Widespread ancient whole-genome duplications in Malpighiales coincide with Eocene global climatic upheaval.

Whole-genome duplications (WGDs) are widespread and prevalent in vascular plants and frequently coincide with major episodes of global and climatic upheaval, including the mass extinction at the Cretaceous-Tertiary boundary (c. 65 Ma) and during more recent periods of global aridification in the Miocene (c. 10-5 Ma). Here, we explore WGDs in the diverse flowering plant clade Malpighiales. Using transcriptomes and complete genomes from 42 species, we applied a multipronged phylogenomic pipeline to identify, locate, and determine the age of WGDs in Malpighiales using three means of inference: distributions of synonymous substitutions per synonymous site (Ks ) among paralogs, phylogenomic (gene tree) reconciliation, and a likelihood-based gene-count method. We conservatively identify 22 ancient WGDs, widely distributed across Malpighiales subclades. Importantly, these events are clustered around the Eocene-Paleocene transition (c. 54 Ma), during which time the planet was warmer and wetter than any period in the Cenozoic. These results establish that the Eocene Climatic Optimum likely represents a previously unrecognized period of prolific WGDs in plants, and lends further support to the hypothesis that polyploidization promotes adaptation and enhances plant survival during episodes of global change, especially for tropical organisms like Malpighiales, which have tight thermal tolerances.

Massive mitochondrial gene transfer in a parasitic flowering plant clade.

Recent studies have suggested that plant genomes have undergone potentially rampant horizontal gene transfer (HGT), especially in the mitochondrial genome. Parasitic plants have provided the strongest evidence of HGT, which appears to be facilitated by the intimate physical association between the parasites and their hosts. A recent phylogenomic study demonstrated that in the holoparasite Rafflesia cantleyi (Rafflesiaceae), whose close relatives possess the world's largest flowers, about 2.1% of nuclear gene transcripts were likely acquired from its obligate host. Here, we used next-generation sequencing to obtain the 38 protein-coding and ribosomal RNA genes common to the mitochondrial genomes of angiosperms from R. cantleyi and five additional species, including two of its closest relatives and two host species. Strikingly, our phylogenetic analyses conservatively indicate that 24%-41% of these gene sequences show evidence of HGT in Rafflesiaceae, depending on the species. Most of these transgenic sequences possess intact reading frames and are actively transcribed, indicating that they are potentially functional. Additionally, some of these transgenes maintain synteny with their donor and recipient lineages, suggesting that native genes have likely been displaced via homologous recombination. Our study is the first to comprehensively assess the magnitude of HGT in plants involving a genome (i.e., mitochondria) and a species interaction (i.e., parasitism) where it has been hypothesized to be potentially rampant. Our results establish for the first time that, although the magnitude of HGT involving nuclear genes is appreciable in these parasitic plants, HGT involving mitochondrial genes is substantially higher. This may represent a more general pattern for other parasitic plant clades and perhaps more broadly for angiosperms.

Developmental origins of the world's largest flowers, Rafflesiaceae.

Rafflesiaceae, which produce the world's largest flowers, have captivated the attention of biologists for nearly two centuries. Despite their fame, however, the developmental nature of the floral organs in these giants has remained a mystery. Most members of the family have a large floral chamber defined by a diaphragm. The diaphragm encloses the reproductive organs where pollination by carrion flies occurs. In lieu of a functional genetic system to investigate floral development in these highly specialized holoparasites, we used comparative studies of structure, development, and gene-expression patterns to investigate the homology of their floral organs. Our results surprisingly demonstrate that the otherwise similar floral chambers in two Rafflesiaceae subclades, Rafflesia and Sapria, are constructed very differently. In Rafflesia, the diaphragm is derived from the petal whorl. In contrast, in Sapria it is derived from elaboration of a unique ring structure located between the perianth and the stamen whorl, which, although developed to varying degrees among the genera, appears to be a synapomorphy of the Rafflesiaceae. Thus, the characteristic features that define the floral chamber in these closely related genera are not homologous. These differences refute the prevailing hypothesis that similarities between Sapria and Rafflesia are ancestral in the family. Instead, our data indicate that Rafflesia-like and Sapria-like floral chambers represent two distinct derivations of this morphology. The developmental repatterning we identified in Rafflesia, in particular, may have provided architectural reinforcement, which permitted the explosive growth in floral diameter that has arisen secondarily within this subclade.

Phylogenomics and a posteriori data partitioning resolve the Cretaceous angiosperm radiation Malpighiales.

