Evidence for Exaptation of the Marchantia polymorpha M20D Peptidase MpILR1 into the Tracheophyte Auxin Regulatory Pathway.

Auxin homeostasis is tightly regulated by several mechanisms, including conjugation of the hormone to specific moieties, such as amino acids or sugar. The inactive phytohormone conjugate is stored in large pools in plants and hydrolyzed to regain full activity. Many conjugate hydrolases (M20D metallopeptidases) have been identified and characterized throughout the plant kingdom. We have traced this regulatory gene family back to liverwort (Marchantia polymorpha), a member of the most ancient extant land plant lineage, which emerged approximately 475 million years ago. We have isolated and characterized a single hydrolase homologue, dubbed M. polymorpha IAA-Leucine Resistant1 (MpILR1), from liverwort. MpILR1 can hydrolyze two auxin (indole acetic acid [IAA]) substrates (IAA-Leucine and IPA-Alanine) at very low levels of activity, but it cannot hydrolyze the two native auxin conjugates of liverwort (IAA-Glycine and IAA-Valine). We conclude from these results that liverwort likely does not employ active auxin conjugate hydrolysis as a regulatory mechanism and that conjugate homeostasis likely takes place in liverwort by passive background degradation. Furthermore, we present evidence that MpILR1 was probably exapted by tracheophytes over evolutionary time into the auxin regulatory pathway.

A phylogenetic examination of the primary anthocyanin production pathway of the Plantae.

BACKGROUND: Anthocyanin pigments aid in reproduction and provide ultraviolet protection to land plants. We have examined the phylogenetic relationships among the five primary enzymes responsible for producing anthocyanin pigment in its three major forms. Dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), Flavonoid 3'glucosyltransferase (F3GT), flavonoid 3'hydroxylase (F3'H), and flavonoid 3'5' hydroxylase (F3'5'H) are responsible for the final steps in anthocyanin pigment production. RESULTS: We were interested in how conserved the anthocyanin pathway genes may be among land plants, and evolutionarily how far back into the plant lineage anthocyanin production may be traced. The DFR, ANS, F3GT, and F3'H genes date back 450 million years to the first land plants. Mosses, spike mosses, and ferns express these four products, although there is no evidence of sequence orthologues for these genes in algae. Additionally, F3'5'H is not evident in organisms that predated gymnosperms. CONCLUSION: Our findings support the hypothesis that "blue" anthocyanin pigments did not evolve until 300-350 mya along with the gymnosperms, although the "red" anthocyanin pigments may be as ancient as the mosses (~450 mya).

Population structure of Zostera marina (eelgrass) on the western Atlantic coast is characterized by poor connectivity and inbreeding.

Zostera marina (eelgrass) can be found in the North Atlantic on the coast of Europe and on the east and west coasts of North America. Over the last 30 years, this once robust species has been reduced to sparse patchy populations due to disease and anthropogenic effects. In order to better understand the consequences of this devastation on the population genetics of the species, we have analyzed the population structure of western Atlantic Z. marina, employing microsatellite DNA polymorphisms. Although high fixation index values suggest moderate genetic differentiation among most of the Z. marina sites, population diversity was low. This lack of diversity was supported by a general dearth of observable heterozygotes in these sites; mean observed heterozygosity values (0.14-0.46) were lower than the mean expected heterozygosity values (0.57-0.81). Additionally, the mean F(IS) (coefficient of local inbreeding) values in these sites were positive, again indicating a surfeit of homozygotes. Allelic richness suggests that Chesapeake Bay has the greatest internal genetic diversity of the sites studied. Inbreeding seems prevalent in these American populations, suggesting possible reproductive fitness problems in the future. There is evidence of demographic bottlenecking and particularly low genetic diversity in Long Island. Northern Maine had the highest effective population size, suggesting a possible use in future restoration projects.

Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli.

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV).

A minimally invasive method of piscine tissue collection and an analysis of long-term field-storage conditions for samples.

BACKGROUND: The acquisition of high-quality DNA for use in phylogenetic and molecular population genetic studies is a primary concern for evolutionary and genetic researchers. Many non-destructive DNA sampling methods have been developed and are used with a variety of taxa in applications ranging from genetic stock assessment to molecular forensics. RESULTS: The authors have developed a field sampling method for obtaining high-quality DNA from sunfish (Lepomis) and other freshwater fish that employs a variation on the buccal swab method and results in the collection of DNA suitable for PCR amplification and polymorphism analysis. Additionally, since the circumstances of storage are always a concern for field biologists, the authors have tested the potential storage conditions of swabbed samples and whether those conditions affect DNA extraction and PCR amplification. It was found that samples stored at room temperature in the dark for over 200 days could still yield DNA suitable for PCR amplification and polymorphism detection. CONCLUSION: These findings suggest that valuable molecular genetic data may be obtained from tissues that have not been treated or stored under optimal field conditions. Furthermore, it is clear that the lack of adequately low temperatures during transport and long term storage should not be a barrier to anyone wishing to engage in field-based molecular genetic research.

