HYPOCOTYL IN FAR-RED Research Articles

Organ-specific transcriptional regulation by HFR1 and HY5 in response to shade in Arabidopsis.

Light is crucial for plants and serves as a signal for modulating their growth. Under shade, where red to far-red light ratio is low, plants exhibit shade avoidance responses (SAR). LONG HYPOCOTYL IN FAR-RED 1 (HFR1) and ELONGATED HYPOCOTYL 5 (HY5) are known to be negative regulators of SAR and physically interact with one another. However, transcriptional regulatory network underlying SAR by these two transcription factors has not been explored. Here, we performed organ-specific transcriptome analyses of Arabidopsis thaliana hfr1-5, hy5-215 and hfr1hy5 to identify genes that are co-regulated by HFR1 and HY5 in hypocotyls and cotyledons. Genes co-regulated by HFR1 and HY5 were enriched in various processes related to cell wall modification and chlorophyll biosynthesis in hypocotyls. Phytohormone (abscisic acid and jasmonic acid) and light responses were significantly regulated by HFR1 and HY5 in both organs, though it is more prominent under shade in cotyledons. HFR1 and HY5 also differentially regulate the expression of the cell wall-related genes for xyloglucan endotransglucosylase/hydrolase, expansin, arabinogalactan protein and class III peroxidase depending on the organs. Furthermore, HFR1 and HY5 cooperatively regulated hypocotyl responsiveness to shade through auxin metabolism. Together, our study illustrates the importance of the HFR1-HY5 module in regulating organ-specific shade responses in Arabidopsis.

LONG HYPOCOTYL IN FAR-RED 1 mediates a trade-off between growth and defense under shade in Arabidopsis.

Plants respond to vegetative shade with developmental and physiological changes that is collectively known as shade avoidance syndrome (SAS). Although LONG HYPOCOTYL IN FAR-RED 1 (HFR1) is known to be a negative regulator of SAS by forming heterodimers with other basic helix-loop-helix (bHLH) transcription factors to inhibit them, its function in genome-wide transcriptional regulation is not fully elucidated. Here, we performed RNA-sequencing analyses of hfr1-5 and HFR1 overexpression line (HFR1(ΔN)-OE) to comprehensively identify HFR1-regulated genes at different time points of shade treatment. We found that HFR1 mediates the trade-off between shade-induced growth and shade-repressed defense, by regulating the expression of relevant genes in shade. Genes involved in promoting growth, such as for auxin biosynthesis, transport, signaling and response were induced by shade but suppressed by HFR1 at both short and long durations of shade. Likewise, most ethylene-related genes were shade-induced and HFR1-repressed. On the other hand, shade suppressed defense-related genes while HFR1 induced their expression, especially under long duration of shade treatment. We demonstrated that HFR1 confers increased resistance to bacterial infection under shade.

HFR1, a bHLH Transcriptional Regulator from Arabidopsis thaliana, Improves Grain Yield, Shade and Osmotic Stress Tolerances in Common Wheat.

Common wheat, Triticum aestivum, is the most widely grown staple crop worldwide. To catch up with the increasing global population and cope with the changing climate, it is valuable to breed wheat cultivars that are tolerant to abiotic or shade stresses for density farming. Arabidopsis LONG HYPOCOTYL IN FAR-RED 1 (AtHFR1), a photomorphogenesis-promoting factor, is involved in multiple light-related signaling pathways and inhibits seedling etiolation and shade avoidance. We report that overexpression of AtHFR1 in wheat inhibits etiolation phenotypes under various light and shade conditions, leading to shortened plant height and increased spike number relative to non-transgenic plants in the field. Ectopic expression of AtHFR1 in wheat increases the transcript levels of TaCAB and TaCHS as observed previously in Arabidopsis, indicating that the AtHFR1 transgene can activate the light signal transduction pathway in wheat. AtHFR1 transgenic seedlings significantly exhibit tolerance to osmotic stress during seed germination compared to non-transgenic wheat. The AtHFR1 transgene represses transcription of TaFT1, TaCO1, and TaCO2, delaying development of the shoot apex and heading in wheat. Furthermore, the AtHFR1 transgene in wheat inhibits transcript levels of PHYTOCHROME-INTERACTING FACTOR 3-LIKEs (TaPIL13, TaPIL15-1B, and TaPIL15-1D), downregulating the target gene STAYGREEN (TaSGR), and thus delaying dark-induced leaf senescence. In the field, grain yields of three AtHFR1 transgenic lines were 18.2–48.1% higher than those of non-transgenic wheat. In summary, genetic modification of light signaling pathways using a photomorphogenesis-promoting factor has positive effects on grain yield due to changes in plant architecture and resource allocation and enhances tolerances to osmotic stress and shade avoidance response.