The angiosperm order Malpighiales includes ~16,000 species and constitutes up to 40% of the understory tree diversity in tropical rain forests. Despite remarkable progress in angiosperm systematics during the last 20 y, relationships within Malpighiales remain poorly resolved, possibly owing to its rapid rise during the mid-Cretaceous. Using phylogenomic approaches, including analyses of 82 plastid genes from 58 species, we identified 12 additional clades in Malpighiales and substantially increased resolution along the backbone. This greatly improved phylogeny revealed a dynamic history of shifts in net diversification rates across Malpighiales, with bursts of diversification noted in the Barbados cherries (Malpighiaceae), cocas (Erythroxylaceae), and passion flowers (Passifloraceae). We found that commonly used a priori approaches for partitioning concatenated data in maximum likelihood analyses, by gene or by codon position, performed poorly relative to the use of partitions identified a posteriori using a Bayesian mixture model. We also found better branch support in trees inferred from a taxon-rich, data-sparse matrix, which deeply sampled only the phylogenetically critical placeholders, than in trees inferred from a taxon-sparse matrix with little missing data. Although this matrix has more missing data, our a posteriori partitioning strategy reduced the possibility of producing multiple distinct but equally optimal topologies and increased phylogenetic decisiveness, compared with the strategy of partitioning by gene. These approaches are likely to help improve phylogenetic resolution in other poorly resolved major clades of angiosperms and to be more broadly useful in studies across the Tree of Life.

Horizontal transfer of expressed genes in a parasitic flowering plant.

BACKGROUND: Recent studies have shown that plant genomes have potentially undergone rampant horizontal gene transfer (HGT). In plant parasitic systems HGT appears to be facilitated by the intimate physical association between the parasite and its host. HGT in these systems has been invoked when a DNA sequence obtained from a parasite is placed phylogenetically very near to its host rather than with its closest relatives. Studies of HGT in parasitic plants have relied largely on the fortuitous discovery of gene phylogenies that indicate HGT, and no broad systematic search for HGT has been undertaken in parasitic systems where it is most expected to occur. RESULTS: We analyzed the transcriptomes of the holoparasite Rafflesia cantleyi Solms-Laubach and its obligate host Tetrastigma rafflesiae Miq. using phylogenomic approaches. Our analyses show that several dozen actively transcribed genes, most of which appear to be encoded in the nuclear genome, are likely of host origin. We also find that hundreds of vertically inherited genes (VGT) in this parasitic plant exhibit codon usage properties that are more similar to its host than to its closest relatives. CONCLUSIONS: Our results establish for the first time a substantive number of HGTs in a plant host-parasite system. The elevated rate of unidirectional host-to- parasite gene transfer raises the possibility that HGTs may provide a fitness benefit to Rafflesia for maintaining these genes. Finally, a similar convergence in codon usage of VGTs has been shown in microbes with high HGT rates, which may help to explain the increase of HGTs in these parasitic plants.

Molecular mechanisms for mammalian melanogenesis. Comparison with insect cuticular sclerotization.

Melanogenesis is an important biochemical process for the production of skin pigments which protect many animals from the damage of solar radiation. The abnormalities in melanogenesis are associated with albinism, vitiligo, as well as malignant melanoma in humans. In the lower forms of animals viz., insects, the exoskeleton is hardened to protect their soft bodies by a process called sclerotization, which is often accompanied by melanization. Recent advances in the biochemistry of sclerotization and melanization reveal remarkable similarity between these two processes. The seven stages of sclerotization are: (a) enzymatic oxidation of N-acyldopamine, (b) Michael-1,4-addition reactions of N-acyldopamine quinone, (c) tautomerization of quinone to quinone methide, (d) Michael-1,6-addition of quinone methides, (e) tautomerization of N-acyldopamine quinone methide to 1,2-dehydro-N-acyldopamine, (f) enzymatic oxidation of 1,2-dehydro-N-acyldopamine, and (g) the reactions of resultant quinonoid compounds. Amazingly, striking similarities in the reaction sequences are found in the melanization process starting from dopa. These comparisons predict a central role for quinone methides as reactive intermediates during melanization. Accordingly, recent studies provide increasing evidence in favor of this proposition.

A novel quinone: quinone methide isomerase generates quinone methides in insect cuticle.

Insoluble cuticle-bound enzyme(s) of Manduca sexta pharate pupae, which is known to convert N-acetyldopamine to N-acetylnorepinephrine through the intermediate formation of quinone methide, also converted exogenously supplied N-acetyldopamine quinone to N-acetylnorepinephrine. The presence of a quinone trap such as N-acetylcysteine in the reaction mixture containing N-acetyldopamine and cuticle prevented the formation of N-acetylnorepinephrine but readily yielded N-acetylcysteine-N-acetyldopamine quinone adduct as a dead-end product. These results indicate the oxidation of N-acetyldopamine to its quinone and its enzyme-catalyzed isomerization to quinone methide before yielding N-acetylnorepinephrine as the stable product. The role of this newly discovered isomerase in sclerotization of insect cuticle is discussed.

Quinone methide as a reactive intermediate formed during the biosynthesis of papiliochrome II, a yellow wing pigment of papilionid butterflies.