A phylogenomic study of the genus Alphavirus employing whole genome comparison.

The phylogenetics of the genus Alphavirus have historically been characterized using partial gene, single gene or partial proteomic data. We have mined cDNA and amino acid sequences from GenBank for all fully sequenced and some partially sequenced alphaviruses and generated phylogenomic analyses of the genus Alphavirus genus, employing capsid encoding structural regions, non-structural coding regions and complete viral genomes. Our studies support the presence of the previously reported recombination event that produced the Western Equine Encephalitis clade, and confirm many of the patterns of geographic radiation and divergence of the multiple species. Our data suggest that the Salmon Pancreatic Disease Virus and Sleeping Disease Virus are sufficiently divergent to form a separate clade from the other alphaviruses. Also, unlike previously reported studies employing limited sequence data for correlation of phylogeny, our results indicate that the Barmah Forest Virus and Middelburg Virus appear to be members of the Semliki Forest clade. Additionally, our analysis indicates that the Southern Elephant Seal Virus is part of the Semliki Forest clade, although still phylogenetically distant from all known members of the genus Alphavirus. Finally, we demonstrate that the whole Rubella viral genome provides an ideal outgroup for phylogenomic studies of the genus Alphavirus.

A novel auxin conjugate hydrolase from wheat with substrate specificity for longer side-chain auxin amide conjugates.

This study investigates how the ILR1-like indole acetic acid (IAA) amidohydrolase family of genes has functionally evolved in the monocotyledonous species wheat (Triticum aestivum). An ortholog for the Arabidopsis IAR3 auxin amidohydrolase gene has been isolated from wheat (TaIAR3). The TaIAR3 protein hydrolyzes negligible levels of IAA-Ala and no other IAA amino acid conjugates tested, unlike its ortholog IAR3. Instead, TaIAR3 has low specificity for the ester conjugates IAA-Glc and IAA-myoinositol and high specificity for the conjugates of indole-3-butyric acid (IBA-Ala and IBA-Gly) and indole-3-propionic-acid (IPA-Ala) so far tested. TaIAR3 did not convert the methyl esters of the IBA conjugates with Ala and Gly. IBA and IBA conjugates were detected in wheat seedlings by gas chromatography-mass spectrometry, where the conjugate of IBA with Ala may serve as a natural substrate for this enzyme. Endogenous IPA and IPA conjugates were not detected in the seedlings. Additionally, crude protein extracts of wheat seedlings possess auxin amidohydrolase activity. Temporal expression studies of TaIAR3 indicate that the transcript is initially expressed at day 1 after germination. Expression decreases through days 2, 5, 10, 15, and 20. Spatial expression studies found similar levels of expression throughout all wheat tissues examined.

MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.

BACKGROUND: The rapid increase in the amount of protein and DNA sequence information available has become almost overwhelming to researchers. So much information is now accessible that high-quality, functional gene analysis and categorization has become a major goal for many laboratories. To aid in this categorization, there is a need for non-commercial software that is able to both align sequences and also calculate pairwise levels of similarity/identity. RESULTS: We have developed MatGAT (Matrix Global Alignment Tool), a simple, easy to use computer application that generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. CONCLUSIONS: The advantages of this program over other software are that it is open-source freeware, can analyze a large number of sequences simultaneously, can visualize both sequence alignment and similarity/identity values concurrently, employs global alignment in calculations, and has been formatted to run under both the Unix and the Microsoft Windows Operating Systems. We are presently completing the Macintosh-based version of the program.

A molecular phylogenomic analysis of the ILR1-like family of IAA amidohydrolase genes.

The ILR1-like family of hydrolase genes was initially isolated in Arabidopsis thaliana and is thought to help regulate levels of free indole-3-acetic-acid.We have investigated how this family has evolved in dicotyledon, monocotyledon and gymnosperm species by employing the GenBank and TIGR databases to retrieve orthologous genes. The relationships among these sequences were assessed employing phylogenomic analyses to examine molecular evolution and phylogeny. The members of the ILR1-like family analysed were ILL1, ILL2, ILL3, ILL6, ILR1 and IAR3. Present evidence suggests that IAR3 has undergone the least evolution and is most conserved. This conclusion is based on IAR3 having the largest number of total interspecific orthologues, orthologous species and unique orthologues. Although less conserved than IAR3, DNA and protein sequence analyses of ILL1 and ILR1 suggest high conservation. Based on this conservation, IAR3, ILL1 and ILR1 may have had major roles in the physiological evolution of 'higher' plants. ILL3 is least conserved, with the fewest orthologous species and orthologues. The monocotyledonous orthologues for most family-members examined have evolved into two separate molecular clades from dicotyledons, indicating active evolutionary change. The monocotyledon clades are: (a) those possessing a putative endoplasmic reticulum localizing signal; and (b) those that are putative cytoplasmic hydrolases. IAR3, ILL1 and ILL6 are all highly orthologous to a gene in the gymnosperm Pinus taeda, indicating an ancient enzymatic activity. No orthologues could be detected in Chlamydomonas, moss and fern databases.