Phytochrome C Research Articles

Molecular phylogenetic studies on Asparagus (Asparagaceae) revealed the position of the highly endangered Irano-Turkish halophytic species A. lycaonicus P.H.Davis related to A. officinalis L.

Asparagus lycaonicus P.H.Davis (Asparagaceae), a highly interesting halophytic species was found in two disjunct populations of ca. 1550 km aerial distance, one in Central Anatolia (Bolluk Salt Lake) and the other in Central Iran (Meyghan Salt Lake). Using one nuclear phytochrome C (PHYC) and two plastid regions (trnH-psbA and trnD-T) the phylogenetic position of this Irano-Turkish endemic species was studied. In contrast to low resolution of plastid sequences, the PHYC provides good resolution for understanding species level phylogeny in SW Asian Asparagus. Molecular data confirms a conspecific position of the two disjunct localities in Turkey and Iran and their position in a clade containing cultivated Asparagus and many other Irano-Turkish species. Asparagus lycaonicus is extremely rare in Iran and since its discovery in 1986, it has never been rediscovered in Iran, even in its initial collection site. Intensive land use activities, water level changes and the passage of Arak wastewater into the Meyghan Salt Lake area has drastically changed the habitat of A. lycaonicus and might have caused its extinction. Since its discovery in 1960 in Turkey, the species is only found in a small area around and inside a sodic extraction factory around Bolluk Lake. Considering the critical condition of its populations in Iran and Turkey, we evaluate the conservation status of Asparagus lycaonicus as Critically Endangered (CR). In conclusion we urge the authorities to implement in situ measures for conservation and restoration of the habitats of A. lycaonicus, a halophytic species related to cultivated Asparagus. The taxonomic status of some newly described Asparagus from Iran is discussed with reducing A. touranensis Hamdi & Assadi to synonym of A. griffithii Baker., A. khorasanensis Hamdi & Assadi to synonym of A. breslerianus Schult. & Schult.f, and Asparagus bojnurdensis Hamdi & Assadi to synonym of A. verticillatus L.

Phytochromes transmit photoperiod information via the evening complex in Brachypodium

BackgroundDaylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as PHOTOPERIOD1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in adapting barley and wheat to northern latitudes. How monocot plants sense photoperiod and integrate this information into growth and development is not well understood.ResultsWe find that phytochrome C (PHYC) is essential for flowering in Brachypodium distachyon. Conversely, ELF3 acts as a floral repressor and elf3 mutants display a constitutive long day phenotype and transcriptome. We find that ELF3 and PHYC occur in a common complex. ELF3 associates with the promoters of a number of conserved regulators of flowering, including PPD1 and VRN1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in short days and is necessary for rapid flowering in response to long days. PHYC is in the active Pfr state at the end of the day, but we observe it undergoes dark reversion over the course of the night.ConclusionsWe propose that PHYC acts as a molecular timer and communicates information on night-length to the circadian clock via ELF3.

Domestication-associated PHYTOCHROME C is a flowering time repressor and a key factor determining Setaria as a short-day plant.

Phytochromes play vital roles in the regulation of flowering time, but little is known in Panicoideae species, especially the C4 model Setaria. Here, genomic variations of PHYTOCHROME C (PHYC) between wild and cultivated Setaria gene pools were analysed and three SiphyC mutants were identified. The function of SiPHYC was verified by CRISPR-Cas9 approach and transcriptome sequencing. Furthermore, efficiency of indoor cultivation of SiphyC mutants were systematically evaluated. An extreme purified selection of PHYC was detected in wild to cultivated domestication process of Setaria. SiphyC mutants and knockout transgenic plants showed an early heading date and a loss of response to short-day photoperiod. Furthermore, variable expression of SiFTa, SiMADS14 and SiMADS15 might be responsible for promoting flowering of SiphyC mutants. Moreover, SiphyC mutant was four times that of the indoor plot ratio of wild-type and produced over 200 seeds within 45 d per individual. Our results suggest that domestication-associated SiPHYC repressed flowering and determined Setaria as a short-day plant, and SiphyC mutants possess the potential for creating efficient indoor cultivation system suitable for research on Setaria as a model, and either for maize or sorghum as well.

