Key Amino Acid Residues Responsible for the Color of Green and Yellow Fluorescent Proteins from the Coral Polyp Zoanthus sp.

Site-directed mutagenesis was used to study the structural basis of color diversity of fluorescent proteins by the example of two closely related proteins from one organism (coral polyp Zoanthus sp.), one of which produces green and the other, yellow fluorescence. As a result, the following conversions of emission colors were performed: from yellow to green, from yellow to a dual color (yellow and green), and from green to yellow. The saltatory character of the spectral transitions and the manifestation of the dual-color fluorescence suggest that chemically different fluorophores are responsible for the green and yellow fluorescence. The simultaneous presence of three residues, Gly63, Lys65, and Asp68, is necessary for the efficient formation of the yellow rather than green fluorophore.     

GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity

Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.     

Role of host fruit color in the behavior of the walnut fly Rhagoletis juglandis

Observations and sticky-trap tests were used to assess the effect of fruit color on the behavior of adult male and female Rhagoletis juglandis Cresson (Diptera: Tephritidae), a tephritid that infests husks of Arizona walnut in southeastern Arizona. In the first experiment, during which flies were observed foraging among walnut models suspended from small walnut trees, models were painted green to appear ripe and uninfested or yellow with brown patches to appear ripe and infested. Flies used for this first experiment were also of two types: prior to observations, one group of flies had access to real walnuts for 1.5 days (prior experience) while the other group of flies was held without real walnut fruits (no prior experience). Regardless of prior experience with real walnut fruits, female flies landed on green models more than yellow/brown models. Experienced males also were more likely to land on green models than on yellow/brown models. More interactions also occurred on green models, because there were more landings.In the field behavioral assay, flies from a natural population given a choice of green, yellow, and yellow/brown models landed most often on green models, and all interactions and oviposition attempts occurred on green models. Flies also distinguished models by color in field sticky trap assays.These results suggest that female response to ripeness cues is innate, while males develop a preference for green based on their encounter rate with females.     

Yellow mutant of the Neotropical green lacewing Chrysoperla externa: trait inheritance and predator performance

The Neotropical green lacewing Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) is a key predator of various small soft‐bodied pest species. Chrysopidae species are known as ‘green lacewings’ due to their overall green body coloration. However, yellow mutant individuals were observed emerging from our lacewing rearing colony. Thus, the mode of inheritance of the yellow trait was studied and the hypothesis of an autosomal recessive allele for yellow color was tested using hybridization and backcrossing techniques. Furthermore, the possible implications of this color variation on specific life‐history characteristics of C. externa and the predation rates of each morph were evaluated. In both yellow and green morphs, basic life‐history characteristics were monitored, including time to hatching and viability of eggs, duration, and viability of larval and pupal stages, emergence rate and survivorship of adults, and fecundity and longevity of females. The yellow and green morphs were indistinguishable with respect to all life‐history traits evaluated and the predation rate of their larvae. Crossing experiments revealed the yellow color to be caused by a homozygous recessive allele, without sex‐linked expression. We conclude that the allele for yellow color is occurring at high frequency in the laboratory colony, supporting the existence of a genetic polymorphism for body ground color.     

青斑蝶Tirumala limniace成虫颜色偏好性研究

【目的】明确青斑蝶Tirumala limniace雌、雄成虫对颜色的偏好性,可以为此蝴蝶人工饲养时的合理环境条件设定提供必要的理论支持。【方法】本研究通过在室内和野外对青斑蝶成虫对6种颜色(白色、红色、绿色、黄色、蓝色和紫色)光源的选择行为进行观察,明确青斑蝶成虫对不同颜色的偏好性。【结果】(1)在室内和在野外雌、雄成虫均优先选择黄色。(2)雌雄成虫在野外无其它光源干扰下对黄色的选择性强于室内。(3)单色光源照明和多种不同颜色光源同时存在下,成虫对颜色的偏好程度存在差异:当只有单色光源照明时,成虫对颜色的偏好程度为黄色>绿色=红色≥蓝色≥紫色;当不同颜色光源同时存在时,成虫对颜色的偏好程度则为黄色>蓝色≥绿色=红色≥紫色。(4)在不同颜色光源同时存在时,配对后的雌成虫表现出了比雄成虫更强的颜色选择性:雌成虫对黄色光源的选择性强于雄成虫,且其未产生颜色选择的成虫数量也少于雄成虫。【结论】青斑蝶成虫存在对黄色选择的颜色恒常性,野外成虫对颜色的选择性强于室内,雌、雄成虫对偏好颜色选择性存在性别差异。     

