Phytochrome-mediated Light Signaling Research Articles

Exciting new discoveries in phytochrome-mediated light signaling pathways

Exciting new discoveries in phytochrome-mediated light signaling pathways

Regulation of Plant Photoresponses by Protein Kinase Activity of Phytochrome A.

Extensive research has been conducted for decades to elucidate the molecular and regulatory mechanisms for phytochrome-mediated light signaling in plants. As a result, tens of downstream signaling components that physically interact with phytochromes are identified, among which negative transcription factors for photomorphogenesis, PHYTOCHROME-INTERACTING FACTORs (PIFs), are well known to be regulated by phytochromes. In addition, phytochromes are also shown to inactivate an important E3 ligase complex consisting of CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and SUPPRESSORs OF phyA-105 (SPAs). This inactivation induces the accumulation of positive transcription factors for plant photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5). Although many downstream components of phytochrome signaling have been studied thus far, it is not fully elucidated which intrinsic activity of phytochromes is necessary for the regulation of these components. It should be noted that phytochromes are autophosphorylating protein kinases. Recently, the protein kinase activity of phytochrome A (phyA) has shown to be important for its function in plant light signaling using Avena sativa phyA mutants with reduced or increased kinase activity. In this review, we highlight the function of phyA as a protein kinase to explain the regulation of plant photoresponses by phyA.

Phytochrome Signaling Networks.

The perception of light signals by the phytochrome family of photoreceptors has a crucial influence on almost all aspects of growth and development throughout a plant's life cycle. The holistic regulatory networks orchestrated by phytochromes, including conformational switching, subcellular localization, direct protein-protein interactions, transcriptional and posttranscriptional regulations, and translational and posttranslational controls to promote photomorphogenesis, are highly coordinated and regulated at multiple levels. During the past decade, advances using innovative approaches have substantially broadened our understanding of the sophisticated mechanisms underlying the phytochrome-mediated light signaling pathways. This review discusses and summarizes these discoveries of the role of the modular structure of phytochromes, phytochrome-interacting proteins, and their functions; the reciprocal modulation of both positive and negative regulators in phytochrome signaling; the regulatory roles of phytochromes in transcriptional activities, alternative splicing, and translational regulation; and the kinases and E3 ligases that modulate PHYTOCHROME INTERACTING FACTORs to optimize photomorphogenesis.

Generation and Characterization of a Specific Polyclonal Antibody against Arabidopsis thaliana Phytochrome-Interacting Factor 3

Phytochrome-interacting factors (PIFs) are a basic helix-loop-helix family of transcriptional regulators that maintain skotomorphogenesis and suppress photomorphogenesis. PIFs are regulated by plant photoreceptors, especially phytochromes. In general, PIFs physically interact with phytochromes, and this interaction induces PIF’s phosphorylation and subsequent degradation, contributing to the initiation of photomorphogenic development. Among the eight members of PIF (PIF1 to PIF8) reported in Arabidopsis thaliana, PIF3 is the first discovered member and plays central roles in de-etiolation and chlorophyll biosynthesis. More recently, PIF3 has been also reported to regulate hormone signaling and cold tolerance in plants. Although PIF3 protein shows dynamic behaviors in plants, its study has been limited due to the lack of an authentic PIF3 antibody. In this study, we produced polyclonal antibodies using inclusion bodies and characterized the PIF3 antibody based on specificity and sensitivity. In addition, we investigated PIF3 phosphorylation and degradation during phytochrome-mediated light signaling in plants. Furthermore, we successfully performed in vitro protein–protein interaction and co-immunoprecipitation assays between phytochrome B (phyB) and PIF3 using the antibody. Therefore, we obtained an authentic PIF3 antibody that could be used as a valuable tool to study the multi-faceted functions of PIF3.