Unraveling the Dynamic Integration of Auxin, Brassinosteroid and Gibberellin in Early Shade-Induced Hypocotyl Elongation.

The online version contains supplementary material available at 10.1007/s43657-022-00044-3.

Comparative phenotypic and transcriptomic analyses unravel conserved and distinct mechanisms underlying shade avoidance syndrome in Brassicaceae vegetables

BackgroundPlants grown under shade are exposed to low red/far-red ratio, thereby triggering an array of altered phenotypes called shade avoidance syndrome (SAS). Shade negatively influences plant growth, leading to a reduction in agricultural productivity. Understanding of SAS is crucial for sustainable agricultural practices, especially for high-density indoor farming. Brassicaceae vegetables are widely consumed around the world and are commonly cultivated in indoor farms. However, our understanding of SAS in Brassicaceae vegetables and their genome-wide transcriptional regulatory networks are still largely unexplored.ResultsShade induced common signs of SAS, including hypocotyl elongation and reduced carotenoids/anthocyanins biosynthesis, in two different Brassicaceae species: Brassica rapa (Choy Sum and Pak Choy) and Brassica oleracea (Kai Lan). Phenotype-assisted transcriptome analysis identified a set of genes induced by shade in these species, many of which were related to auxin biosynthesis and signaling [e.g. YUCCA8 (YUC8), YUC9, and INDOLE-3-ACETIC ACID INDUCIBLE (IAAs)] and other phytohormones signaling pathways including brassinosteroids and ethylene. The genes functioning in plant defense (e.g. MYB29 and JASMONATE-ZIM-DOMAIN PROTEIN 9) as well as in biosynthesis of anthocyanins and glucosinolates were repressed upon shade. Besides, each species also exhibited distinct SAS phenotypes. Shade strongly reduced primary roots and elongated petioles of B. oleracea, Kai Lan. However, these SAS phenotypes were not clearly recognized in B. rapa, Choy Sum and Pak Choy. Some auxin signaling genes (e.g. AUXIN RESPONSE FACTOR 19, IAA10, and IAA20) were specifically induced in B. oleracea, while homologs in B. rapa were not up-regulated under shade. Contrastingly, shade-exposed B. rapa vegetables triggered the ethylene signaling pathway earlier than B. oleracea, Kai Lan. Interestingly, shade induced the transcript levels of LONG HYPOCOTYL IN FAR-RED 1 (HFR1) homolog in only Pak Choy as B. rapa. As HFR1 is a key negative regulator of SAS in Arabidopsis, our finding suggests that Pak Choy HFR1 homolog may also function in conferring higher shade tolerance in this variety.ConclusionsOur study shows that two Brassicaceae species not only share a conserved SAS mechanism but also exhibit distinct responses to shade, which will provide comprehensive information to develop new shade-tolerant cultivars that are suitable for high-density indoor farms.

The CIB1 transcription factor regulates light- and heat-inducible cell elongation via a two-step HLH/bHLH system.

Light and high temperature promote plant cell elongation. PHYTOCHROME INTERACTING FACTOR4 (PIF4, a typical basic helix-loop-helix [bHLH] transcriptional activator) and the non-DNA binding atypical HLH inhibitors PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) competitively regulate cell elongation in response to light conditions and high temperature. However, the underlying mechanisms have not been fully clarified. Here, we show that in Arabidopsis thaliana, the bHLH transcription factor CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) positively regulates cell elongation under the control of PIF4, PAR1, and HFR1. Furthermore, PIF4 directly regulates CIB1 expression by interacting with its promoter, and PAR1 and HFR1 interfere with PIF4 binding to the CIB1 promoter. CIB1 activates genes that function in cell elongation, and PAR1 interferes with the DNA binding activity of CIB1, thus suppressing cell elongation. Hence, two antagonistic HLH/bHLH systems, the PIF4-PAR1/HFR1 and CIB1-PAR1 systems, regulate cell elongation in response to light and high temperature. We thus demonstrate the important role of non-DNA binding small HLH proteins in the transcriptional regulation of cell elongation in plants.

