Evolution: The art of deceptive pollination.

Beetle daisies evolved floral spots that mimic female bee flies to entice mate-seeking males for pollination. A new study shows that these deceptive spots emerged through stepwise co-option of multiple genetic elements, shedding light on the origin of complex phenotypic novelties.

Taxon-specific, phased siRNAs underlie a speciation locus in monkeyflowers.

Taxon-specific small RNA loci are widespread in eukaryotic genomes, yet their role in lineage-specific adaptation, phenotypic diversification, and speciation is poorly understood. Here, we report that a speciation locus in monkeyflowers (Mimulus), YELLOW UPPER (YUP), contains an inverted repeat region that produces small interfering RNAs (siRNAs) in a phased pattern. Although the inverted repeat is derived from a partial duplication of a protein-coding gene that is not involved in flower pigmentation, one of the siRNAs targets and represses a master regulator of floral carotenoid pigmentation. YUP emerged with two protein-coding genes that control other aspects of flower coloration as a "superlocus" in a subclade of Mimulus and has contributed to subsequent phenotypic diversification and pollinator-mediated speciation in the descendant species.

To stripe or not to stripe: the origin of a novel foliar pigmentation pattern in monkeyflowers (Mimulus).

The origin of phenotypic novelty is one of the most challenging problems in evolutionary biology. Although genetic regulatory network rewiring or co-option has been widely recognised as a major contributor, in most cases how such genetic rewiring/co-option happens is completely unknown. We have studied a novel foliar pigmentation pattern that evolved recently in the monkeyflower species Mimulus verbenaceus. Through genome-wide association tests using wild-collected samples, experimental crosses of laboratory inbred lines, gene expression analyses, and functional assays, we identified an anthocyanin-activating R2R3-MYB gene, STRIPY, as the causal gene triggering the emergence of the discrete, mediolateral anthocyanin stripe in the M. verbenaceus leaf. Chemical mutagenesis revealed the existence of upstream activators and repressors that form a 'hidden' prepattern along the leaf proximodistal axis, potentiating the unique expression pattern of STRIPY. Population genomics analyses did not reveal signatures of positive selection, indicating that nonadaptive processes may be responsible for the establishment of this novel trait in the wild. This study demonstrates that the origin of phenotypic novelty requires at least two separate phases, potentiation and actualisation. The foliar stripe pattern of M. verbenaceus provides an excellent platform to probe the molecular details of both processes in future studies.

Red Fluorescence of European Hedgehog (Erinaceus europaeus) Spines Results from Free-Base Porphyrins of Potential Microbial Origin.

Bioluminescence has been recognized as an important means for inter- and intra-species communication. A growing number of reports of red fluorescence occurring in keratinaceous materials have become available. The fluorophore(s) in these cases were shown to be, or suspected to be, free base porphyrins. The red fluorescence found in the downs of bustards was associated with inter-species signaling in mate selection. First reported in 1925, we confirm that spines of the European hedgehog (Erinaceus europaeus) when irradiated with UV (365-395 nm) light display red fluorescence localized in the light-colored sections of their proximal ends. Using reflectance fluorescence spectroscopy, we confirmed that the fluorophores responsible for the emission are free-base porphyrins, as suspected in the original report. Base-induced degradation of the spine matrix and subsequent HPLC, UV-vis, and ESI+ mass spectrometry analysis revealed the presence of a mixture of coproporphyrin III and uroporphyrin III as predominant porphyrins and a minor fraction of protoporphyrin IX. Investigation of the spine microbiome uncovered the abundant presence of bacteria known to secrete and/or interconvert porphyrins and that are not present on the non-fluorescing quills of the North American porcupine (Erethizon dorsatum). Given this circumstantial evidence, we propose the porphyrins could originate from commensal bacteria. Furthermore, we hypothesize that the fluorescence may be incidental and of no biological function for the hedgehog.

Repressors of anthocyanin biosynthesis.

Anthocyanins play a variety of adaptive roles in both vegetative tissues and reproductive organs of plants. The broad functionality of these compounds requires sophisticated regulation of the anthocyanin biosynthesis pathway to allow proper localization, timing, and optimal intensity of pigment deposition. While it is well-established that the committed steps of anthocyanin biosynthesis are activated by a highly conserved MYB-bHLH-WDR (MBW) protein complex in virtually all flowering plants, anthocyanin repression seems to be achieved by a wide variety of protein and small RNA families that function in different tissue types and in response to different developmental, environmental, and hormonal cues. In this review, we survey recent progress in the identification of anthocyanin repressors and the characterization of their molecular mechanisms. We find that these seemingly very different repression modules act through a remarkably similar logic, the so-called 'double-negative logic'. Much of the double-negative regulation of anthocyanin production involves signal-induced degradation or sequestration of the repressors from the MBW protein complex. We discuss the functional and evolutionary advantages of this logic design compared with simple or sequential positive regulation. These advantages provide a plausible explanation as to why plants have evolved so many anthocyanin repressors.