Requirement for cysteine in the color silver staining of proteins in polyacrylamide gels

To determine whether cysteine residues have a contribution to the mechanism of color silver staining, we silver stained sodium dodecylsulfate polyacrylamide gel electrophoresis separations of proteins which have few or no cysteines. Proteins without cysteine stained negatively (yellow against a yellow background) with silver. Proteins with one or more cysteines stained orange, red, brown, or green/gray depending on the mole percentage of cysteine and whether they contained covalently attached lipids. The colors could not be correlated with the mole percentages of cysteine of these proteins indicating that some components other than cysteine affect the staining color of cysteine-containing proteins. Silver staining of amino acids, sugars, nucleotide bases, or lipopolysaccharide dot-blotted onto nitrocellulose paper implicated adenine, lipids, the basic amino acids, and glutamine, but not sugars or other amino acids in silver/protein complexes.     

Fluorescence microscopic differentiation of monoamines in the hypothalamus and spinal cord of the lamprey,using a new filter system

Summary Using a new filter system for fluorescence microscopy, in the hypothalamic area and spinal cord of the lamprey the yellow fluorescent cells and varicosities could clearly be differentiated from the blue-green fluorescent cells and varicosities. On the basis of the criteria for monoamines, the blue-green fluorescence and the yellow one were due to catecholamine and indolealkylamine (most probably 5-hydroxytryptamine), respectively. This filter system can specially be recommended for observations and color microphotography of monoamine fluorescence in trasmitted-light darkfield fluorescence microscopy.     

Cloning of anthozoan fluorescent protein genes

Many cnidarians display vivid fluorescence under proper lighting conditions. In general, these colors are due to the presence of fluorescent proteins similar to the green fluorescent protein (GFP) originally isolated from the hydrozoan medusa Aequorea victoria (Cnidaria: Hydrozoa). To optimize the search for new fluorescent proteins (FPs), a technique was developed that allows for the rapid cloning and screening of FP genes without the need for a prior knowledge of gene sequence. Using this method, four new FP genes were cloned, a green from Montastraea cavernosa (Anthozoa: Scleractinia: Faviidae), a cyan from Pocillopora damicornis (Anthozoa: Scleractinia: Pocilloporidae), a cyan from Discosoma striata (Anthozoa: Corallimorpharia), and a red from a second Discosoma species. Two additional green FPs were cloned, one from M. cavernosa and one from its congener Montastraea faveolata, from purified cDNA using PCR primers designed for the first M. cavernosa green FP. Each FP has recognizable amino acid sequence motifs that place them conclusively in the GFP protein family. Mutation of these products using a low-stringency PCR protocol followed by screening of large numbers of bacterial colonies allowed rapid creation of mutants with a variety of characteristics, including changes in color, maturation time, and brightness. An enhanced version of the new red FP, DspR1+, matures faster at 30 degrees C than the commercially available DsRed but matures slower than DsRed at 37 degrees C. One of the M. cavernosa green FPs, McaG2, is highly resistant to photobleaching and has a fluorescence quantum yield approximately twice that of EGFP-1.     