A Gas-and-Brake Mechanism of bHLH Proteins Modulates Shade Avoidance.

Plants detect proximity of competitors through reduction in the ratio between red and far-red light that triggers the shade avoidance syndrome, inducing responses such as accelerated shoot elongation and early flowering. Shade avoidance is regulated by PHYTOCHROME INTERACTING FACTORs, a group of basic helix-loop-helix (bHLH) transcription factors. Another (b)HLH protein, KIDARI (KDR), which is non-DNA-binding, was identified in de-etiolation studies and proposed to interact with LONG HYPOCOTYL IN FAR-RED1 (HFR1), a (b)HLH protein that inhibits shade avoidance. Here, we established roles of KDR in regulating shade avoidance in Arabidopsis (Arabidopsis thaliana) and investigated how KDR regulates the shade avoidance network. We showed that KDR is a positive regulator of shade avoidance and interacts with several negative growth regulators. We identified KDR interactors using a combination of yeast two-hybrid screening and dedicated confirmations with bimolecular fluorescence complementation. We demonstrated that KDR is translocated primarily to the nucleus when coexpressed with these interactors. A genetic approach confirmed that several of these interactions play a functional role in shade avoidance; however, we propose that KDR does not interact with HFR1 to regulate shade avoidance. Based on these observations, we propose that shade avoidance is regulated by a three-layered gas-and-brake mechanism of bHLH protein interactions, adding a layer of complexity to what was previously known.

Genetic Interaction Among Phytochrome, Ethylene and Abscisic Acid Signaling During Dark-Induced Senescence in Arabidopsis thaliana.

Leaf senescence is induced by various internal and external stimuli. Dark-induced senescence has been extensively investigated, but the detailed mechanism underlying it is not well understood. The red light/far-red light receptor phytochrome B and its downstream transcription factors, PYHTOCHROME INTERACTING FACTORs (PIFs) 4 and 5, are known to play an important role in dark-induced senescence. Furthermore, the senescence-inducing phytohormones, ethylene and abscisic acid (ABA) are reported to be involved in dark-induced senescence. In this study, we analyzed the relationship between ethylene, ABA and PIFs in dark-induced leaf senescence. A triple mutant of the core ABA signaling components; SNF1-related protein kinases 2D (SRK2D), SRK2E, and SRK2I, displayed an ABA insensitive phenotype in ABA-induced senescence, whilst the ethylene insensitive mutant ein2 demonstrated low sensitivity to ABA, suggesting that ethylene signaling is involved in ABA-induced senescence. However, the pif4 pif5 mutant did not display low sensitivity to ABA, suggesting that PIF4 and PIF5 act upstream of ABA signaling. Although PIF4 and PIF5 reportedly regulate ethylene production, the triple mutant ein2 pif4 pif5 showed a stronger delayed senescence phenotype than ein2 or pif4 pif5, suggesting that EIN2 and PIF4/PIF5 partially regulate leaf senescence independently of each other. While direct target genes for PIF4 and PIF5, such as LONG HYPOCOTYL IN FAR-RED1 (HFR1) and PHYTOCHROME INTERACTING FACTOR 3-LIKE 1 (PIL1), showed transient upregulation under dark conditions (as is seen in the shade avoidance response), expression of STAY GREEN1 (SGR1), ORESARA1 (ORE1) and other direct target genes of PIF5, continued to increase during dark incubation. It is possible that transcription factors other than PIF4 and PIF5 are involved in the upregulation of SGR1 and ORE1 at a later stage of dark-induced senescence. Possible candidates are senescence-induced senescence regulators (SIRs), which include the NAC transcription factors ORE1 and AtNAP. In fact, ORE1 is known to bind to the SGR1 promoter and promotes its expression. It is therefore inferred that the phytochrome-PIF pathway regulates initial activation of senescence and subsequently, induced SIRs reinforce leaf senescence during dark-induced senescence.

UVR8-mediated inhibition of shade avoidance involves HFR1 stabilization in Arabidopsis.