Mutational loss of carotenoids in alkaliphilic Bacillus pseudofirmus OF4 results in sensitivity to oxidative stress and growth at high pH.

Alkaliphilic Bacillus pseudofirmus OF4, which has a broad pH growth range of 7.5 to above 10.5, is yellow-pigmented due to carotenoids. Carotenoids contribute to membrane rigidity and can alleviate cellular oxidative stress. This study was undertaken to gain insight into the roles carotenoids play in alkaliphile physiology. Carotenoid content was high in stationary phase and in cells grown nonfermentatively at pH 10.5 A colourless mutant was isolated by the in-frame deletion of a key carotenogenic gene, crtM. In cells grown to stationary phase in a pH 10.5 medium with a suboptimal concentration of Na+, the ∆crtM strain exhibited lower resistance to paraquat and hydrogen peroxide. Preincubation of the mutant in a nutrient-free pH 10.5 buffer revealed a pronounced sensitivity to hydrogen peroxide in growth at pH 7.5. In growth curves in media with optimal or suboptimal nutrient concentrations conducted at 37°, the mutant grew identically to the wild-type at pH 7.5 but its lag time was longer than the wild-type at pH 10.5 and growth was slower when the carbon source, malate, was limiting. When the temperature of the growth curves was lowered to 25°, the mutant no longer had a pH 10.5 phenotype, implicating the effect of carotenoids on membrane rigidity for the pH 10.5 growth phenotype. These results suggest that carotenoids in B. pseudofirmus OF4 play a role in managing oxidative stress when cells are adapting to other stressful conditions such as nutrient limitation while also helping to maintain membrane fluidity/rigidity balance for membrane-linked functions.

Carotenoid composition and conformation in retinal oil droplets of the domestic chicken.

Carotenoid-containing oil droplets in the avian retina act as cut-off filters to enhance colour discrimination. We report a confocal resonance Raman investigation of the oil droplets of the domestic chicken, Gallus gallus domesticus. We show that all carotenoids present are in a constrained conformation, implying a locus in specific lipid binding sites. In addition, we provide proof of a recent conclusion that all carotenoid-containing droplets contain a mixture of all carotenoids present, rather than only a subset of them-a conclusion that diverges from the previously-held view. Our results have implications for the mechanism(s) giving rise to these carotenoid mixtures in the differently-coloured droplets.

Emergence of a floral colour polymorphism by pollinator-mediated overdominance.

Maintenance of polymorphism by overdominance (heterozygote advantage) is a fundamental concept in evolutionary biology. In most examples known in nature, overdominance is a result of homozygotes suffering from deleterious effects. Here we show that overdominance maintains a non-deleterious polymorphism with black, red and white floral morphs in the Alpine orchid Gymnadenia rhellicani. Phenotypic, metabolomic and transcriptomic analyses reveal that the morphs differ solely in cyanidin pigments, which are linked to differential expression of an anthocyanidin synthase (ANS) gene. This expression difference is caused by a premature stop codon in an ANS-regulating R2R3-MYB transcription factor, which is heterozygous in the red colour morph. Furthermore, field observations show that bee and fly pollinators have opposite colour preferences; this results in higher fitness (seed set) of the heterozygous morph without deleterious effects in either homozygous morph. Together, these findings demonstrate that genuine overdominance exists in nature.

Addition of a Carbonyl End Group Increases the Rate of Excited-State Decay in a Carotenoid via Conjugation Extension and Symmetry Breaking.

Steady-state absorption, transient absorption, and transient grating spectroscopies were employed to elucidate the role of a conjugated carbonyl group in the photophysics of carotenoids. Spheroidenone and spheroidene have similar molecular structures and differ only in an additional carbonyl group in spheroidenone. Comparison of the optical responses of these two molecules under similar experimental conditions was used to understand the role of this carbonyl group in the structure. It was found that the carbonyl group has two main effects: first, it dramatically increases the depopulation rate of the excited states of the molecule. The lifetimes of all the excited states of spheroidenone were found to be almost half of the ones for spheroidene. Second, the presence of the carbonyl group in the chain alters the decay mechanism to the symmetry-forbidden S1 state of the molecule, so that the higher vibrational levels of the S1 state are populated much more effectively. It was also revealed that for both molecules, the S2/S x → S1(hot) → S1 decay process is not purely sequential and follows a branched model.