Mushroom tyrosinase and the recently identified, 4-alkyl-o-benzoquinone: 2-hydroxy-p-quinone methide isomerase were used to investigate the mechanism of biosynthesis of papiliochrome II pigment found in the yellow scales of the papilionid butterflies. Incubation of N-beta-alanyldopamine (NBAD) and L-kynurenine with mushroom tyrosinase resulted in the formation of adducts tentatively characterized as NBAD quinone-L-kynurenine adducts. If quinone isomerase was included in this reaction mixture, the formation of two new products could be witnessed. These two products exhibited the same retention time and the same UV and visible spectral properties as those of papiliochrome II diastereoisomers. Since quinone isomerase catalyzes the conversion of quinones to quinone methides, the above studies indicate that papiliochrome II biosynthesis involves non-enzymatic and hence non-stereoselective condensation of enzymatically generated NBAD quinone methide with L-kynurenine.

Protease inhibitor controls prophenoloxidase activation in Manduca sexta.

Prophenoloxidase from the hemolymph of tobacco hornworm Manduca sexta can be activated by a specific activating enzyme found in the cuticle. Inhibition studies with benzamidine, diisopropyl phosphofluoridate and p-nitrophenyl-p'-guanidinobenzoate indicate that the activating enzyme is a trypsin-like serine protease. An endogenous protease inhibitor, isolated from the hemolymph of Manduca larvae, inhibits the prophenoloxidase activation mediated by this enzyme. These results indicate that the probable physiological role of endogenous protease inhibitor is to control the undesired activation of prophenoloxidase in the hemolymph.

o-quinone/quinone methide isomerase: a novel enzyme preventing the destruction of self-matter by phenoloxidase-generated quinones during immune response in insects.

Melanization and encapsulation of invading foreign organisms observed during the immune response in insects is known to be due to the action of activated phenoloxidase. Phenoloxidase-generated quinones are deposited either directly or after self-polymerization on foreign objects accounting for the observed reactions. Since the reactions of quinones are nonenzymatic, they do not discriminate self from nonself and hence will also destroy self-matter. In this report we present evidence for the presence of a novel quinone/quinone methide isomerase in the hemolymph of Sarcophaga bullata which destroys long-lived quinones and hence acts to protect the self-matter. Quinone methides, formed by the action of this enzyme on physiologically important quinones, being unstable undergo rapid hydration to form nontoxic metabolites.

Trapping of transiently formed quinone methide during enzymatic conversion of N-acetyldopamine to N-acetylnorepinephrine.

We have demonstrated that quinone methide formation is an important aspect of insect physiology and proposed that enzymatically generated quinone methides react nonenzymatically with water or other nucleophiles to form Michael-1,6-addition products [(1988) Adv. Insect Physiol. 21, 179-231; (1989) J. Cell. Biochem. suppl. 13C, 58]. Using a purified o-quinone isomerase from the larval cuticle of Sacrophaga bullata and mushroom tyrosinase, we now demonstrate that transiently formed N-acetyldopamine quinone methide from N-acetyldopamine can be trapped by methanol to produce beta-methoxy N-acetyldopamine. The methanol adduct thus formed was found to be a racemic mixture and can be resolved into the optical isomers on cyclodextrin chiral column. These results confirm our contention that enzymatically generated quinone methides are nonenzymatically and nonstereoselectively transformed to Michael-1,6-adducts by reaction with water or other nucleophiles.

N-acetyldopamine quinone methide/1,2-dehydro-N-acetyl dopamine tautomerase. A new enzyme involved in sclerotization of insect cuticle.

The enzyme system causing the side chain desaturation of the sclerotizing precursor, N-acetyldopamine (NADA), was solubilized from the larval cuticle of Sarcophaga bullata and resolved into three components. The first enzyme, phenoloxidase, catalyzed conversion of NADA to NADA quinone and provided it for the second enzyme (NADA quinone isomerase), which makes the highly unstable NADA quinone methide. Quinone methide was hydrated rapidly and nonenzymatically to form N-acetylnorepinephrine. In addition, it also served as the substrate for the last enzyme, quinone methide tautomerase, which converted it to 1,2-dehydro-NADA. Reconstitution of NADA side chain desaturase activity was achieved by mixing the last enzyme fraction with NADA quinone isomerase, obtained from the hemolymph of the same organism, and mushroom tyrosinase. Therefore, NADA side chain desaturation observed in insects is caused by the combined action of three enzymes rather than the action of a single specific NADA desaturase, as previously thought.

Characterization of a new enzyme system that desaturates the side chain of N-acetyldopamine.

