Leslie R. Goertzen

The CRF domain defines Cytokinin Response Factor proteins in plants

BackgroundCytokinin Response Factors (CRFs) are a small subset of AP2/ERF transcription factor genes shown in Arabidopsis to regulate leaf development as part of the cytokinin signal transduction pathway. This study examines the phylogenetic distribution of CRF genes in other plant species, and attempts to identify the extent of sequence conservation and potential gene function among all CRF genes.ResultsWe identified CRF genes in representatives of all major land plant lineages, including numerous flowering plant taxa in addition to the model systems in which ERF genes have been catalogued. Comparative analysis across this broader sampling has identified strongly conserved amino acid motifs other than the AP2/ERF domain for all CRF proteins as well as signature sequences unique to specific clades of CRF genes. One of these motifs, here designated as the CRF domain, is conserved in and unique to CRF proteins distinguishing them from related genes. We show that this novel domain of approximately 65 amino acids is found in CRF proteins from all groups of land plants and only in CRF genes. Phylogenetic analyses suggest that the evolution of CRF genes has included numerous duplication events. In this phylogenetic context we examine protein evolution including the gain and loss of accessory domains, correlate these molecular evolutionary events with experimental data on cytokinin regulation and speculate on the function and evolution of the CRF domain within AP2/ERF transcription factor proteins. We also tested a prediction drawn from the phylogenetic analyses that four CRF domain containing genes from Tomato, previously unexamined for cytokinin response, are transcriptionally inducible by cytokinin, supporting the link between CRF genes, CRF-specific domains and cytokinin regulation.ConclusionCRF genes can be identified in all lineages of land plants, as a distinct subset of AP2/ERF proteins containing a specific and unique CRF domain. The CRF domain can be used to identify previously unclassified predicted genes or genes identified only as members of the AP2/ERF protein family. CRF domain presence and phylogenetic relatedness to known Arabidopsis CRF genes predicts gene function to some extent.

Expression, in vivo localization and phylogenetic analysis of a pyridoxine 5′-phosphate oxidase in Arabidopsis thaliana

Pyridoxal phosphate (PLP), a vitamin B(6) vitamer, is an essential cofactor for numerous enzymes. Pyridoxine/pyridoxamine phosphate oxidase (PPOX) catalyzes the synthesis of pyridoxal phosphate from pyridoxine phosphate (PNP) and/or pyridoxamine phosphate (PMP). The At5g49970 locus in Arabidopsis thaliana encodes an AtPPOX, a PNP/PMP oxidase. The expression of the AtPPOX gene varied in different tissues of Arabidopsis examined, being up-regulated by light, heat shock, ABA, and ethylene treatments, and down-regulated by exposure to drought and NaCl. Monoclonal antibodies raised against two different domains of AtPPOX recognized different sizes of AtPPOX, suggesting that AtPPOX proteins are produced as splice variants of the AtPPOX gene in Arabidopsis. Phylogenetic analysis of AtPPOX across all domains of life demonstrated that plant AtPPOX homologs have an additional Yjef_N domain preceding the Pyridox_Oxidase domain at the C-terminal end of the protein, while AtPPOX homologs from bacteria, fungi and animals have only Pyridox_Oxidase domain. The presence of the Yjef_N domain in plant AtPPOX homologs suggests that acquisition of this domain, and its fusion with the pyridox_oxidase domain began with the endosymbiotic acquisition of the chloroplast. Bioinformatic analysis suggested that AtPPOX is localized in chloroplast, but the monoclonal antibody could not be used for subcellular localization of this protein. A GFP-AtPPOX fusion construct introduced into the Arabidopsis protoplast confirmed localization of AtPPOX into the chloroplast. An RNAi mutant of AtPPOX showed sensitivity to high light suggesting a role for PPOX in resistance to photooxidative damage, and alteration in root growth in the presence of sucrose suggests a role for PPOX in root development.