Structural Tuning of Quantum Decoherence and Coherent Energy Transfer in Photosynthetic Light Harvesting.

Photosynthetic organisms capture energy from solar photons by constructing light-harvesting proteins containing arrays of electronic chromophores. Collective excitations (excitons) arise when energy transfer between chromophores is coherent, or wavelike, in character. Here we demonstrate experimentally that coherent energy transfer to the lowest-energy excitons is principally controlled in a light-harvesting protein by the temporal persistence of quantum coherence rather than by the strength of vibronic coupling. In the peridinin-chlorophyll protein from marine dinoflagellates, broad-band two-dimensional electronic spectroscopy reveals that replacing the native chlorophyll a acceptor chromophores with chlorophyll b slows energy transfer from the carotenoid peridinin to chlorophyll despite narrowing the donor-acceptor energy gap. The formyl substituent on the chlorophyll b macrocycle hastens decoherence by sensing the surrounding electrostatic noise. These findings demonstrate how quantum coherence enhances the efficiency of energy transfer despite being very short lived in light-harvesting proteins at physiological temperatures.

Quantum Coherent Excitation Energy Transfer by Carotenoids in Photosynthetic Light Harvesting.

It remains an open question whether quantum coherence and molecular excitons created by delocalization of electronic excited states are essential features of the mechanisms that enable efficient light capture and excitation energy transfer to reaction centers in photosynthetic organisms. The peridinin-chlorophyll a protein from marine dinoflagellates is an example of a light-harvesting system with tightly clustered antenna chromophores in which quantum coherence has long been suspected, but unusually it features the carotenoid peridinin as the principal light absorber for mid-visible photons. We report that broad-band two-dimensional electronic spectroscopy indeed reveals the initial presence of exciton relaxation pathways that enable transfer of excitation from peridinin to chlorophyll a in <20 fs, but the quantum coherence that permits this is very short-lived. Strongly coupled excited-state vibrational distortions of the peridinins trigger a dynamic transition of the electronic structure of the system and a rapid conversion to incoherent energy transfer mechanisms.

Molecular Basis of Overdominance at a Flower Color Locus.

Single-gene overdominance is one of the major mechanisms proposed to explain heterosis (i.e., hybrid vigor), the phenomenon that hybrid offspring between two inbred lines or varieties show superior phenotypes to both parents. Although sporadic examples of single-gene overdominance have been reported over the decades, the molecular nature of this phenomenon remains poorly understood and it is unclear whether any generalizable principle underlies the various cases. Through bulk segregant analysis, chemical profiling, and transgenic experiments, we show that loss-of-function alleles of the FLAVONE SYNTHASE (FNS) gene cause overdominance in anthocyanin-based flower color intensity in the monkeyflower species Mimulus lewisii FNS negatively affects flower color intensity by competing with the anthocyanin biosynthetic enzymes for the same substrates, yet positively affects flower color intensity by producing flavones, the colorless copigments required for anthocyanin stabilization, leading to enhanced pigmentation in the heterozyote (FNS/fns) relative to both homozygotes (FNS/FNS and fns/fns). We suggest that this type of antagonistic pleiotropy (i.e., alleles with opposing effects on different components of the phenotypic output) might be a general principle underlying single-gene overdominance.

Chlorophyll a with a farnesyl tail in thermophilic cyanobacteria.

Photosystem II (PSII) of oxygenic photosynthetic organisms normally contains exclusively chlorophyll a (Chl a) as its major light-harvesting pigment. Chl a canonically consists of the chlorin headgroup with a 20-carbon, 4-isoprene unit, phytyl tail. We have examined the 1.9 Å crystal structure of PSII from thermophilic cyanobacteria reported by Shen and coworkers in 2012 (PDB accession of 3ARC/3WU2). A newly refined electron density map from this structure, presented here, reveals that some assignments of the cofactors may be different from those modeled in the 3ARC/3WU2 structure, including a specific Chl a that appears to have a truncated tail by one isoprene unit. We provide experimental evidence using high-performance liquid chromatography and mass spectrometry for a small population of Chl a esterified to a 15-carbon farnesyl tail in PSII of thermophilic cyanobacteria.

Excitation Energy Transfer by Coherent and Incoherent Mechanisms in the Peridinin-Chlorophyll a Protein.