Sun-loving plants perceive the proximity of potential light-competing neighboring plants as a reduction in the red:far-red ratio (R:FR), which elicits a suite of responses called the "shade avoidance syndrome" (SAS). Changes in R:FR are primarily perceived by phytochrome B (phyB), whereas UV-B perceived by UV RESISTANCE LOCUS 8 (UVR8) elicits opposing responses to provide a counterbalance to SAS, including reduced shade-induced hypocotyl and petiole elongation. Here we show at the genome-wide level that UVR8 broadly suppresses shade-induced gene expression. A subset of this gene regulation is dependent on the UVR8-stabilized atypical bHLH transcription regulator LONG HYPOCOTYL IN FAR-RED 1 (HFR1), which functions in part redundantly with PHYTOCHROME INTERACTING FACTOR 3-LIKE 1 (PIL1). In parallel, UVR8 signaling decreases protein levels of the key positive regulators of SAS, namely the bHLH transcription factors PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5, in a COP1-dependent but HFR1-independent manner. We propose that UV-B antagonizes SAS via two mechanisms: degradation of PIF4 and PIF5, and HFR1- and PIL1-mediated inhibition of PIF4 and PIF5 function. This work highlights the importance of typical and atypical bHLH transcription regulators for the integration of light signals from different photoreceptors and provides further mechanistic insight into the crosstalk of UVR8 signaling and SAS.

Genomic evidence reveals SPA-regulated developmental and metabolic pathways in dark-grown Arabidopsis seedlings.

Photomorphogenesis is repressed in the dark mainly by an E3 ubiquitin ligase complex comprising CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and four homologous proteins called SUPPRESSOR OF PHYA-105 (SPA1-SPA4) in Arabidopsis. This complex induces the ubiquitination and subsequent degradation of positively acting transcription factors (TFs; e.g. ELONGATED HYPOCOTYL (HY5), LONG HYPOCOTYL IN FAR-RED 1 (HFR1), PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) and others] in the dark to repress photomorphogenesis. Genomic evidence showed a large number of genes regulated by COP1 in the dark, of which many are direct targets of HY5. However, the genomic basis for the constitute photomorphogenic phenotype of spaQ remains unknown. Here, we show that >7200 genes are differentially expressed in the spaQ background compared to wild-type in the dark. Comparison of the RNA sequencing (RNA-Seq) data between cop1 and spaQ revealed a large overlapping set of genes regulated by the COP1-SPA complex. In addition, many of the genes coordinately regulated by the COP1-SPA complex are also regulated by HY5 directly and indirectly. Taken together, our data reveal that SPA proteins repress photomorphogenesis by controlling gene expression in concert with COP1, likely through regulating the abundance of downstream TFs in light signaling pathways. Moreover, SPA proteins may function both in a COP1-dependent and -independent manner in regulating many biological processes and developmental pathways in Arabidopsis.

A PIF7-CONSTANS-Centered Molecular Regulatory Network Underlying Shade-Accelerated Flowering

To compete with their neighbors for light and escape shaded environments, sun-loving plants have developed the shade-avoidance syndrome (SAS), a set of responses including alteration of plant architecture and initiation of early flowering and seed set. Previous studies on SAS mainly focused on dissecting molecular basis of hypocotyl elongation in seedlings under shade light; however, the molecular mechanisms underlying shade-accelerated flowering in adult plants remain unknown. In this study, we found that CONSTANS (CO) and PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) have an additive effect on shade-induced flowering, but that LONG HYPOCOTYL IN FAR-RED1 (HFR1) represses early flowering by binding to CO and PIF7 and preventing the binding of CO to the promoter of FLOWERING LOCUS T (FT) and the binding of PIF7 to the promoter of pri-MIR156E/F. Under shade, de-phosphorylated PIF7 and accumulated CO, balanced by HFR1, upregulate the expression of FT, TSF, SOC1, and SPLs to accelerate flowering. Moreover, we found that the function of PIF7 in flowering time is independent of phyA. Collectively, these regulatory interactions establish a crucial link between the light signal and genetic network that regulates flowering transition under shade.

Arabidopsis histone deacetylase HDA15 directly represses plant response to elevated ambient temperature.

