Phytochromes are a widespread family of red/far-red responsive photoreceptors first discovered in plants, where they constitute one of the three main classes of photomorphogenesis regulators. All phytochromes utilize covalently attached bilin chromophores that enable photoconversion between red-absorbing (P r ) and far-red-absorbing (P fr ) forms. Phytochromes are thus photoswitchable photosensors; canonical phytochromes have a conserved N-terminal photosensory core and a Cterminal regulatory region which typically includes a histidine-kinase-related domain. The discovery of new bacterial and cyanobacterial members of the phytochrome family within the last decade has greatly aided biochemical and structural characterization of this family, with the first crystal structure of a bacteriophytochrome photosensory core appearing in 2005. This structure and other recent biochemical studies have provided exciting new insights into the structure of phytochrome, the photoconversion process that is central to light sensing, and the mechanism of signal transfer by this important family of photoreceptors. Keywordsphytochrome; biochemistry; biliprotein; photoreceptor; light signaling; photochemistry GENERAL INTRODUCTIONPhytochrome was first discovered in plants in 1959 as the photoreceptor that mediates plant growth and development in response to long-wavelength visible light (9). Phytochrome measures the ratio of red light (R) to far-red light (FR), thereby allowing the plant to assess the quantity of photosynthetically active light and trigger shade avoidance responses (89). Phytochromes are found in all flowering plants and cryptophytes, and this important family of developmental regulators constitutes one of the three major classes of photoreceptors in higher plants, with the others being cryptochromes and phototropins (3,8,91). *Corresponding author: Telephone: 530-752-1865; FAX: 530-752-3085; E-mail: jclagarias@ucdavis.edu. SIDE BAR Phytochromes as Sensors of Oxygen-Dependent Heme Catabolism. The bilin chromophores incorporated by all phytochromes are synthesized from heme in two steps. First, a heme oxygenase converts heme into BV, which is directly incorporated as the chromophore of BphP and Fph phytochromes. In plants and cyanobacteria, however, BV is further reduced to yield PΦB in higher plants and PCB in cyanobacteria and green algae. Conversion of BV to PΦB is carried out by HY-2 in the chloroplast, while reduction of BV to yield PCB is instead carried out by PcyA. Both HY-2 and PcyA belong to a conserved family of ferredoxin-dependent bilin reductases. The kinase activity and regulatory signaling state of many phytochromes are regulated not only by light but by the presence or absence of chromophore. The synthesis of chromophore is itself dependent on the heme metabolism of the cell, because chromophore will only be produced sparingly if cells are starved for heme or oxygen. Hence, phytochrome signaling is sensitive to heme metabolism and oxygen levels. Phytochromes therefore integrate both the light en...

The biliprotein phytochrome regulates plant growth and developmental responses to the ambient light environment through an unknown mechanism. Biochemical analyses demonstrate that phytochrome is an ancient molecule that evolved from a more compact light sensor in cyanobacteria. The cyanobacterial phytochrome Cph1 is a light-regulated histidine kinase that mediates red, far-red reversible phosphorylation of a small response regulator, Rcp1 (response regulator for cyanobacterial phytochrome), encoded by the adjacent gene, thus implicating protein phosphorylation-dephosphorylation in the initial step of light signal transduction by phytochrome.

Cyanobacteria are unique among bacteria in performing oxygenic photosynthesis, often together with nitrogen fixation and, thus, are major primary producers in many ecosystems. The cyanobacterium, Leptolyngbya sp. strain JSC-1, exhibits an extensive photoacclimative response to growth in far-red light that includes the synthesis of chlorophylls d and f. During far-red acclimation, transcript levels increase more than twofold for ~900 genes and decrease by more than half for ~2000 genes. Core subunits of photosystem I, photosystem II, and phycobilisomes are replaced by proteins encoded in a 21-gene cluster that includes a knotless red/far-red phytochrome and two response regulators. This acclimative response enhances light harvesting for wavelengths complementary to the growth light (λ = 700 to 750 nanometers) and enhances oxygen evolution in far-red light.

Phytochromes are well-known as photoactive red-and near IRabsorbing chromoproteins with cysteine-linked linear tetrapyrrole (bilin) prosthetic groups. Phytochrome photoswitching regulates adaptive responses to light in both photosynthetic and nonphotosynthetic organisms. Exclusively found in cyanobacteria, the related cyanobacteriochrome (CBCR) sensors extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. Blue/green light sensing by a wellstudied subfamily of CBCRs proceeds via a photolabile thioether linkage to a second cysteine fully conserved in this subfamily. In the present study, we show that dual-cysteine photosensors have repeatedly evolved in cyanobacteria via insertion of a second cysteine at different positions within the bilin-binding GAF domain (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) shared by CBCRs and phytochromes. Such sensors exhibit a diverse range of photocycles, yet all share ground-state absorbance of near-UV to blue light and a common mechanism of light perception: reversible photoisomerization of the bilin 15,16 double bond. Using site-directed mutagenesis, chemical modification and spectroscopy to characterize novel dual-cysteine photosensors from the cyanobacterium Nostoc punctiforme ATCC 29133, we establish that this spectral diversity can be tuned by varying the lightdependent stability of the second thioether linkage. We also show that such behavior can be engineered into the conventional phytochrome Cph1 from Synechocystis sp. PCC6803. Dual-cysteine photosensors thus allow the phytochrome superfamily in cyanobacteria to sense the full solar spectrum at the earth surface from near infrared to near ultraviolet.biliprotein | optogenetics | photoreceptor | UV-A sensor