Vascular Expression and C-terminal Sequence Divergence of Cytokinin Response Factors in Flowering Plants

Cytokinin response factors (CRFs) are important transcription factors that form a side branch of the cytokinin signaling pathway and have been linked to cytokinin-regulated processes during development. CRF proteins are defined as belonging to a specific transcription factor family by the presence of an AP2/ERF DNA-binding domain and can be distinguished within this family by a group-specific CRF domain involved in protein-protein interactions. Here we further delimit CRFs into five distinct clades (I-V) represented across all major angiosperm lineages. Protein sequences within each clade contain a clade-specific C-terminal region distinct from other CRFs, suggesting ancient evolutionary divergence and specialization within this gene family. Conserved patterns of transcriptional regulation support these clade divisions. Despite these important differences, CRFs appear to show preferential localization or targeting to vascular tissue in quantitative real-time PCR and reporter line analyses of Arabidopsis thaliana and Solanum lycopersicum (tomato). Phloem tissue expression within the vasculature often appears the strongest in CRF reporter lines, and an analysis of CRF promoter sequences revealed conservation and significant enrichment of phloem targeting cis-elements, suggesting a potential role for CRFs in this tissue. An examination of CRF loss-of-function mutants from cytokinin-regulated clades revealed alterations in higher order vein patterning. This supports both the general link of CRFs to vascular tissue and clade-specific differences between CRFs, since alterations in vascular patterning appear to be clade specific. Together these findings indicate that CRFs are potential regulators of developmental processes associated with vascular tissues.

Invasive Wisteria in the Southeastern United States: genetic diversity, hybridization and the role of urban centers

The increasing numbers and negative impacts of invasive species have prompted research on the relationship between human activities and the success of invasive horticultural plants. In this study, we use population genetic relationships to model the escape of a common garden vine, exotic Wisteria, into natural habitats. Urban and naturalized Wisteria populations in Charleston, South Carolina and Tallahassee, Florida were investigated using a combination of chloroplast, mitochondrial and nuclear DNA markers. Fifty-nine of 72 (81.9%) Wisteria collections were hybrids of Wisteria sinensis and W. floribunda. Chi-square analysis of the distribution of shared W. floribunda haplotypes among naturalized and urban populations supports the relationship of time with invasion success. Naturalized populations were more similar to those in historic neighborhoods. The most common haplotype, F1, was encountered 22 times but its distribution was not significantly different between urban and naturalized populations. In contrast, a significantly higher proportion of haplotype F2 found in naturalized populations suggests that selection may also be acting within these populations. Finally, due to extensive human dispersal, there is no relationship between genetic distance and geographical distance among the populations sampled. We conclude that Wisteria’s long history of horticulture, rampant hybridization, and human-aided dispersal are all implicated in the successful ability of these plants to invade natural habitats throughout the USA.

Digitization Workflows for Flat Sheets and Packets of Plants, Algae, and Fungi

Effective workflows are essential components in the digitization of biodiversity specimen collections. To date, no comprehensive, community-vetted workflows have been published for digitizing flat sheets and packets of plants, algae, and fungi, even though latest estimates suggest that only 33% of herbarium specimens have been digitally transcribed, 54% of herbaria use a specimen database, and 24% are imaging specimens. In 2012, iDigBio, the U.S. National Science Foundations (NSF) coordinating center and national resource for the digitization of public, nonfederal U.S. collections, launched several working groups to address this deficiency. Here, we report the development of 14 workflow modules with 7–36 tasks each. These workflows represent the combined work of approximately 35 curators, directors, and collections managers representing more than 30 herbaria, including 15 NSF-supported plant-related Thematic Collections Networks and collaboratives. The workflows are provided for download as Portable Document Format (PDF) and Microsoft Word files. Customization of these workflows for specific institutional implementation is encouraged.

