Cold Hardiness and Acclimation Intensity in Flower Buds for Fall Bloom and Spring Bloom Clones in Rhododendron kiusianum, a Dwarf Evergreen Azalea

The relationship between the degree of cold hardiness (supercoolingability of florets) and the acclimation intensity in flowerbuds was investigated in the fall bloom and the spring bloom(typical) clones of Rhododendron kiusianum, a hardy dwarf evergreenazalea. Supercooling ability or exotherm temperature distribution(ETD) in florets was determined by differential thermal analysis(DTA) and the intensity of bud acclimation or the rate of deacclimationwas judged by the changes in ETD profiles resulting from thedehardening temperature treatment. Although the two clone typesshowed no significant differences in ETDs and water contentsin florets, they differed in their rates of bud deacclimation.The flower buds of fall bloom clones generally tend to deacclimatemore quickly than the spring bloom ones throughout the seasons.It is concluded that the degree of cold hardiness in flowerbuds of R. kiusianum does not differ between the fall bloomand spring bloom clones but the intensity of bud acclimationdoes; acclimation intensity is higher in the spring bloom clonesand the rate of deacclimation is greater in the fall bloom ones. (Received October 14, 1985; Accepted February 5, 1986)     

Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion

In this work, we examined photoreactivity of synthetic eumelanins, formed by autooxidation of DOPA, or enzymatic oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid and synthetic pheomelanins obtained by enzymatic oxidation of 5‐S‐cysteinyldopa or 1:1 mixture of DOPA and cysteine. Electron paramagnetic resonance oximetry and spin trapping were used to measure oxygen consumption and formation of superoxide anion induced by irradiation of melanin with blue light, and time‐resolved near‐infrared luminescence was employed to determine the photoformation of singlet oxygen between 300 and 600 nm. Both superoxide anion and singlet oxygen were photogenerated by the synthetic melanins albeit with different efficiency. At 450‐nm, quantum yield of singlet oxygen was very low (~10^?4) but it strongly increased in the UV region. The melanins quenched singlet oxygen efficiently, indicating that photogeneration and quenching of singlet oxygen may play an important role in aerobic photochemistry of melanin pigments and could contribute to their photodegradation and photoaging.     

Proteomic analysis for protein carbonyl as an indicator of oxidative damage in senescence-accelerated mice

The senescence-accelerated prone mouse strain 8 (SAMP8) exhibits a remarkable age-accelerated deterioration in learning and memory. In this study, we identified carbonyl modification, a marker of protein oxidation, in liver and brain of SAMP8 from peptide mass fingerprints using MALDI-TOF mass spectrometry in combination with LC-MS/MS analysis. Carbonyl modification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in liver at 3 month and hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp) in brain at 9 month were higher in SAMP8 compared with control SAMR1. We demonstrated carbonyl modification of purified Cu,Zn-SOD increased by the reaction with H₂O₂. Therefore, progressive accumulation of oxidative damage to Cu,Zn-SOD, may cause dysfunction of defense systems against oxidative stress in SAMP8 with a higher oxidative states, leading to acceleration of aging. Furthermore, carbonyl modification of HCNP-pp may be involved in pathophysiological alterations associated with deterioration in the learning and memory in the brain seen in SAMP8.     

Spiny Mice Modulate Eumelanin to Pheomelanin Ratio to Achieve Cryptic Coloration in “Evolution Canyon,” Israel

Background

Coat coloration in mammals is an explicit adaptation through natural selection. Camouflaging with the environment is the foremost evolutionary drive in explaining overall coloration. Decades of enquiries on this topic have been limited to repetitive coat color measurements to correlate the morphs with background/habitat blending. This led to an overwhelming endorsement of concealing coloration as a local phenotypic adaptation in animals, primarily rodents to evade predators. However, most such studies overlooked how rodents actually achieve such cryptic coloration. Cryptic coloration could be attained only through optimization between the yellow- to brown-colored “pheomelanin” and gray to black-colored “eumelanin” in the hairs. However, no study has explored this conjecture yet. “Evolution Canyon” (EC) in Israel is a natural microscale laboratory where the relationship between organism and environment can be explored. EC is comprised of an “African” slope (AS), which exhibits a yellow-brownish background habitat, and a “European” slope (ES), exhibiting a dark grayish habitat; both slopes harbor spiny mice (Acomys cahirinus). Here, we examine how hair melanin content of spiny mice living in the opposing slopes of EC evolves toward blending with their respective background habitat.