Growth and Physiological Responses of Norway Spruce (Picea abies (L.) H. Karst) Supplemented with Monochromatic Red, Blue and Far-Red Light

Monochromatic red light (R) supplementation is more efficient than blue light (B) in promoting Norway spruce (Picea abies (L.) H. Karst) growth. Transcriptome analysis has revealed that R and B may regulate stem growth by regulating phytohormones and secondary metabolites; however, the effects of light qualities on physiological responses and related gene expression in Norway spruce require further study. In the present study, three-year-old Norway spruce seedlings received sunlight during the daytime were exposed to monochromatic B (460 mm), monochromatic R (660 nm), monochromatic far-red light (FR, 730 nm), and a combination of three monochromatic lights (control, R:FR:B = 7:1:1) using light-emitting diode (LED) lamps for 12 h after sunset for 90 day. Growth traits, physiological responses, and related gene expression were determined. The results showed that light quality significantly affected Norway spruce growth. The stem height, root collar diameter, and current-year shoot length of seedlings treated with R were 2%, 10% and 12% higher, respectively, than those of the control, whereas seedlings treated with B and FR showed significantly lower values of these parameters compared with that of the control. The net photosynthetic rate (Pn) of seedlings under R treatment was 10% higher than that of the control, whereas the Pn values of seedlings treated with FR and B were 22% and 33%, respectively, lower than that of the control. The ratio of phosphoenolpyruvate carboxylase to ribulose-1,5-bisphosphate carboxylase/oxygenase (PEPC/Rubisco) of seedlings after the R treatment (0.581) was the highest and 3.98 times higher than that of the seedlings treated with B. Light quality significantly affected the gibberellic acid (GAs) levels, which was 13% higher in seedlings treated with R (6.4 g/100 ng) than that of the control, whereas, the GAs level of seedlings treated with B and FR was 17% and 19% lower, respectively, than that of the control. In addition, seedlings treated with R achieved the lowest ratio of leaf chlorophyll content to fresh weight (8.7). Compared to the R and control treatments, seedlings received FR treatment had consistently lower values of the quantum yield of electron transport beyond QA− (primary quinone, ϕEo) and efficiency, with which a trapped exciton moves an electron into the electron transport chain beyond QA− (ψo), while higher values of the relatively variable fluorescence at the J step and normalized relatively variable fluorescence at the K step (Wk). The values of ϕEo, ψO, VJ and Wk in seedlings treated with B were similar to those in the control group. The expression of genes associated with light signal transduction, such as PHYTOCHROME C (PHYC), ELONGATED HYPOCOTYL5 (HY5), CONSTITUTIVE PHOTOMORPHOGENIC 1-2 (COP1-2), and PHYTOCHROMEINTERACTING FACTOR 3 (PIF3), was significantly higher in seedlings under B treatment than those under other light treatments. Nevertheless, significant differences were not observed in the expression of COP1-2, HY5, and PIF3 between the R treatment and the control. The expression value of COP1-2 was significantly lower in R than FR light treatments. In conclusion, compared with the control, R promotes, whereas B and FR inhibit Norway spruce growth, which was accompanied by physiological changes and genes expression regulation that may be relate to a changing phytochrome photostationary state (PSS) with the supplemental R in seedlings.

Transcriptome Profiling of Light-Regulated Anthocyanin Biosynthesis in the Pericarp of Litchi.

Light is a key environmental factor that affects anthocyanin biosynthesis. To enhance our understanding of the mechanisms involved in light-regulated anthocyanin biosynthesis in the pericarp of litchi, we performed transcriptomic analyses on the basis of Illumina sequencing. Fruit clusters were bagged with double-layer Kraft paper bags at 42 days after anthesis. The bags were removed after 2 weeks. Under light conditions, anthocyanins accumulated rapidly in the pericarp. RNA sequences were de novo assembled into 75,935 unigenes with an average length of 913 bp. Approximately 74.5% of unigenes (56,601) were annotated against four public protein databases. A total of 16,622 unigenes that significantly differed in terms of abundance were identified. These unigenes are implicated in light signal perception and transduction, flavonoid biosynthesis, carotenoid biosynthesis, plant hormone signal transduction, and photosynthesis. In photoreceptors, the expression levels of UV RESISTANCE LOCUS 8 (UVR8), Phototropin 2 (PHOT2), Phytochrome B (PHYB), and Phytochrome C (PHYC) increased significantly when the fruits were exposed to light. This result indicated that they likely play important roles in anthocyanin biosynthesis regulation. After analyzed digital gene expression (DGE), we found that the light signal transduction elements of COP1 and COP10 might be responsible for anthocyanin biosynthesis regulation. After the bags were removed, nearly all structural and regulatory genes, such as UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT), MYB, basic helix-loop-helix (bHLH), and WD40, involved in the anthocyanin biosynthetic pathway were upregulated. In addition to MYB-bHLH-WD40 transcription complex, ELONGATED HYPOCOTYL (HY5), NAM/ATAF/CUC (NAC), homeodomain leucine zipper proteins (ATHBs), and FAR-RED ELONGATED HYPOCOTYL (FHY) possibly participate in light-induced responses. On the basis of DGEs and qRT-PCR validation, we observed a light-induced anthocyanin biosynthesis and regulation pattern in litchi pericarp. This study enhanced our understanding of the molecular mechanisms governing light-induced anthocyanin biosynthesis in litchi pericarp.