Excitation energy transfer from peridinin to chlorophyll (Chl) a is unusually efficient in the peridinin-chlorophyll a protein (PCP) from dinoflagellates. This enhanced performance is derived from the long intrinsic lifetime of 4.4 ps for the S2 (11Bu+) state of peridinin in PCP, which arises from the electron-withdrawing properties of its carbonyl substituent. Results from heterodyne transient grating spectroscopy indicate that S2 serves as the donor for two channels of energy transfer: a 30 fs process involving quantum coherence and delocalized peridinin-Chl states and an incoherent, 2.5 ps process initiated by dynamic exciton localization, which accompanies the formation of a conformationally distorted intermediate in 45 fs. The lifetime of the S2 state is lengthened in PCP by its intramolecular charge-transfer character, which increases the system-bath coupling and slows the torsional motions that promote nonradiative decay to the S1 (21Ag-) state.

Torsional Dynamics and Intramolecular Charge Transfer in the S2 (1(1)Bu(+)) Excited State of Peridinin: A Mechanism for Enhanced Mid-Visible Light Harvesting.

Of the carotenoids known in photosynthetic organisms, peridinin exhibits one of the highest quantum efficiencies for excitation energy transfer to chlorophyll (Chl) a acceptors. The mechanism for this enhanced performance involves an order-of-magnitude slowing of the S2 (1(1)Bu(+)) → S1 (2(1)Ag(-)) nonradiative decay pathway compared to carotenoids lacking carbonyl substitution. Using femtosecond transient grating spectroscopy with optical heterodyne detection, we have obtained the first evidence that the nonradiative decay of the S2 state of peridinin is promoted by large-amplitude torsional motions. The decay of an intermediate state termed Sx, which we assign to a twisted form of the S2 state, is substantially slowed by solvent friction in peridinin due to its intramolecular charge transfer (ICT) character.

Femtosecond Heterodyne Transient Grating Studies of Nonradiative Deactivation of the S2 (1(1)Bu(+)) State of Peridinin: Detection and Spectroscopic Assignment of an Intermediate in the Decay Pathway.

Femtosecond heterodyne transient grating spectroscopy was employed to investigate the nonradiative decay pathway from the S2 (1(1)Bu(+)) state to the S1 (2(1)Ag(-)) state of peridinin in methanol solution. Just as previously observed by this laboratory for ß-carotene in benzonitrile, the real (absorption) and imaginary (dispersion) components of the transient grating signal obtained with Fourier transform spectral interferometry from peridinin exhibit ultrafast responses indicating that S2 state decays in 12 fs to produce an intermediate state, Sx. The excited state absorption spectrum from the Sx state of peridinin, however, is found to be markedly blue-shifted from that of ß-carotene because it makes a substantial contribution to the signal observed with 40 fs, 520 nm pulses. The results of a global target analysis and numerical simulations using nonlinear response functions and the multimode Brownian oscillator model support the assignment of Sx to a displaced conformation of the S2 state rather than to a vibrationally excited (or hot) S1 state. The Sx state in peridinin is assigned to a structure with a distorted conjugated polyene backbone moving past an activation-energy barrier between planar and twisted structures on the S2 potential surface. The lengthened lifetime of the Sx state of peridinin in methanol, 900 ± 100 fs, much longer than that typically observed for carotenoids lacking carbonyl substituents, ∼150 fs, can be attributed to the slowing of torsional motions by solvent friction. In peridinin, the system-bath coupling is significantly enhanced over that in ß-carotene solution most likely due to the intrinsic intramolecular charge transfer character it derives from the electron withdrawing nature of the carbonyl substituent. An important additional implication is that the Sx state, and the distorted structures reached subsequently along the torsional gradient on the S2 potential surface, may serve as the principal excitation energy transfer donors to chlorophyll a in the peridinin-chlorophyll a protein from dinoflagellates.

Spectroscopic Investigation of the Carotenoid Deoxyperidinin: Direct Observation of the Forbidden S0 → S1 Transition.

This paper presents a spectroscopic investigation of deoxyperidinin, a synthetic peridinin analogue in which the carbonyl functional group in peridinin was replaced by a nonconjugated methylene group. Steady-state and ultrafast time-resolved absorption and fluorescence spectroscopic experiments are carried out on deoxyperidinin in n-hexane and acetonitrile at room temperature and in 2-methyltetrahydrofuran at 77 K. The spectra of deoxyperidinin have higher vibronic resolution compared to those of peridinin. The higher resolution is due to a substantial reduction in both molecular conformational disorder and inhomogeneous broadening of the spectra of deoxyperidinin compared to peridinin. Features in the steady-state absorption spectrum of deoxyperidinin that are not evident in the spectrum of peridinin are unambiguously assigned to the forbidden S0 (1(1)Ag(-)) → S1 (2(1)Ag(-)) absorption transition. The characteristics of both the steady-state and time-resolved spectra are interpreted using EOM-CCSD, SAC-CI, and MNDO-PSDCI quantum computational formalisms that provided a theoretical framework for understanding the photophysical properties of the molecules.