卷蛾分索赤眼蜂雌蜂的颜色偏好性

为了确定卷蛾分索赤眼蜂Trichogrammatoidea bactrae Nagaraja 雌蜂的颜色偏好性, 在室内通过在培养皿底部黏贴彩纸的方法测定卷蛾分索赤眼蜂雌蜂对红、 黄、 黑、 紫、 绿、 白、 蓝7种颜色的行为趋性反应。结果表明, 卷蛾分索赤眼蜂雌蜂在红、 黄、 紫、 绿和蓝5种颜色上的滞留时间都极显著地高于对照（P<0.01）, 在黑和白2种颜色上的滞留时间与对照没有显著差异（P>0.05）; 对黄色的首次选择率极显著高于对照(P< 0.01), 对红、 紫、 绿和蓝色的首次选择率均显著高于对照(P＜0.05), 对黑色和白色的首次选择率与对照没有显著差异。当雌蜂分别在黄与红、 紫、 绿和蓝两两颜色之间选择时, 雌蜂在黄色彩纸上的滞留时间显著长于其他4种颜色。当雌蜂对红、 紫、 绿、 蓝和黄色5种颜色一起选择时, 在首次选择率、 滞留次数上5种颜色间都没有明显差异(P＞0.05); 但在红色和蓝色上的滞留时间显著长于紫色(P＜0.05), 在这3种颜色上的滞留时间与在黄色和绿色上的滞留时间均无显著差异(P＞0.05)。卷蛾分索赤眼蜂雌蜂在7种颜色卵卡上分别与透明纸（对照）上的米蛾卵的选择寄生时, 在黄色卵卡上的寄生卵量极显著多于对照(P＜0.01), 黑色卵卡上的寄生卵量极显著少于对照(P＜0.01), 其他5种颜色的卵卡上的寄生卵量与对照没有显著差异(P＞0.05)。结果说明, 卷蛾分索赤眼蜂雌蜂对黄色最为偏好, 其次偏好红、 紫、 绿和蓝色, 较不喜好白色和黑色。     

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

Bimolecular fluorescence complementation (BiFC) is an approach used to analyze protein–protein interaction in vivo, in which non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein are reconstituted to emit fluorescence only when they are brought together by interaction of two proteins to fuse both fragments. A method for simultaneous visualization of two protein complexes by multicolor BiFC with fragments from green fluorescent protein (GFP) and its variants such as cyan and yellow fluorescent proteins (CFP and YFP) was recently reported in animal cells. In this paper we describe a new strategy for simultaneous visualization of two protein complexes in plant cells using the multicolor BiFC with fragments from CFP, GFP, YFP and a red fluorescent protein variant (DsRed-Monomer). We identified nine different BiFC complexes using fragments of CFP, GFP and YFP, and one BiFC complex using fragments of DsRed-Monomer. Fluorescence complementation did not occur by combinations between fragments of GFP variants and DsRed-Monomer. Based on these findings, we achieved simultaneous visualization of two protein complexes in a single plant cell using two colored fluorescent complementation pairs (cyan/red, green/red or yellow/red).     

Yellow–green color polymorphism in males of a pea aphid clone and its genetic pattern

Although most aphid species living on leaves have a green body color, little is known regarding the biosynthetic pathways of green pigments. We found that a clone of the pea aphid, Acyrthosiphon pisum (Harris) produced both green- and yellow-colored males. The females of this clone were green in color, while 8.4% of the males produced were yellow. To date, yellow body color has been reported only in a single mutant clone in A. pisum. To explore the genetic pattern of yellow body color, green or yellow males were mated with green females of the same clone. The hatchability of the eggs sired by yellow males (26.2%) was less than half that of the eggs sired by green males (79.0%). The hatched foundresses of both groups were all green, with no yellow foundresses. Because aphids have an XX-XO sex determination system, color polymorphism in males suggests that male body color may be governed by an X-linked locus. If females possess heterozygosity at the putative locus, they can produce alternative phenotypes in males. The small proportion of yellow males and absence of yellow foundresses imply that the allele responsible for yellow body color has a deleterious effect. The present study suggests that this clone could be used to elucidate the biosynthetic pathways and underlying genetics of green pigments in aphids.     

Color Differential Staining of Nor-Associated Heterochromatic Segments Using Acridine Orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 × SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoicordum fragrans. Fluorescence on other heterocnromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.     