Methodology/Principal Findings

We measured hair-melanin (both eumelanin and pheomelanin) contents of 30 spiny mice from the EC using high-performance liquid chromatography (HPLC) that detects specific degradation products of eumelanin and pheomelanin. The melanin pattern of A. cahirinus approximates the background color of the slope on which they dwell. Pheomelanin is slightly (insignificantly) higher in individuals found on the AS to match the brownish background, whereas individuals of the ES had significantly greater eumelanin content to mimic the dark grayish background. This is further substantiated by a significantly higher eumelanin and pheomelanin ratio on the ES than on the AS.

Conclusion/Significance

It appears that rodents adaptively modulate eumelanin and pheomelanin contents to achieve cryptic coloration in contrasting habitats even at a microscale.     

Biochemical and Domain Analyses of FSUAxe6B,a Modular Acetyl Xylan Esterase,Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85

Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser₄₄, His₂₇₃, Glu₁₉₄, and Asp₂₇₀, with both Glu₁₉₄ and Asp₂₇₀ functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.The development of strategies for biomass conversion to fuels (biofuels) is a subject of keen interest as we search for energy resources alternative to fossil fuels (39). Plant cell matter accounts for 150 to 200 billion tons of biomass on our planet annually (31). It is technically possible, but economically far from realization, to convert plant cell wall to biofuels (41). Thus, currently, plant cell wall utilization as a source of biofuels is mostly at the laboratory scale, although there is a great impetus to move production to the industrial scale.The main components of the plant cell wall are cellulose, hemicellulose, and lignin. These components form complex structures that provide the plant with physical strength (42). Biologically, there are two major steps in the production of alcohols from plant-based feedstock. The first step is an enzymatic hydrolysis of the plant cell wall components to fermentable sugars, and the second step is fermentation of the resultant sugars into alcohols. A major limitation of the process is the lack of highly efficient biocatalysts required for the first step. However, it is known that microbes, either as individuals or consortia, that harbor genes encoding enzymes that hydrolyze plant cell wall polysaccharides abound in nature. Research efforts directed at deepening knowledge of how multiple enzymes participate synergistically to degrade the plant cell wall will accelerate the capacity to achieve the goal of converting biomass to biofuels on a large scale (12, 27). However, improvement of “enzyme cocktails” developed for depolymerization of lignocellulosic biomass will be dependent on a better understanding of the structure/function of individual enzymes that together constitute the arsenal of enzymes (hydrolyzome) used by naturally occurring organisms known to be highly efficient in plant cell wall degradation.Ruminant animals harbor a variety of plant cell wall-degrading bacteria in their first stomach or rumen (26). These animals digest forages with the aid of a microbial consortium that is able to metabolize plant cell wall polysaccharides to short-chain fatty acids, the main energy source for the ruminant host. Fibrobacter succinogenes is a ubiquitous rumen bacterium and has been estimated in previous reports to occupy 0.1% to 1.0% of the microbial population in the cattle rumen, based on the quantification of 16S rRNA genes as a marker (25, 43). F. succinogenes is a significant cellulolytic rumen bacterium, and it has the ability to grow on crystalline cellulose as a sole source of carbon and energy (17). Additionally, it has been demonstrated that this bacterium can solubilize hemicelluloses, although it only partially utilized the constituent monosaccharides released (34). As further evidence, F. succinogenes failed to grow on xylose (33), a constituent of most hemicelluloses. Since F. succinogenes is a highly versatile microbe capable of degrading both cellulose and hemicellulose, strains of this bacterium are attractive models to study natural strategies for efficient deconstruction of plant cell wall polysaccharides.Through analysis of the genome sequence of F. succinogenes S85, a gene cluster that encodes more than 10 hemicellulose-targeting enzymes was identified. Most of the enzymes in the cluster are modular polypeptides, a common feature in many carbohydrate-active enzymes. Kam and coworkers (23) previously identified two acetyl xylan esterases (Axe6A and Axe6B) in this cluster and predicted that each gene encoded a polypeptide composed of two domains: an esterase catalytic domain and a family 6 carbohydrate-binding module (CBM6). Whereas Axe6A was fairly well characterized, difficulties in expression of recombinant Axe6B restricted its characterization (23). In this report, overproduction of recombinant F. succinogenes S85 Axe6B (FSUAxe6B) is demonstrated, and furthermore, it is shown that rather than having two domains, the polypeptide harbors three domains composed of an esterase, CBM6, and a region of unknown function. Bioinformatics analysis suggested that the unknown domain observed in FSUAxe6B is, so far, distributed only in F. succinogenes S85; thus, it was designated F. succinogenes-specific paralogous module 1 or FPm-1. Twenty-four polypeptides, with the majority containing glycoside hydrolase family motifs and CBMs, were found to harbor this peptide at the extreme C-terminal region. In addition to assigning a carbohydrate binding function to FPm-1, critical residues that confer esterase activity to the N-terminal half of FSUAxe6B were also identified through site-directed mutagenesis.     