파이토크롬 광수용체에 의한 식물의 광신호전달

Light is essential for plant growth and development, which serves as an energy source for photosynthesis and as environmental cues for photomorphogenesis (i.e., light-mediated development). Plants continually adapt to light environments to optimize their growth and development by monitoring with help of various photoreceptors including phytochromes. As red (R) and far-red (FR) light-absorbing photoreceptors, they function as a molecular switch with R-absorbing Pr and FR-absorbing Pfr forms. In the dark, phytochromes are biosynthesized as the inactive Pr form, which can be phototransformed into the physiologically active Pfr form upon exposure to light. The Pr-to-Pfr photoactivation of phytochromes induces a highly regulated signaling network for photomorphogenesis in plants, which includes translocation of phytochromes into the nucleus, interaction of phytochromes with a wide array of downstream signal transducers, regulated proteolysis of signaling targets, and transcriptional regulation of various photoresponsive genes. Moreover, several key components in the phytochrome-mediated light signaling have been identified from extensive research over several decades, which includes phytochrome-interacting factor (PIF), constitutively photomorphogenic 1 (COP1), suppressor of <italic>phyA-105</italic> (SPA), and elongated hypocotyl 5 (HY5). In this review, we provide a highlighted view for the molecular mechanisms of phytochromes in plant light signaling based on recent advances.

Decreased R:FR Ratio in Incident White Light Affects the Composition of Barley Leaf Lipidome and Freezing Tolerance in a Temperature-Dependent Manner

In cereals, C-repeat binding factor genes have been defined as key components of the light quality-dependent regulation of frost tolerance by integrating phytochrome-mediated light and temperature signals. This study elucidates the differences in the lipid composition of barley leaves illuminated with white light or white light supplemented with far-red light at 5 or 15 °C. According to LC-MS analysis, far-red light supplementation increased the amount of monogalactosyldiacylglycerol species 36:6, 36:5, and 36:4 after 1 day at 5 °C, and 10 days at 15 °C resulted in a perturbed content of 38:6 species. Changes were observed in the levels of phosphatidylethanolamine, and phosphatidylserine under white light supplemented with far-red light illumination at 15 °C, whereas robust changes were observed in the amount of several phosphatidylserine species at 5 °C. At 15 °C, the amount of some phosphatidylglycerol species increased as a result of white light supplemented with far-red light illumination after 1 day. The ceramide (42:2)-3 content increased regardless of the temperature. The double-bond index of phosphatidylglycerol, phosphatidylserine, phosphatidylcholine ceramide together with total double-bond index changed when the plant was grown at 15 °C as a function of white light supplemented with far-red light. white light supplemented with far-red light increased the monogalactosyldiacylglycerol/diacylglycerol ratio as well. The gene expression changes are well correlated with the alterations in the lipidome.

Effect of phytochrome-mediated red light signaling on phosphorus uptake and accumulation in rice

ABSTRACT Phosphorus (P) is a macronutrient essential for plant growth and productivity. Plants uptake P as inorganic phosphate (Pi); however, in the natural ecosystem, Pi availability is frequently a severe limiting factor for plant growth. Thus, plants have evolved several mechanisms, such as the expression of Pi starvation-responsive genes, to adapt to Pi deficient conditions. Although we recently reported that phytochrome (Phy)-mediated red light signaling promotes Pi uptake by increasing expression levels of such genes in the model plant Arabidopsis, it remains elusive whether a similar mechanism exists in agricultural crops. In the present study, we analyzed the effects of red light signaling on Pi uptake in rice (Oryza sativa L.) using osphyA and osphyB single mutants, and the osphyA osphyB double mutant. Unlike osphyA seedlings, osphyB seedlings showed a reduction in Pi uptake and Pi content. Furthermore, illumination of wild-type seedlings with red light significantly promoted Pi uptake, whereas illumination with blue or far-red light did not. The osphyB mutant showed reduced expression levels of several Pi starvation-responsive genes including Pi transporter genes. Additionally, these phenotypes of osphyB knockout mutants were much more evident under Pi deficient conditions than under Pi sufficient conditions. Moreover, red light promoted Pi uptake in seedlings of other plant species including broccoli (Brassica oleracea L. var. italica) and lettuce (Latuca sativa L.). These results suggest that OsPhyB-mediated red light signaling promotes Pi uptake in rice by up-regulating the expression of Pi starvation response-associated genes, and this phenomenon may be conserved in a wide range of plant species.