A novel enzyme system that desaturates the side chain of the catecholamine derivative, N-acetyldopamine (NADA), was isolated and characterized from the larval cuticle of Sarcophaga bullata. The NADA desaturase system which converts NADA to 1,2-dehydro-NADA, surprisingly, does not resemble dehydrogenases such as succinate dehydrogenase. It uniquely performs the desaturation reaction by oxidizing NADA to its corresponding quinone and subsequently converting the resultant quinone to 1,2-dehydro-NADA via NADA quinone methide. Accordingly, desaturase enzyme preparation contained both o-diphenoloxidase activity and NADA quinone:NADA quinone methide isomerase activity. In addition, inhibition studies as well as trapping experiments also confirmed the obligatory formation of NADA quinone as the transient intermediate of the NADA desaturation. It is the first report of a cell-free system causing the side chain desaturation of any catecholamine derivative.

Nonenzymatic transformations of enzymatically generated N-acetyldopamine quinone and isomeric dihydrocaffeiyl methyl amide quinone.

We have recently demonstrated that the side chain hydroxylation of N-acetyldopamine and related compounds observed in several insects is caused by a two-enzyme system catalyzing the initial oxidation of catecholamine derivatives and subsequent isomerization of the resultant quinones to isomeric quinone methides, which undergo rapid nonenzymatic hydration to yield the observed products [Saul, S.J. and Sugumaran, M. (1989) FEBS Lett. 249, 155-158]. During our studies on o-quinone/p-quinone methide tautomerase, we observed that quinone methides are also produced nonenzymatically slowly, under physiological conditions. The quinone methide derived from N-acetyldopamine was hydrated to yield N-acetylnorepinephrine as the stable product as originally shown by Senoh and Witkop [(1959) J. Am. Chem. Soc. 81, 6222-6231], while the isomeric quinone methide from dihydrocaffeiyl methylamide exhibited a new reaction to form caffeiyl amide as the stable product. The identity of this product was established by UV and IR spectral studies and by chemical synthesis. We could not find any evidence of intramolecular cyclization of N-acetyldopamine quinone to iminochrome-type compound(s). The importance of quinone methides in these reactions is discussed.

Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata.

Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (K(a)=40 microM) was achieved by the cationic detergent, cetyl pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserved C-terminal region common to all known PPOs.

Biological and toxicological consequences of quinone methide formation.

Quinone methides are a class of reactive, electrophilic compounds which are capable of alkylating cellular macromolecules. They are formed during xenobiotic biotransformation reactions and are hypothesized to mediate the toxicity of a large number of quinone antitumor drugs as well as several alkylphenols. In addition, oxidation of specific endogenous alkylphenols (e.g. coniferyl alcohol) and alkylcatechols (e.g. N-acetyldopamine, dopa) to quinone methides plays an important role in the synthesis of several complex plant and animal polymers, including lignin, cuticle and melanin. The role of quinone methides in these various processes is reviewed.

On the presence of prophenoloxidase in the hemolymph of the horseshoe crab, Limulus.

Melanization, and hence the participation of phenoloxidase, in defense mechanism of arthropods is well established. However, in the living fossil, horseshoe crab, it has been claimed that the prophenoloxidase system widely found in the hemolymph of most arthropods is absent. On the contrary, we present evidence for the presence of a prophenoloxidase system in the hemolymph of Limulus and a method to study its activation. Activation of prophenoloxidase was achieved by treatment with either the anionic detergent, SDS, or the cationic detergent, cetylpyridinium chloride. The detergents seemed to bind to the proenzyme below their critical micellar concentration and induce conformational changes that cause the activation of prophenoloxidase. In addition, a number of fatty acids and phospholipids also activated the prophenoloxidase. Proteases such as trypsin activated the enzyme only marginally. The approximate molecular weight of the proenzyme was found to be 70,000. Substrate specificity studies, product analysis and inhibition experiments revealed that the Limulus enzyme is a typical o-diphenoloxidase. The possible reasons for the failure to detect the phenoloxidase activity by earlier workers are discussed.

Observations on life cycle of certain spiders from Western Ghats of Tamil Nadu.

Survey was conducted, in the different forests ecosystems of Western Ghats of Tamil Nadu in Nilgris, Coimbatore, Erode, Virudhunagar and Tirunelveli districts to collect the adult spiders and study them taxonomically. Fifty-six species of spider collections were made. From the fifty six, biology was studied for six spider species, such as Micrommata virescens n.sp., Oxyopes javanus, Peucetia virridana, Agelena kariansholensis n.sp., Heteropoda venatoria and Olios hampsoni. Biology studies with Peucetia virridana and Micrommata virescens showed that both species took more than 350 days to complete their life cycles. Heteropoda venetoria and Oxyopes javanus took more than 250 days to complete their life cycle. Agelena kariansholensis took 381 days and Olios hampsoni took 345 days to complete their life cycles. 30% of Peucetia virridana and more than 20% of Heteropoda venatoria and Micrommata virescens and 7% of Oxyopes javanus developed into adults in captivity.