Elevated ambient temperatures affect plant growth and substantially impact biomass and crop yield. Recent results have indicated that chromatin remodelling is critical in plant thermal responses but howhistone modification dynamics affectsplant thermal response has not been clearly demonstarted. Here we show that Arabidopsis histone deacetylase genes HDA9, HDA15 and HDA19 play distinct roles in plant response to elevated ambient temperature. hda9 and hda19 mutants showed a warm-temperature-insensitive phenotype at 27°C, whereas hda15 plants displayed a constitutive warm-temperature-induced phenotype at 20°C and an enhanced thermal response at 27°C. The hda19 mutation led to upregulation of genes mostly related to stress response at both 20 and 27°C. The hda15 mutation resulted in upregulation of many warm temperature-responsive as well as metabolic genes at 20 and 27°C, while hda9 led to differential expression of a large number of genes at 20°C and impaired induction of warm-temperature-responsive genes at 27°C. HDA15 is associated with thermosensory mark genes at 20°C and that the association is decreased after shifting to 27°C, indicating that HDA15 is a direct repressor of plant thermal-responsive genes at normal temperature. In addition, as hda9, the hda15 mutation also led to upregulation of many metabolic genes and accumulation of primary metabolites. Furthermore, we show that HDA15 interacts with the transcription factor HFR1 (long Hypocotyl in Far Red1) to cooperatively repress warm-temperature response. Our study demonstrates that the histone deacetylases target to different sets of genes and play distinct roles in plant response to elevated ambient temperature.

KAI2-KL signaling intersects with light-signaling for photomorphogenesis

ABSTRACTLight is an important environmental cue, causing a high degree of developmental plasticity in higher plants. The outcome of light-regulated developmental response is determined by not only photo-sensory systems but also endogenous physiological contexts in plants. KARRIKIN-INSENSITIVE2 (KAI2) functions as a receptor of karrikin and endogenous, as yet to be identified, KAI2 ligand (KL). The loss-of-function of KAI2 caused light-hyposensitive photomorphogenesis, affecting the expression light-responsive genes under the light conditions. However, it remains still unclear how KAI2-KL signaling interacts with light-signaling. Here, we show that the ply2 mutation, a severe loss-of-function allele of KAI2 affected the expression of a subset of light-responsive genes, irrespectively of light condition. The results implied that the overlapping set of light- and KAI2-responsive genes may serve as an integrating node between light- and KAI2-KL signaling. Further, the results of double mutant analyses between the ply2 mutant and mutants of CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) or LONG HYPOCOTYL IN FAR-RED (HFR1) implicated that KAI2-KL signaling acts at downstream of COP1, largely independently of HFR1. Together, these results suggest that KAI2-KL signaling intersects with a subset of the light-regulatory network, by which plants adjust their photomorphogenic development.

Nitric oxide promotes light-initiated seed germination by repressing PIF1 expression and stabilizing HFR1

Seed germination is a crucial stage in the life cycle of plants and is tightly controlled by internal and external signals. Phytochrome photoreceptors perceive light stimulation to promote seed germination. Previous studies have shown that PHYTOCHROME-INTERACTION FACTOR 1 (PIF1) is a negative regulatory factor and represses seed germination, while LONG HYPOCOTYL IN FAR-RED (HFR1) sequesters PIF1 by forming a heterodimer to relieve the inhibitory effect of seed germination during the initial phase. Nitric oxide (NO) has been reported to break seed dormancy, but the underlying mechanism is not well understood. Here, we report that NO signal enhances phytochrome B (PHYB)-dependent seed germination, and PHYB perceives red light stimulation to activate NR activity and NO accumulation. NO signal not only downregulates the transcription of PIF1, but also stabilize HFR1 proteins to intensify the interaction of the HFR1-PIF1 heterodimer, and compensate for the inhibitory effect of PIF1 on its target genes associated with hormone metabolism and cell wall loosening, consequently initiating seed germination. Thus, our results reveal a new mechanism for NO signals in modulating PHYB-mediated seed germination by repressing PIF1 expression at the transcriptional level as well as preventing PIF1 activity by stabilizing HFR1 protein.