Color differential staining of NOR-associated heterochromatic segments using acridine orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 x SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoscordum fragrans. Fluorescence on other heterochromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.     

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.     

Live visualization of protein synthesis in axonal growth cones by microinjection of photoconvertible Kaede into Xenopus embryos

Photoconvertible fluorescent proteins, such as Kaede, can be switched irreversibly from their native color to a new one. This property can be exploited to visualize de novo mRNA translation, because newly synthesized proteins can be distinguished from preexisting ones by their color. In this protocol, Kaede cDNA linked to the 3' untranslated region (UTR) of beta-actin is delivered into cells fated to become the retina by injection into Xenopus blastomeres. Brief exposure (6-10 s) to UV light (350-410 nm) of Kaede-positive retinal axons/growth cones efficiently converts Kaede from its native green fluorescence to red. The reappearance of the green signal reports the synthesis of new Kaede protein. This approach can be used to investigate the spatiotemporal control of translation of specific mRNAs in response to external stimuli and to test the efficiency of full-length versus mutant UTRs. The 3-d protocol can be adapted for broad use with other photoactivatable fluorescent proteins.     

Discovery of androecium color polymorphism in Epimedium pubescens with habitat preference of anther/pollen color in the genus

Aims Interspecific and intraspecific variation in flower color in natural populations provides an opportunity for us to understand the evolution and maintenance of diversity of floral traits. Compared to corolla color, little is known about the color polymorphism of sexual organs in flowering plants. To explore evolutionary transitions of androecium color and polymorphism within species, interspecific and intraspecific variation in androecium (anther and pollen) color in the genus Epimedium (Berberidaceae) was investigated.Methods To explore the geographical patterns of anther/pollen color variation in Epimedium species, data of 45 species were collected and their phylogeny was constructed based on available DNA sequences. To investigate whether intraspecific variation in androecium color relates to habitat preference, three environmental factors were measured in the field population of Epimedium pubescens in northeastern Sichuan, China, which plants had green or yellow androecia. Vegetative and reproductive traits of this species were compared between the two color morphs.Important findings Androecium (anther and pollen) color polymorphism in field populations of Epimedium pubescens is reported here where nine populations are monomorphic with a green androecium but three populations are dimorphic with individuals having either a green or a yellow androecium. Inflorescence stalk height, stalk diameter, leaf number, flower number and spur length (as well as spur and nectar volume) were not significantly different between two morphs. Compared to the yellow morph, the green morph had relatively larger leaves and anthers, but smaller sepals. The green morph produced more pollen and larger seeds, but the same number of ovules. Seed set was not significantly different between green and yellow morph. Investigations of environmental factors in the color dimorphic populations of E. pubescens indicated that the green morph was more likely to occur in habitats with relatively lower light intensity. The distribution survey of 45 Epimedium species showed that species with a green androecium tended to appear at lower elevations. Comparative phylogenetic analysis showed that transitions from yellow to a green androecium or to androecial color dimorphism occurred at least seven times. This genus, characterized by anther color diversity and containing some species with anther color polymorphism, provides a model system in which to study the evolution and maintenance of colorful sexual organs in flowering plants.     

Landing Surface Color Preferences of Spathius agrili (Hymenoptera: Braconidae), a Parasitoid of Emerald Ash Borer, Agrilus planipennis (Coleoptera: Buprestidae)

The color preferences for landing surfaces were examined for Spathius agrili (Hymenoptera: Braconidae), a parasitic wasp introduced for biocontrol of emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae). Lures with the 3-component pheromone blend of male S. agrili were used to activate upwind flight by virgin female S. agrili in a laminar flow wind tunnel. Paper discs with halves of two different colors (combination pairs of black, white, red, yellow, green, or purple), with the pheromone lure in the center, were tested to quantify preferences for landing on one color over another. Females landed preferentially on green, yellow, and white surfaces, and landed the least frequently on red, black, and purple surfaces. Changes in color preferences due to adjacent colors were observed and discussed.     

mEosFP-based green-to-red photoconvertible subcellular probes for plants

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.