Taphonomic experiments resolve controls on the preservation of melanosomes and keratinous tissues in feathers

Fossils are a key source of data on the evolution of feather structure and function through deep time, but their ability to resolve macroevolutionary questions is compromised by an incomplete understanding of their taphonomy. Critically, the relative preservation potential of two key feather components, melanosomes and keratinous tissue, is not fully resolved. Recent studies suggesting that melanosomes are preferentially preserved conflict with observations that melanosomes preserve in fossil feathers as external moulds in an organic matrix. To date, there is no model to explain the latter mode of melanosome preservation. We addressed these issues by degrading feathers in systematic taphonomic experiments incorporating decay, maturation and oxidation in isolation and combination. Our results reveal that the production of mouldic melanosomes requires interactions with an oxidant and is most likely to occur prior to substantial maturation. This constrains the taphonomic conditions under which melanosomes are likely to be fossilized. Critically, our experiments also confirm that keratinous feather structures have a higher preservation potential than melanosomes under a range of diagenetic conditions, supporting hitherto controversial hypotheses that fossil feathers can retain degraded keratinous structures.     

The mouse ruby‐eye 2d (ru2d/Hps5ru2‐d) allele inhibits eumelanin but not pheomelanin synthesis

The novel mutation named ru2^d/Hps5^ru2‐d, characterized by light‐colored coats and ruby‐eyes, prohibits differentiation of melanocytes by inhibiting tyrosinase (Tyr) activity, expression of Tyr, Tyr‐related protein 1 (Tyrp1), Tyrp2, and Kit. However, it is not known whether the ru2^d allele affects pheomelanin synthesis in recessive yellow (e/Mc1r^e) or in pheomelanic stage in agouti (A) mice. In this study, effects of the ru2^d allele on pheomelanin synthesis were investigated by chemical analysis of melanin present in dorsal hairs of 5‐week‐old mice from F₂ generation between C57BL/10JHir (B10)‐co‐isogenic ruby‐eye 2^d and B10‐congenic recessive yellow or agouti. Eumelanin content was decreased in ruby‐eye 2^d and ruby‐eye 2^d agouti mice, whereas pheomelanin content in ruby‐eye 2^d recessive yellow and ruby‐eye 2^d agouti mice did not differ from the corresponding Ru2^d/‐ mice, suggesting that the ru2^d allele inhibits eumelanin but not pheomelanin synthesis.     

Adaptation of Pelage Color and Pigment Variations in Israeli Subterranean Blind Mole Rats,Spalax Ehrenbergi

Background

Concealing coloration in rodents is well established. However, only a few studies examined how soil color, pelage color, hair-melanin content, and genetics (i.e., the causal chain) synergize to configure it. This study investigates the causal chain of dorsal coloration in Israeli subterranean blind mole rats, Spalax ehrenbergi.

Methods

We examined pelage coloration of 128 adult animals from 11 populations belonging to four species of Spalax ehrenbergi superspecies (Spalax galili, Spalax golani, Spalax carmeli, and Spalax judaei) and the corresponding coloration of soil samples from the collection sites using a digital colorimeter. Additionally, we quantified hair-melanin contents of 67 animals using HPLC and sequenced the MC1R gene in 68 individuals from all four mole rat species.

Results

Due to high variability of soil colors, the correlation between soil and pelage color coordinates was weak and significant only between soil hue and pelage lightness. Multiple stepwise forward regression revealed that soil lightness was significantly associated with all pelage color variables. Pelage color lightness among the four species increased with the higher southward aridity in accordance to Gloger''s rule (darker in humid habitats and lighter in arid habitats). Darker and lighter pelage colors are associated with darker basalt and terra rossa, and lighter rendzina soils, respectively. Despite soil lightness varying significantly, pelage lightness and eumelanin converged among populations living in similar soil types. Partial sequencing of the MC1R gene identified three allelic variants, two of which were predominant in northern species (S. galili and S. golani), and the third was exclusive to southern species (S. carmeli and S. judaei), which might have caused the differences found in pheomelanin/eumelanin ratio.

Conclusion/Significance

Darker dorsal pelage in darker basalt and terra rossa soils in the north and lighter pelage in rendzina and loess soils in the south reflect the combined results of crypsis and thermoregulatory function following Gloger''s rule.