Transcriptomic network analyses shed light on the regulation of cuticle development in maize leaves

Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal that phyB1 phyB2 double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

Phytochrome-mediated light signal perception and responses elucidated by researches using rice phytochrome mutants and application potentiality of the knowledge to breeding rice plants

Phytochrome-mediated light signal perception and responses elucidated by researches using rice phytochrome mutants and application potentiality of the knowledge to breeding rice plants

Functional identification of MdPIF1 as a Phytochrome Interacting Factor in Apple

Light plays a central role in regulating both apple plant yield and fruit quality formation; however, the Phytochrome interacting factors (PIFs), which are the main components of Phytochrome-mediated light signal transduction in apple, have rarely been characterized. Here, we isolated and identified a PIF-like protein(MdPIF1) in apple, which is similar to AtPIF1. MdPIF1 was constitutively expressed at different levels in various apple tissues, and the transcription level of MdPIF1 was significantly induced during seed germination. A functional complementation assay in the Arabidopsis PIF1-deletion mutant pil5 suggested that MdPIF1 was a negative regulator in the Phy-mediated inhibition of hypocotyl elongation under far-red light conditions. MdPIF1-overexpression promoted hypocotyl elongation, while inhibiting seed germination and PIF1 deletion-induced the bleaching phenotype in the pil5 mutant. In addition, expression analysis indicated that MdPIF1 was involved in the germination of apple seeds and dormancy breaking of apple buds. Moreover, MdPIF1 inhibited the growth of apple calli via Phy-mediated pathways. These findings build a solid foundation for studies on Phytochrome-mediated light signal transduction and molecular breeding in apple.

Light and Temperature Signalling at the Level of CBF14 Gene Expression in Wheat and Barley

The wheat and barley CBF14 genes have been newly defined as key components of the light quality-dependent regulation of the freezing tolerance by the integration of phytochrome-mediated light and temperature signals. To further investigate the wavelength dependence of light-induced CBF14 expression in cereals, we carried out a detailed study using monochromatic light treatments at an inductive and a non-inductive temperature. Transcript levels of CBF14 gene in winter wheat Cheyenne, winter einkorn G3116 and winter barley Nure genotypes were monitored. We demonstrated that (1) CBF14 is most effectively induced by blue light and (2) provide evidence that this induction does not arise from light-controlled CRY gene expression. (3) We demonstrate that temperature shifts induce CBF14 transcription independent of the light conditions and that (4) the effect of temperature and light treatments are additive. Based on these data, it can be assumed that temperature and light signals are relayed to the level of CBF14 expression via separate signalling routes.

Phytochrome B Negatively Affects Cold Tolerance by Regulating OsDREB1 Gene Expression through Phytochrome Interacting Factor-Like Protein OsPIL16 in Rice

Cross talk between light signaling and cold signaling has been elucidated in the model plant Arabidopsis and tomato, but little is known about their relationship in rice. Here, we report that phytochrome B (phyB) mutants exhibit improved cold tolerance compared with wild type (WT) rice (Oryza sativa L. cv. Nipponbare). The phyB mutants had a lower electrolyte leakage index and malondialdehyde concentration than the WT, suggesting that they had greater cell membrane integrity and less lipid peroxidation. Real-time PCR analysis revealed that the expression levels of dehydration-responsive element binding protein 1 (OsDREB1) family genes, which functions in the cold stress response in rice, were increased in the phyB mutant under normal and cold stress conditions. PIFs are central players in phytochrome-mediated light signaling networks. To explore the relationship between rice PIFs and OsDREB1 gene expression, we produced overexpression lines of rice PIF genes. OsDREB1 family genes were up-regulated in OsPIL16-overexpression lines, which had improved cold tolerance relative to the WT. Chromatin immunoprecipitation (ChIP)-qPCR assay revealed that OsPIL16 can bind to the N-box region of OsDREB1B promoter. Expression pattern analyses revealed that OsPIL16 transcripts were induced by cold stress and was significantly higher in the phyB mutant than in the WT. Moreover, yeast two-hybrid assay showed that OsPIL16 can bind to rice PHYB. Based on these results, we propose that phyB deficiency positively regulates OsDREB1 expression through OsPIL16 to enhance cell membrane integrity and to reduce the malondialdehyde concentration, resulting in the improved cold tolerance of the phyB mutants.