Evolution of Asparagus L. (Asparagaceae): Out-of-South-Africa and multiple origins of sexual dimorphism

In the most comprehensive study to date we explored the phylogeny and evolution of the genus Asparagus, with emphasis on the southern African species. We included 211 accessions, representing 77 (92%) of the southern African, 6 (17%) of the tropical African, 10 (56%) of the strictly European and 6 (9%) of the Eurasian species. We analyzed DNA sequences from three plastid regions (trnH-psbA, trnD-T, ndhF) and from the nuclear region phytochrome C (PHYC) with parsimony and maximum likelihood methods, and recovered a monophyletic Asparagus. The phylogeny conflicts with all previous infra-generic classifications. It has many strongly supported clades, corroborated by morphological characters, which may provide a basis for a revised taxonomy. Additionally, the phylogeny indicates that many of the current species delimitations are problematic. Using biogeographic analyses that account for phylogenetic uncertainty (S-DIVA) and take into account relative branch lengths (Lagrange) we confirm the origin of Asparagus in southern Africa, and find no evidence that the dispersal of Asparagus follow the Rand flora pattern. We find that all truly dioecious species of Asparagus share a common origin, but that sexual dimorphism has arisen independently several times.

Phylogenetics, divergence times and diversification from three genomic partitions in monocots

Resolution of evolutionary relationships among some monocot orders remains problematic despite the application of various taxon and molecular locus sampling strategies. In this study we sequenced and analysed a fragment of the low-copy, nuclear phytochrome C (PHYC) gene and combined these data with a previous multigene data set (four plastid, one mitochondrial, two nuclear ribosomal loci) to determine if adding this marker improved resolution and support of relationships among major lineages of monocots. Our results indicate the addition of PHYC to the multigene dataset increases support along the backbone of the monocot tree, although relationships among orders of commelinids remain elusive. We also estimated divergence times in monocots by applying newly evaluated fossil calibrations to our resolved phylogenetic tree. Inclusion of early-diverging angiosperm lineages confirmed the origin of extant monocots c. 131 Mya and strengthened the hypothesis of recent divergence times for some lineages, although current divergence time estimation methods may inadequately model rate heterogeneity in monocots. We note significant shifts in diversification in at least two monocot orders, Poales and Asparagales. We describe patterns of diversification in the context of radiation of other relevant plant and animal lineages. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178, 375–393.

PHYTOCHROME C is an essential light receptor for photoperiodic flowering in the temperate grass, Brachypodium distachyon.

We show that in the temperate grass, Brachypodium distachyon, PHYTOCHROME C (PHYC), is necessary for photoperiodic flowering. In loss-of-function phyC mutants, flowering is extremely delayed in inductive photoperiods. PHYC was identified as the causative locus by utilizing a mapping by sequencing pipeline (Cloudmap) optimized for identification of induced mutations in Brachypodium. In phyC mutants the expression of Brachypodium homologs of key flowering time genes in the photoperiod pathway such as GIGANTEA (GI), PHOTOPERIOD 1 (PPD1/PRR37), CONSTANS (CO), and florigen/FT are greatly attenuated. PHYC also controls the day-length dependence of leaf size as the effect of day length on leaf size is abolished in phyC mutants. The control of genes upstream of florigen production by PHYC was likely to have been a key feature of the evolution of a long-day flowering response in temperate pooid grasses.

Phytochrome C plays a major role in the acceleration of wheat flowering under long-day photoperiod.

Phytochromes are dimeric proteins that function as red and far-red light sensors influencing nearly every phase of the plant life cycle. Of the three major phytochrome families found in flowering plants, phytochrome C (PHYC) is the least understood. In Arabidopsis and rice, PHYC is unstable and functionally inactive unless it heterodimerizes with another phytochrome. However, when expressed in an Arabidopsis phy-null mutant, wheat PHYC forms signaling active homodimers that translocate into the nucleus in red light to mediate photomorphogenic responses. Tetraploid wheat plants homozygous for loss-of-function mutations in all PHYC copies (phyC(AB)) flower on average 108 d later than wild-type plants under long days but only 19 d later under short days, indicating a strong interaction between PHYC and photoperiod. This interaction is further supported by the drastic down-regulation in the phyC(AB) mutant of the central photoperiod gene photoperiod 1 (PPD1) and its downstream target flowering locus T1, which are required for the promotion of flowering under long days. These results implicate light-dependent, PHYC-mediated activation of PPD1 expression in the acceleration of wheat flowering under inductive long days. Plants homozygous for the phyC(AB) mutations also show altered profiles of circadian clock and clock-output genes, which may also contribute to the observed differences in heading time. Our results highlight important differences in the photoperiod pathways of the temperate grasses with those of well-studied model plant species.

