Yuri N. Zhuravlev

NUCLEAR LOCI AND COALESCENT METHODS SUPPORT ANCIENT HYBRIDIZATION AS CAUSE OF MITOCHONDRIAL PARAPHYLY BETWEEN GADWALL AND FALCATED DUCK (ANAS SPP.)

Abstract Many species have mitochondrial DNA lineages that are phylogenetically intermixed with other species, but studies have rarely tested the cause of such paraphyly. In this study, we tested two hypotheses that could explain mitochondrial paraphyly of Holarctic gadwalls (Anas strepera) with respect to Asian falcated ducks (A. falcata). First, hybridization could have resulted in falcated duck mitochondrial DNA (mtDNA) introgressing into the gadwall gene pool. Second, gadwalls and falcated ducks could have diverged so recently that mtDNA lineages have not sorted to reciprocal monophyly. We used coalescent analyses of three independent loci to distinguish between these two hypotheses. Two lines of evidence support introgression. First, analyses of the three loci combined show that some introgression is necessary to explain current genetic diversity in gadwalls. Second, we generated alternative predictions regarding time since divergence estimated from mtDNA: falcated ducks and gadwalls would have diverged between 65,000 and 700,000 years before present (ybp) under the introgression hypothesis and between 11,000 and 76,000 ybp under the incomplete lineage sorting hypothesis. The two independent nuclear introns indicated that these species diverged between 210,000 and 5,200,000 ybp, which did not overlap the predicted time for incomplete lineage sorting. These analyses also suggested that ancient introgression (∼14,000 ybp) has resulted in the widespread distribution and high frequency of falcated-like mtDNA (5.5% of haplotypes) in North America. This is the first study to use a rigorous quantitative framework to reject incomplete lineage sorting as the cause of mitochondrial paraphyly.

Primary structures of two ribonucleases from ginseng calluses - New members of the PR-10 family of intracellular pathogenesis-related plant proteins

The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined. The two sequences differ at 26% of the amino acid positions. Homology was found with a large family of intracellular pathogenesis‐related proteins, food allergens and tree pollen allergens from both dicotyledonous and monocotyledonous plant species. There is about 30% sequence difference with proteins from species belonging to the same plant order (Apiales: parsley and celery), 60% with those from four other dicotyledonous plant orders and about 70% from that of the monocotyledonous asparagus. More thorough evolutionary analyses of sequences lead to the conclusion that the general biological function of members of this protein family may be closely related to the ability to cleave intracellular RNA and that they have an important role in cell metabolism. As the three‐dimensional structure of one of the members of this protein family has been determined recently [Gajhede et al., Nature Struct Biol 3 (1996) 1040–1045], it may be possible to assign active‐site residues in the enzyme molecule and make hypotheses about its mode of action. Structural features in addition to the cellular site of biosynthesis indicate that this family of ribonucleases is very different from previously investigated ones.

The impact of plant rolC oncogene on ginsenoside production by ginseng hairy root cultures

Plasmid constructions containing rolA, rolB and rolC genes, isolated earlier from the TL-DNA of Agrobacterium rhizogenes were used to transform a cell culture (strain 1c) of Panax ginseng. The levels of ginsenosides were measured in the resulting transgenic tissues to evaluate the possible role of rol genes in ginsenoside formation. The ginsenoside content of the hairy root culture of P. ginseng, transformed by wild type A4 plasmid DNA and containing all rol loci, was higher than that of the control 1c culture (5.12-8.92 mg g ˇ1 dry wt), being in the range of 13.23-21.27 mg g ˇ1 dry wt. Ginseng tissue, transgenic for the rolA gene appeared to lose the ability to synthesize ginsenosides since only a trace amount of Re ginse- noside was found in 1c-rolA tissue. 1c-rolB cultures contained at least five times lower ginsenoside levels compared to the initial 1c culture. The ginsenoside content of rolC transgenic roots was about three times higher than that of the respective control. Taking into account the diAerences in cell diAerentiation levels in tissues transformed by rol genes, we compared the ginsenoside levels in rolC roots and tumours. It was found that ginsenoside production in tissues with diAerent levels of diAerentiation is nearly the same. We have concluded that the plant oncogene rolC is responsible for increased ginsenoside formation in ginseng hairy root cultures. # 1998 Elsevier Science Ltd. All rights reserved

Movements of Birds and Avian Influenza from Asia into Alaska

Despite involvement of large numbers of birds, the delivery rate of Asian-origin viruses to North America through Alaska is apparently low.