Arabidopsis RSS1 Mediates Cross-Talk Between Glucose and Light Signaling During Hypocotyl Elongation Growth

Plants possess exuberant plasticity that facilitates its ability to adapt and survive under challenging environmental conditions. The developmental plasticity largely depends upon cellular elongation which is governed by a complex network of environmental and phytohormonal signals. Here, we report role of glucose (Glc) and Glc-regulated factors in controlling elongation growth and shade response in Arabidopsis. Glc controls shade induced hypocotyl elongation in a dose dependent manner. We have identified a Glc repressed factor REGULATED BY SUGAR AND SHADE1 (RSS1) encoding for an atypical basic helix-loop-helix (bHLH) protein of unknown biological function that is required for normal Glc actions. Phenotype analysis of mutant and overexpression lines suggested RSS1 to be a negative regulator of elongation growth. RSS1 affects overall auxin homeostasis. RSS1 interacts with the elongation growth-promoting proteins HOMOLOG OF BEE2 INTERACTING WITH IBH 1 (HBI1) and BR ENHANCED EXPRESSION2 (BEE2) and negatively affects the transcription of their downstream targets such as YUCs, INDOLE-3-ACETIC ACID INDUCIBLE (IAAs), LONG HYPOCOTYL IN FAR-RED1 (HFR1), HOMEOBOX PROTEIN 2 (ATHB2), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASES (XTHs) and EXPANSINS. We propose, Glc signals might maintain optimal hypocotyl elongation under multiple signals such as light, shade and phytohormones through the central growth regulatory bHLH/HLH module.

UV-B Perceived by the UVR8 Photoreceptor Inhibits Plant Thermomorphogenesis

SummarySmall increases in ambient temperature can elicit striking effects on plant architecture, collectively termed thermomorphogenesis [1]. In Arabidopsis thaliana, these include marked stem elongation and leaf elevation, responses that have been predicted to enhance leaf cooling [2, 3, 4, 5]. Thermomorphogenesis requires increased auxin biosynthesis, mediated by the bHLH transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) [6, 7, 8], and enhanced stability of the auxin co-receptor TIR1, involving HEAT SHOCK PROTEIN 90 (HSP90) [9]. High-temperature-mediated hypocotyl elongation additionally involves localized changes in auxin metabolism, mediated by the indole-3-acetic acid (IAA)-amido synthetase Gretchen Hagen 3 (GH3).17 [10]. Here we show that ultraviolet-B light (UV-B) perceived by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8) [11] strongly attenuates thermomorphogenesis via multiple mechanisms inhibiting PIF4 activity. Suppression of thermomorphogenesis involves UVR8 and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)-mediated repression of PIF4 transcript accumulation, reducing PIF4 abundance. UV-B also stabilizes the bHLH protein LONG HYPOCOTYL IN FAR RED (HFR1), which can bind to and inhibit PIF4 function. Collectively, our results demonstrate complex crosstalk between UV-B and high-temperature signaling. As plants grown in sunlight would most likely experience concomitant elevations in UV-B and ambient temperature, elucidating how these pathways are integrated is of key importance to the understanding of plant development in natural environments.

Convergence of CONSTITUTIVE PHOTOMORPHOGENESIS 1 and PHYTOCHROME INTERACTING FACTOR signalling during shade avoidance.

Shade-avoidance responses require CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) but the mechanisms of action of COP1 under shade have not been elucidated. Using simulated shade and control conditions, we analysed: the transcriptome and the auxin levels of cop1 and phytochrome interacting factor 1 (pif1) pif3 pif4 pif5 (pifq) mutants; the dynamics of ELONGATED HYPOCOTYL 5 (HY5) and LONG HYPOCOTYL IN FAR-RED (HFR1) proteins; and the epistatic relationships between cop1 and pif3, pif4, pif5, hy5 and hfr1 mutations in Arabidopsis thaliana. Despite severely impaired shade-avoidance responses, only a few genes that responded to shade in the wild-type failed to do so in cop1. Shade enhanced the convergence between cop1 and pifq transcriptomes, mainly on shade-avoidance marker genes. Shade failed to increase auxin levels in cop1. Residual shade avoidance in cop1 was not further reduced by the pif3, pif4 or pif5 mutations, suggesting convergent pathways. HFR1 stability decreased under shade in a COP1-dependent manner but shade increased HY5 stability. The cop1 mutant retains responses to shade and is more specifically impaired in shade avoidance. COP1 promotes the degradation of HFR1 under shade, thus increasing the ability of PIFs to control gene expression, increase auxin levels and promote stem growth.

Regulation of Carotenoid Biosynthesis by Shade Relies on Specific Subsets of Antagonistic Transcription Factors and Cofactors.