Phylogeny and proposed circumscription of Breynia, Sauropus and Synostemon (Phyllanthaceae), based on chloroplast and nuclear DNA sequences

Previous estimates of phylogeny in the Phyllanthaceae, Phyllantheae, have been hampered by undersampling of species from morphologically distinctive groups and using too few gene regions. To increase the phylogenetic resolution, sequences of two nuclear (ITS1–5.8S–ITS2) and Phytochrome C (PHYC)) and two non-coding chloroplast (accD–psaI, trnS–trnG) DNA markers were analysed using maximum parsimony and Bayesian inference with expanded sampling in Breynia, Glochidion, Sauropus and Synostemon. Our results supported reinstatement of Synostemon, previously included in Sauropus s.str., to generic rank, and provided evidence towards its future infrageneric classification. The results also indicated expansion of Breynia to include Sauropus s.str.; this combined monophyletic group consists of two strongly supported clades. Finally, we showed monophyly for Glochidion, which is sister to Phyllanthus subg. Phyllanthodendron, both still remaining undersampled. Morphological features characteristic of Breynia, Sauropus and Synostemon are discussed, as well as the desirability of dividing Phyllanthus into smaller genera.

Altitudinal and climatic adaptation is mediated by flowering traits and FRI, FLC, and PHYC genes in Arabidopsis.

Extensive natural variation has been described for the timing of flowering initiation in many annual plants, including the model wild species Arabidopsis (Arabidopsis thaliana), which is presumed to be involved in adaptation to different climates. However, the environmental factors that might shape this genetic variation, as well as the molecular bases of climatic adaptation by modifications of flowering time, remain mostly unknown. To approach both goals, we characterized the flowering behavior in relation to vernalization of 182 Arabidopsis wild genotypes collected in a native region spanning a broad climatic range. Phenotype-environment association analyses identified strong altitudinal clines (0-2600 m) in seven out of nine flowering-related traits. Altitudinal clines were dissected in terms of minimum winter temperature and precipitation, indicating that these are the main climatic factors that might act as selective pressures on flowering traits. In addition, we used an association analysis approach with four candidate genes, FRIGIDA (FRI), FLOWERING LOCUS C (FLC), PHYTOCHROME C (PHYC), and CRYPTOCHROME2, to decipher the genetic bases of this variation. Eleven different loss-of-function FRI alleles of low frequency accounted for up to 16% of the variation for most traits. Furthermore, an FLC allelic series of six novel putative loss- and change-of-function alleles, with low to moderate frequency, revealed that a broader FLC functional diversification might contribute to flowering variation. Finally, environment-genotype association analyses showed that the spatial patterns of FRI, FLC, and PHYC polymorphisms are significantly associated with winter temperatures and spring and winter precipitations, respectively. These results support that allelic variation in these genes is involved in climatic adaptation.

Hydatellaceae identified as a new branch near the base of the angiosperm phylogenetic tree

Although the relationship of angiosperms to other seed plants remains controversial, great progress has been made in identifying the earliest extant splits in flowering-plant phylogeny, with the discovery that the New Caledonian shrub Amborella trichopoda, the water lilies (Nymphaeales), and the woody Austrobaileyales constitute a basal grade of lines that diverged before the main radiation in the clade. By focusing attention on these ancient lines, this finding has re-written our understanding of angiosperm structural and reproductive biology, physiology, ecology and taxonomy. The discovery of a new basal lineage would lead to further re-evaluation of the initial angiosperm radiation, but would also be unexpected, as nearly all of the approximately 460 flowering-plant families have been surveyed in molecular studies. Here we show that Hydatellaceae, a small family of dwarf aquatics that were formerly interpreted as monocots, are instead a highly modified and previously unrecognized ancient lineage of angiosperms. Molecular phylogenetic analyses of multiple plastid genes and associated noncoding regions from the two genera of Hydatellaceae identify this overlooked family as the sister group of Nymphaeales. This surprising result is further corroborated by evidence from the nuclear gene phytochrome C (PHYC), and by numerous morphological characters. This indicates that water lilies are part of a larger lineage that evolved more extreme and diverse modifications for life in an aquatic habitat than previously recognized.