PHYLOGEOGRAPHY OF THE MALLARD (ANAS PLATYRHYNCHOS): HYBRIDIZATION, DISPERSAL, AND LINEAGE SORTING CONTRIBUTE TO COMPLEX GEOGRAPHIC STRUCTURE

Abstract Population genetic variation in Mallards (Anas platyrhynchos; n = 152) from Western Russia, North Asia, the Aleutian Islands, and mainland Alaska was investigated using 667 base pairs of the 5′-end of the mitochondrial DNA (mtDNA) control region. DNA sequencing revealed two clades that correspond to Avise et al.’s (1990) group A and B mtDNA haplotypes. Group A haplotypes (80.3%) were wide- spread in all localities from Western Russia to Alaska. Group B haplotypes (19.7%), by contrast, were found primarily in mainland Alaska, where they occurred at high frequency (77.4%), but they also occurred at low frequencies (declining east to west) in the Aleutian Islands (11.8%) and the Primorye region of North Asia (4.4%). Group B haplotypes were not observed in Western Russia or elsewhere in North Asia outside Primorye. Consequently, Mallards exhibited substantial genetic structure between Old World and New World (ΦST = 0.4112–0.4956) but possessed little genetic structure within the Old World continental area (ΦST = 0.0018). Nonetheless, when only group A haplotypes were included in the analysis, Mallards from the Aleutian Islands differed (albeit with low levels of divergence) from each of the other three sampled regions in the Old World and New World (ΦST = 0.0728–0.1461, P < 0.05). Mallards inhabit the Aleutian Islands year-round, so these insular populations may be isolated from Asian and North American populations that occur in the Aleutian Islands only during migration. Overall weak phylogeographic structure and low genetic differentiation within Asia, and between Asia and North America when only group A haplotypes were evaluated, is probably explained by large long-term population sizes and significant intra-continental dispersal. The coexistence and nonrandom distribution of two divergent mtDNA haplotype lineages in Alaska, the Aleutian Islands, and the Primorye region of North Asia, but not in Western Russia or elsewhere in North Asia, is consistent with historical and contemporary hybridization and incomplete sorting of A and B mtDNA haplotype lineages in Mallards and closely related species inhabiting the Old World and New World.

MULTILOCUS PHYLOGEOGRAPHY OF A HOLARCTIC DUCK: COLONIZATION OF NORTH AMERICA FROM EURASIA BY GADWALL (ANAS STREPERA)

Abstract More than 100 species of birds have Holarctic distributions extending across Eurasia and North America, and many of them likely achieved these distributions by recently colonizing one continent from the other. Mitochondrial DNA (mtDNA) and five nuclear introns were sequenced to test the direction and timing of colonization for a Holarctic duck, the gadwall (Anas strepera). Three lines of evidence suggest gadwalls colonized North America from Eurasia. First, New World (NW) gadwalls had fewer alleles at every locus and 61% of the allelic richness found in Old World (OW) gadwalls. Second, NW gadwalls had lower mtDNA allelic richness than other NW ducks. Third, coalescent analysis suggested that less than 5% of the ancestral population contributed to NW gadwalls at the time of divergence. Gadwalls likely colonized North America during the Late Pleistocene (∼81,000 years ago), but the confidence interval on that estimate was large (8500–450,000 years ago). Intercontinental gene flow and selection also likely contributed to genetic diversity in gadwalls. This study illustrates the use of multiple loci and coalescent analyses for critically testing a priori hypotheses regarding dispersal and colonization and provides an independent datapoint supporting an OW to NW bias in the direction of colonization.

Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in rolC transgenic cell cultures of Vitis amurensis

Resveratrol, a naturally occurring polyphenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of Vitis amurensis Rupr. with the oncogene rolC of Agrobacterium rhizogenes increased resveratrol production in the two transformed callus cultures 3.7 and 11.9 times. The rolC-transformed calli were capable of producing 0.099% and 0.144% dry weight of resveratrol. We characterized phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in the two rolC transgenic callus cultures of V. amurensis. In the rolC transgenic culture with higher resveratrol content, expression of VaPAL3, VaSTS3, VaSTS4, VaSTS5, VaSTS6, VaSTS8, VaSTS9, and VaSTS10 was increased; while in the rolC culture with lower resveratrol content, expression of VaPAL3 and VaSTS9 was increased. We suggest that transformation of V. amurensis calli with the rolС gene induced resveratrol accumulation via selective enhancement of expression of individual PAL and STS genes involved in resveratrol biosynthesis. We compared the data on PAL and STS gene expression in rolC transgenic calli with the previously obtained results for rolB transgenic calli of V. amurensis. We propose that the transformation of V. amurensis with the rolC and rolB genes of A. rhizogenes increased resveratrol accumulation through different regulatory pathways.

Suppression of Reactive Oxygen Species and Enhanced Stress Tolerance in Rubia cordifolia Cells Expressing the rolC Oncogene

It is known that expression of the Agrobacterium rhizogenes rolC gene in transformed plant cells causes defense-like reactions, such as increased phytoalexin production and expression of pathogenesis-related proteins. In the present study, we examined whether this phenomenon is associated with increased production of reactive oxygen species (ROS). Single-cell assays based on confocal microscopy and fluorogenic dyes (2,7-dichlorofluorescein diacetate and dihydrorhodamine 123) showed reduced steady-state levels of ROS in rolC-expressing Rubia cordifolia cells as compared with normal cells. Paraquat, a ROS inducer, caused significant ROS elevation in normal cells but had little effect on rolC-transformed cells. Likewise, ROS elevation triggered by a light stress was suppressed in transformed cells. Our results indicate that the rolC gene acts as a ROS suppressor in unstressed cells and its expression prevents stress-induced ROS elevations. We detected a two- to threefold increase in tolerance of rolC-transformed cells to salt, heat, and cold treatments. Simultaneously, rolC-transformed cells maintained permanently active defensive status, as found by measuring isochorismate synthase gene expression and anthraquinone production. Thus, the oncogene provoked multiple effects in which ROS production and phytoalexin production were clearly dissociated.

Enhanced resveratrol accumulation in rolB transgenic cultures of Vitis amurensis correlates with unusual changes in CDPK gene expression.

It has been established that transformation of Vitis amurensis callus culture with the plant oncogene rolB of Agrobacterium rhizogenes results in a high level of resveratrol production in the transformed culture. In the present report, we investigated two rolB transgenic V. amurensis cell cultures with different levels of rolB expression and resveratrol production. We examined whether the calcium ion flux and later steps of the calcium-mediated signal transduction pathway play a role in resveratrol biosynthesis in the rolB transgenic cultures. It has been shown that the calcium channel blockers, LaCl(3), verapamil, and niflumic acid, significantly reduced the accumulation of resveratrol in the rolB transgenic cultures. The number of the calcium-dependent protein kinase (CDPK) transcript variants and abundance of some of the transcripts were considerably altered in the rolB transgenic cell cultures, as revealed by frequency analysis of RT-PCR products and real-time PCR. Some unusual CDPK transcripts with deletions and insertions in the kinase domain were isolated from cDNA probes of rolB-transformed cells. These results suggest that active resveratrol biosynthesis in rolB transgenic cultures of V. amurensis is Ca2+ dependent. We propose that the rolB gene has an important role in regulation of calcium-dependent transduction pathways in transformed cells.

The rolB Gene Suppresses Reactive Oxygen Species in Transformed Plant Cells through the Sustained Activation of Antioxidant Defense

The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.