Phytochrome Interacting Factors (PIFs) in Solanum lycopersicum: Diversity, Evolutionary History and Expression Profiling during Different Developmental Processes.

Although the importance of light for tomato plant yield and edible fruit quality is well known, the PHYTOCHROME INTERACTING FACTORS (PIFs), main components of phytochrome-mediated light signal transduction, have been studied almost exclusively in Arabidopsis thaliana. Here, the diversity, evolution and expression profile of PIF gene subfamily in Solanum lycopersicum was characterized. Eight tomato PIF loci were identified, named SlPIF1a, SlPIF1b, SlPIF3, SlPIF4, SlPIF7a, SlPIF7b, SlPIF8a and SlPIF8b. The duplication of SlPIF1, SlPIF7 and SlPIF8 genes were dated and temporally coincided with the whole-genome triplication event that preceded tomato and potato divergence. Different patterns of mRNA accumulation in response to light treatments were observed during seedling deetiolation, dark-induced senescence, diel cycle and fruit ripening. SlPIF4 showed similar expression profile as that reported for A. thaliana homologs, indicating an evolutionary conserved function of PIF4 clade. A comprehensive analysis of the evolutionary and transcriptional data allowed proposing that duplicated SlPIFs have undergone sub- and neofunctionalization at mRNA level, pinpointing the importance of transcriptional regulation for the maintenance of duplicated genes. Altogether, the results indicate that genome polyploidization and functional divergence have played a major role in diversification of the Solanum PIF gene subfamily.

Illuminating Progress in Phytochrome-Mediated Light Signaling Pathways.

Light signals regulate a plethora of plant responses throughout their life cycle, especially the red and far-red regions of the light spectrum perceived by the phytochrome family of photoreceptors. However, the mechanisms by which phytochromes regulate gene expression and downstream responses remain elusive. Several recent studies have unraveled the details on how phytochromes regulate photomorphogenesis. These include the identification of E3 ligases that degrade PHYTOCHROME INTERACTING FACTOR (PIF) proteins, key negative regulators, in response to light, a better view of how phytochromes inhibit another key negative regulator, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), and an understanding of why plants evolved multiple negative regulators to repress photomorphogenesis in darkness. These advances will surely fuel future research on many unanswered questions that have intrigued plant photobiologists for decades.

The effects of phytochrome-mediated light signals on the developmental acquisition of photoperiod sensitivity in rice.

Plants commonly rely on photoperiodism to control flowering time. Rice development before floral initiation is divided into two successive phases: the basic vegetative growth phase (BVP, photoperiod-insensitive phase) and the photoperiod-sensitive phase (PSP). The mechanism responsible for the transition of rice plants into their photoperiod-sensitive state remains elusive. Here, we show that se13, a mutation detected in the extremely early flowering mutant X61 is a nonsense mutant gene of OsHY2, which encodes phytochromobilin (PΦB) synthase, as evidenced by spectrometric and photomorphogenic analyses. We demonstrated that some flowering time and circadian clock genes harbor different expression profiles in BVP as opposed to PSP, and that this phenomenon is chiefly caused by different phytochrome-mediated light signal requirements: in BVP, phytochrome-mediated light signals directly suppress Ehd2, while in PSP, phytochrome-mediated light signals activate Hd1 and Ghd7 expression through the circadian clock genes' expression. These findings indicate that light receptivity through the phytochromes is different between two distinct developmental phases corresponding to the BVP and PSP in the rice flowering process. Our results suggest that these differences might be involved in the acquisition of photoperiod sensitivity in rice.