The PHYTOCHROME C photoreceptor gene mediates natural variation in flowering and growth responses of Arabidopsis thaliana.

Light has an important role in modulating seedling growth and flowering time. We show that allelic variation at the PHYTOCHROME C (PHYC) photoreceptor locus affects both traits in natural populations of A. thaliana. Two functionally distinct PHYC haplotype groups are distributed in a latitudinal cline dependent on FRIGIDA, a locus that together with FLOWERING LOCUS C explains a large portion of the variation in A. thaliana flowering time. In a genome-wide scan for association of 65 loci with latitude, there was an excess of significant P values, indicative of population structure. Nevertheless, PHYC was the most strongly associated locus across 163 strains, suggesting that PHYC alleles are under diversifying selection in A. thaliana. Our work, together with previous findings, suggests that photoreceptor genes are major agents of natural variation in plant flowering and growth response.

Structural characterization, expression analysis and evolution of the red/far-red sensing photoreceptor gene, phytochrome C (PHYC), localized on the ‘B’ genome of hexaploid wheat (Triticum aestivum L.)

Phytochromes are a family of red/far-red light perceiving photoreceptors. The monocot phytochrome family is represented by three members, PHYA, PHYB and PHYC. We have isolated and characterized the first PHY gene member (TaPHYC) from common wheat, Triticum aestivum var. CPAN1676. It codes for a species of the photoreceptor, phyC, which is known to be light-stable in all plants analyzed so far. A sequence of 7.2 kb has been determined, which includes 3.42 kb of coding region. This is the second full-length PHYC gene sequenced from a monocot (first was from rice). TaPHYC gene shares structural similarities with the rice PHYC containing four exons and three introns in the coding region. The 5' UTR is 1.0-kb-long and harbors an upstream open reading frame (URF) encoding 28 aa. Southern blot analysis of TaPHYC indicates that it represents single locus in the wheat genome, although the possibility of additional loci cannot be completely ruled out. Chromosomal localization using nullisomic-tetrasomic lines of Triticum aestivum var. Chinese Spring places TaPHYC on chromosome 4B. PHYC represents a constitutively expressed gene in all the organs tested and under light/dark conditions. However, PHYC was found to be developmentally regulated showing maximal expression in 3-day-old dark-grown seedlings, which declined thereafter. In silico analysis has also been done to compare TaPHYC gene with the partial sequences known from other wheat species and cultivars. The presence of a topoisomerase gene immediately downstream of the PHYC gene, both in rice and wheat genomes, presents yet another example of synteny in cereals and its possible significance has been discussed.

Isolation and characterization of PHYC gene from Stellaria longipes: differential expression regulated by different red/far-red light ratios and photoperiods.

We have cloned and characterized the phytochrome C ( PHYC) gene from Stellaria longipes. The PHYC gene is composed of a 110-bp 5'-untranslated leader sequence, a 3,342-bp coding region, and a 351-bp 3'-untranslated sequence. The Stellaria PHYC contains three long introns within the coding region at conserved locations as in most angiosperm PHY genes. DNA blot analysis indicates that the Stellaria genome contains a single copy of PHYC. Stellaria PHYC shares 60%, 58%, and 57% deduced amino acid identities with rice, Sorghum, and Arabidopsis PHYC, respectively. Phylogenetic analysis indicates that Stellaria PHYC is located in the dicot branch, but is divergent from Arabidopsis PHYC. The Stellaria PHYC is constitutively expressed in different plant organs, though the level of PHYC gene transcript in roots is slightly higher than in flowers, leaves, and stems. When 2-week old seedlings grown in the dark were exposed to constant white light, PHYC mRNA quickly accumulates within 1-12 h. When plants grown in darkness for 7 days were exposed to different red/far-red light (R/FR) ratios, the levels of PHYC mRNA at R/FR = 0.7 are much lower than under R/FR = 3.5. The levels of PHYC mRNA under short-day (SD) photoperiod are higher than under long-day (LD) photoperiod. Plants under SD conditions do not elongate, and are only about 1.7 cm tall at 19 days. In contrast, plants under LD conditions elongate with an average height of 21.2 cm at 19 days. The plants do not flower under SD conditions, but do so at 18-19 days under LD conditions. These results indicate that under SD conditions the high level of PHYC mRNA may inhibit stem elongation and flower initiation. In contrast, under LD conditions the high level of PHYC mRNA may promote stem elongation and flowering.