Carotenoids are photosynthetic pigments essential for the protection against excess light. During deetiolation, their production is regulated by a dynamic repression-activation module formed by PHYTOCHROME-INTERACTING FACTOR1 (PIF1) and LONG HYPOCOTYL5 (HY5). These transcription factors directly and oppositely control the expression of the gene encoding PHYTOENE SYNTHASE (PSY), the first and main rate-determining enzyme of the carotenoid pathway. Antagonistic modules also regulate the responses of deetiolated plants to vegetation proximity and shade (i.e. to the perception of far-red light-enriched light filtered through or reflected from neighboring plants). These responses, aimed to adapt to eventual shading from plant competitors, include a reduced accumulation of carotenoids. Here, we show that PIF1 and related photolabile PIFs (but not photostable PIF7) promote the shade-triggered decrease in carotenoid accumulation. While HY5 does not appear to be required for this process, other known PIF antagonists were found to modulate the expression of the Arabidopsis (Arabidopsis thaliana) PSY gene and the biosynthesis of carotenoids early after exposure to shade. In particular, PHYTOCHROME-RAPIDLY REGULATED1, a transcriptional cofactor that prevents the binding of true transcription factors to their target promoters, was found to interact with PIF1 and hence directly induce PSY expression. By contrast, a change in the levels of the transcriptional cofactor LONG HYPOCOTYL IN FAR RED1, which also binds to PIF1 and other PIFs to regulate shade-related elongation responses, did not impact PSY expression or carotenoid accumulation. Our data suggest that the fine-regulation of carotenoid biosynthesis in response to shade relies on specific modules of antagonistic transcriptional factors and cofactors.

Arabidopsis DET1 degrades HFR1 but stabilizes PIF1 to precisely regulate seed germination

Seed is an essential propagation organ and a critical strategy adopted by terrestrial flowering plants to colonize the land. The ability of seeds to accurately respond to light is vital for plant survival. However, the underlying mechanism is largely unknown. In this study, we reveal a circuit of triple feed-forward loops adopted by Arabidopsis seeds to exclusively repress germination in dark conditions and precisely initiate germination under diverse light conditions. We identify that de-etiolated 1 (DET1), an evolutionarily conserved protein, is a central repressor of light-induced seed germination. Genetic analysis demonstrates that DET1 functions upstream of long hypocotyl in far-red 1 (HFR1) and phytochrome interacting factor 1 (PIF1), the key positive and negative transcription regulators in seed germination. We further find that DET1 and constitutive photomorphogenic 10 (COP10) target HFR1 for protein degradation by assembling a COP10-DET1-damaged DNA binding protein 1-cullin4 E3 ligase complex. Moreover, DET1 and COP10 directly interact with and promote the protein stability of PIF1. Computational modeling reveals that phytochrome B (phyB)-DET1-HFR1-PIF1 and phyB-DET1-Protease-PIF1 are new signaling pathways, independent of the previously identified phyB-PIF1 pathway, respectively mediating the rapid and time-lapse responses to light irradiation. The model-simulated results are highly consistent with their experimental validations, suggesting that our mathematical model captures the essence of Arabidopsis seed germination networks. Taken together, this study provides a comprehensive molecular framework for light-regulated seed germination, improving our understanding of how plants respond to changeable environments.

14-3-3 proteins participate in light signaling through association with PHYTOCHROME INTERACTING FACTORs.

14-3-3 proteins are regulatory proteins found in all eukaryotes and are known to selectively interact with phosphorylated proteins to regulate physiological processes. Through an affinity purification screening, many light-related proteins were recovered as 14-3-3 candidate binding partners. Yeast two-hybrid analysis revealed that the 14-3-3 kappa isoform (14-3-3κ) could bind to PHYTOCHROME INTERACTING FACTOR3 (PIF3) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). Further analysis by in vitro pull-down assay confirmed the interaction between 14-3-3κ and PIF3. Interruption of putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 was not sufficient to inhibit 14-3-3κ from binding or to disturb nuclear localization of PIF3. It was also indicated that 14-3-3κ could bind to other members of the PIF family, such as PIF1 and PIF6, but not to LONG HYPOCOTYL IN FAR-RED1 (HFR1). 14-3-3 mutants, as well as the PIF3 overexpressor, displayed longer hypocotyls, and a pif3 mutant displayed shorter hypocotyls than the wild-type in red light, suggesting that 14-3-3 proteins are positive regulators of photomorphogenesis and function antagonistically with PIF3. Consequently, our results indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to a light signal.