Overexpression of an S-like ribonuclease gene, OsRNS4, confers enhanced tolerance to high salinity and hyposensitivity to phytochrome-mediated light signals in rice

S-like ribonucleases (S-like RNases) are homologous to S-ribonucleases (S-RNases), but are not involved in self-incompatibility. In dicotyledonous plants, S-like RNases play an important role in phosphate recycling during senescence and are induced by inorganic phosphate-starvation and in response to defense and mechanical wounding. However, little information about the functions of the S-like RNase in monocots has been reported. Here, we investigated the expression patterns and roles of an S-like RNase gene, OsRNS4, in abscisic acid (ABA)-mediated responses and phytochrome-mediated light responses as well as salinity tolerance in rice. The OsRNS4 gene was expressed at relatively high levels in leaves although its transcripts were detected in various organs. OsRNS4 expression was regulated by salt, PEG and ABA. The seedlings overexpressing OsRNS4 had longer coleoptiles and first leaves than wild-type seedlings under red light (R) and far-red light (FR), suggesting negative regulation of OsRNS4 in photomorphogenesis in rice seedlings. Moreover, ABA-induced growth inhibition of rice seedlings was significantly increased in the OsRNS4-overexpression (OsRNS4-OX) lines compared with that in WT, suggesting that OsRNS4 probably acts as a positive regulator in ABA responses in rice seedlings. In addition, our results demonstrate that OsRNS4-OX lines have enhanced tolerance to high salinity compared to WT. Our findings supply new evidence on the functions of monocot S-like RNase in regulating photosensitivity and abiotic stress responses.

The homeodomain‐leucine zipper ATHB23, a phytochrome B‐interacting protein, is important for phytochrome B‐mediated red light signaling

Phytochromes are red (R)/far-red (FR) photoreceptors that are central to the regulation of plant growth and development. Although it is well known that photoactivated phytochromes are translocated into the nucleus where they interact with a variety of nuclear proteins and ultimately regulate genome-wide transcription, the mechanisms by which these photoreceptors function are not completely understood. In an effort to enhance our understanding of phytochrome-mediated light signaling networks, we attempted to identify novel proteins interacting with phytochrome B (phyB). Using affinity purification in Arabidopsis phyB overexpressor, coupled with mass spectrometry analysis, 16 proteins that interact with phyB in vivo were identified. Interactions between phyB and six putative phyB-interacting proteins were confirmed by bimolecular fluorescence complementation (BiFC) analysis. Involvement of these proteins in phyB-mediated signaling pathways was also revealed by physiological analysis of the mutants defective in each phyB-interacting protein. We further characterized the athb23 mutant impaired in the homeobox protein 23 (ATHB23) gene. The athb23 mutant displayed altered hypocotyl growth under R light, as well as defects in phyB-dependent seed germination and phyB-mediated cotyledon expansion. Taken together, these results suggest that the ATHB23 transcription factor is a novel component of the phyB-mediated R light signaling pathway.

Expression Patterns of OsPIL11, a Phytochrome-Interacting Factor in Rice, and Preliminary Analysis of Its Roles in Light Signal Transduction

The expression patterns of OsPIL11, one of six putative phytochrome-interacting factors, were analyzed in different organs of transgenic tobacco (Nicotiana tabacum). The expression of OsPIL11 was organ-specific and was regulated by leaf development, abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA). To further explore the role of OsPIL11 in plant light signal transduction, a plant expression vector of OsPIL11 was constructed and introduced into tobacco. When grown under continuous red light, OsPIL11-overexpressed transgenic tobacco exhibited shorter hypocotyls and larger cotyledons and leaves compared to wild-type seedlings. When grown under continuous far-red light, however, transgenic and wild-type seedlings showed similar phenotypes. These results indicate that OsPIL11 is involved in red light induced de-etiolation, but not in far-red light induced de-etiolation in transgenic tobacco, which lays the foundation for dissecting the function of OsPIL11 in phytochrome-mediated light signal transduction in rice.