Photosensory proteins enable living things to detect the quantity and quality of their light environment and to transduce that physical signal into biochemical outputs which entrain their metabolism with the ambient light environment. Phytochromes, which photoconvert between red-absorbing Pr and far-red-absorbing Pfr states, have been the most extensively studied of these interesting proteins. Critical regulators of a number of key adaptive processes in higher plants, including photomorphogenesis and shade avoidance, phytochromes are widespread in photosynthetic and nonphotosynthetic bacteria and even in fungi. Cyanobacterial genomes also possess a plethora of more distant relatives of phytochromes known as cyanobacteriochromes (CBCRs). Biochemical characterization of representative CBCRs has demonstrated that this class of photosensors exhibit a broad range of wavelength sensitivities, spanning the entire visible spectrum. Distinct protein-bilin interactions are responsible for this astonishing array of wavelength sensitivities. Despite this spectral diversity, all members of the extended family of phytochrome photosensors appear to share a common photochemical mechanism for light sensing: photoisomerization of the 15/16 double bond of the bilin chromophore.

Phytochromes are red/far-red photosensory proteins that regulate adaptive responses to light via photoswitching of cysteine-linked linear tetrapyrrole (bilin) chromophores. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. CBCRs and phytochromes share a conserved Cys residue required for bilin attachment. In one CBCR subfamily, often associated with a blue/green photocycle, a second Cys lies within a conserved Asp-Xaa-Cys-Phe (DXCF) motif and is essential for the blue/green photocycle. Such DXCF CBCRs use isomerization of the phycocyanobilin (PCB) chromophore into the related phycoviolobilin (PVB) to shorten the conjugated system for sensing green light. We here use recombinant expression of individual CBCR domains in Escherichia coli to survey the DXCF subfamily from the cyanobacterium Nostoc punctiforme. We describe ten new photoreceptors with well-resolved photocycles and three additional photoproteins with overlapping dark-adapted and photoproduct states. We show that the ability of this subfamily to form PVB or retain PCB provides a powerful mechanism for tuning the photoproduct absorbance, with blue-absorbing dark states leading to a broad range of photoproducts absorbing teal, green, yellow, or orange light. Moreover, we use a novel green/teal CBCR that lacks the blue-absorbing dark state to demonstrate that PVB formation requires the DXCF Cys residue. Our results demonstrate that this subfamily exhibits much more spectral diversity than had been previously appreciated.

Plants constantly monitor their light environment in order to grow and develop optimally, in part through use of the phytochromes, which sense red/far-red light. A phytochrome binding protein, PKS1 (phytochrome kinase substrate 1), was identified that is a substrate for light-regulated phytochrome kinase activity in vitro. In vivo experiments suggest that PKS1 is phosphorylated in a phytochrome-dependent manner and negatively regulates phytochrome signaling. The data suggest that phytochromes signal by serine-threonine phosphorylation.

Photochemical interconversion between the red-absorbing (Pr) and the far-red-absorbing (P fr) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The Pr-to-Pfr transition commences with a rapid Z-to-E photoisomerization at the C 15AC16 methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C15-H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted CAC stretch and NOH in-plane rocking modes within 500 fs, reveal that initial distortion of the C 15AC16 bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to Pr in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C 15-H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C15AC16 Z-to-E isomerization occurs on the excited-state surface.photochemistry ͉ photoisomerization ͉ photosensory proteins ͉ plant signal transduction ͉ time-resolved vibrational spectroscopy L ight sensing and signaling responses mediated by photoreceptors are critical for the survival and growth of all life forms. Phytochromes are a class of biliprotein photoreceptors found in plants, bacteria, and fungi that are capable of sensing red/far-red light via interconversion between red-absorbing (P r ) and far-redabsorbing (P fr ) forms ( Fig. 1) (1). Light absorption by phytochrome triggers a rapid and reversible Z-to-E isomerization of the C 15 AC 16 methine bridge between the C and D rings of its bilin chromophore (2). This photochemistry subsequently drives changes in protein conformation that lead to changes in gene expression that influence growth and development (1, 3). Temporal resolution of the structure of the bilin chromophore during the photoisomerization process is important, not only to unravel common themes underlying ultrafast dynamics of biological reactions, but also for designing synthetic light-harvesting systems with rapid response times, efficient sensing, and energy storage.In the past, ultrafast pump-probe electronic spectroscopy has been used to probe the excited-state dynamics of plant and cyanobacterial (Cph1) phytochromes. Such studies reveal that formation of the isomerized primary photoproduct Lumi-R, characterized by a red-shifted electronic absorption maximum at 700 nm, occurs 25-40 ps after excitation (4-10). The P r excited state exhibits multiexponential fluorescence decay dynamics with at least two lifetimes (10 ps and 45 ps) (5), thereby implicating the presence of at least two excited states. The two-state model for P r * decay has also received support from ultrafast transient absorption measurements on the plant phytochrome phyA, the cyanobacterial phytochrome Cph1, and the bacteriophytochrome Agp1, all of which exhibit two ...