the transfer to darkness does not affect PIF4 target gene mRNAs (Fig. 7D). These results indicate high ambient temperature accelerates the inactivation of Pfr, making phytochromes less active and therefore less able to suppress senescence.
The phytochromes, however, are not the only thermosensors regulating leaf senescence via PIF4 as high ambient temperature increases
High ambient temperature increases PIF4 protein levels both phy-dependently and phy-independently. Since
Currently, no other thermosensors are known to regulate senescence via PIF4. Previous studies have shown that other plant photoreceptors, including phototropin (phot), cryptochrome (cry: cry1 and cry2), and UVR8, also undergo thermal reversion from an active state to an inactive state, making them good thermosensor candidates (Findlay and Jenkins, 2016; Fujii et al., 2017; Herbel et al., 2013). However, phot, cry, and UVR8 are unlikely to be the thermosensor that regulates senescence via PIF4 in prolonged darkness. First, phot has a short half-life (~30 s at 22°C), making it difficult to function in a prolonged darkness (Christie, 2007). Phot also does not regulate
ELF3 and COP1 could be closely associated with a thermosensor regulating high temperature-induced
This work was supported by the National Research Foundation of Korea (NRF-2018R1A3B1052617).
C.K., P.O.L., Y.I.P., and G.C. designed the experiments. C.K., S.J.K., J.J., E.P., and E.O. performed the experiments. C.K., E.O., P.O.L., and G.C. wrote the manuscript. All authors discussed the results and reviewed the manuscript.
The authors have no potential conflicts of interest to disclose.
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