光敏色素B介导光信号影响水稻的脱落酸途径

Several evidences revealed the interaction between phytochrome-mediated light signals and plant hormones abscisic acid (ABA) pathway in Arabidopsis . However, interaction between ABA signaling and phytochrome-mediated light signaling in mediating rice growth and development remain unclear. In the present study, we analyzed effects of PhyB -mediated light signals on ABA metabolism and ABA responses using rice wild type (WT) and the phyB mutant. It was observed that transcript levels of ABA biosynthetic genes (including OsNCED1 , OsNCED2 , OsNCED3 and OsNCED4 ) were higher in the phyB mutant than those in WT, whereas transcript level of ABA deactivating gene OsABA8OX1 was lower in the phyB mutant than that in WT, which probably contributed to the relatively high ABA content in the phyB mutant. ABA treatment inhibited germination of rice seeds grown either in the dark or under light. However, inhibitory effects of ABA treatment on seed germination were more obvious in phyB mutants relative to that in WT when seeds were grown under light conditions, suggesting that PhyB -mediated light signals attenuated the inhibitory effects triggered by ABA. Meantime, we compared the expression patterns of genes related to seed germination in WT and the phyB mutant grown in the medium with or without ABA. It was deduced that these genes is unlikely to contribute for the promotive effects of phyB-mediated light signals on seed germination. In addition, ABA treatment inhibited growth of both above-ground part and seminal root in rice seedlings. PhyB -mediated light signals did not affect the ABA-induced inhibition of above-ground part growth, but negatively regulate the inhibition of root growth. Taken together, our results suggest that PhyB -mediated light signals negatively regulate ABA accumulation and ABA responses in rice. This work reveals the influence of PhyB -mediated light signals on ABA pathway, which lays the foundation for dissecting the molecular mechanism of coordinated regulation of rice development by light and ABA in rice.

Overexpression of a phytochrome-regulated tandem zinc finger protein gene, OsTZF1, confers hypersensitivity to ABA and hyposensitivity to red light and far-red light in rice seedlings

Tandem zinc finger proteins (TZFs) in plants are involved in gene regulation, developmental responses, and hormone-mediated environmental responses in Arabidopsis. However, little information about the functions of the TZF family in monocots has been reported. Here, we investigated a cytoplasmic TZF protein, OsTZF1, which is involved in photomorphogenesis and ABA responses in rice seedlings. The OsTZF1 gene was expressed at relatively high levels in leaves and shoots, although its transcripts were detected in various organs. Red light (R)- and far-red light (FR)-mediated repression of OsTZF1 gene expression was attributed to phytochrome B (phyB) and phytochrome C (phyC), respectively. In addition, OsTZF1 expression was regulated by salt, PEG, and ABA. Overexpression of OsTZF1 caused a long leaf sheath relative to wild type (WT) under R and FR, suggesting that OsTZF1 probably acts as a negative regulator of photomorphogenesis in rice seedlings. Moreover, ABA-induced growth inhibition of rice seedlings was marked in the OsTZF1-overexpression lines compared with WT, suggesting the positive regulation of OsTZF1 to ABA responses. Genome-wide expression analysis further revealed that OsTZF1 also functions in other hormone or stress responses. Our findings supply new evidence on the functions of monocot TZF proteins in phytochrome-mediated light and hormone responses. OsTZF1 encodes a cytoplasm-localized tandem zinc finger protein and is regulated by both ABA and phytochrome-mediated light signaling. OsTZF1 functions in phytochrome-mediated light and ABA responses in rice.