Ex Situ Conservation of the Federally Endangered Plant Species Clematis socialis Kral (Ranunculaceae)

ABSTRACT: The Center for Plant Conservation (CPC) has created sampling guidelines for the ex situ conservation of rare plant species. These guidelines estimate the number of individuals needed to maximize the genetic diversity of the collection according to population genetic theory. For many clonal plant species, knowledge of the number of unique individuals is not easily discerned and application of these guidelines must be based on molecular genetic data. In this paper, we discuss the steps taken in order to meet CPC guidelines for the conservation of a rare clonal plant, Clematis socialis. Due to limited seed availability, methods were developed for successful in vitro propagation and cryopreservation of C. socialis shoot tips. Inter-simple sequence repeat (ISSR) analysis identified fifteen unique genotypes in the ex situ in vitro collection. One genotype in this collection has been conserved from a population that is now presumed extinct. Although the initial sampling protocol managed to capture considerable genetic diversity, an additional 97 genotypes are needed to meet CPC guidelines. The information and experience gained through the initial C. socialis ex situ conservation efforts form the basis for a strategy to improve ex situ conservation activities for this endangered species. We recommend that additional in vitro collections be made from each of the five extant populations and placed in cryostorage.

Grand challenges in organismal biology: The need to develop both theory and resources

This contribution is fifth in a series of articles in Integrative and Comparative Biology that was initiated by Schwenk et al. (2009) and followed by Satterlie et al. (2009), Denny and Helmuth (2009), and Denver et al. (2009). Here, our intent is to be provocative and to stimulate further discourse. Like other contributors we have our own biases, and as it should be clear to the readers, we approach this task as evolutionary biologists, specifically systematists.

Spliceosomal intron size expansion in domesticated grapevine (Vitis vinifera)

BackgroundSpliceosomal introns are important components of eukaryotic genes as their structure, sizes and contents reflect the architecture of gene and genomes. Intron size, determined by both neutral evolution, repetitive elements activities and potential functional constraints, varies significantly in eukaryotes, suggesting unique dynamics and evolution in different lineages of eukaryotic organisms. However, the evolution of intron size, is rarely studied. To investigate intron size dynamics in flowering plants, in particular domesticated grapevines, a survey of intron size and content in wine grape (Vitis vinifera Pinot Noir) genes was conducted by assembling and mapping the transcriptome of V. vinifera genes from ESTs to characterize and analyze spliceosomal introns.ResultsUncommonly large size of spliceosomal intron was observed in V. vinifera genome, otherwise inconsistent with overall genome size dynamics when comparing Arabidopsis, Populus and Vitis. In domesticated grapevine, intron size is generally not related to gene function. The composition of enlarged introns in grapevines indicated extensive transposable element (TE) activity within intronic regions. TEs comprise about 80% of the expanded intron space and in particular, recent LTR retrotransposon insertions are enriched in these intronic regions, suggesting an intron size expansion in the lineage leading to domesticated grapevine, instead of size contractions in Arabidopsis and Populus. Comparative analysis of selected intronic regions in V. vinifera cultivars and wild grapevine species revealed that accelerated TE activity was associated with grapevine domestication, and in some cases with the development of specific cultivars.ConclusionsIn this study, we showed intron size expansion driven by TE activities in domesticated grapevines, likely a result of long-term vegetative propagation and intensive human care, which simultaneously promote TE proliferation and repress TE removal mechanisms such as recombination. The intron size expansion observed in domesticated grapevines provided an example of rapid plant genome evolution in response to artificial selection and propagation, and may shed light on the important genomic changes during domestication. In addition, the transcriptome approach used to gather intron size data significantly improved annotations of the V. vinifera genome.

The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs

MicroRNAs (miRNAs) and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs) are two distinct subfamilies of small RNAs (sRNAs) that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs) are processed from longer RNA precursors by DICER-LIKE proteins (DCLs). Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs) and 108 novel lineage-specific miRNAs (ls-miRNAs). Along with miRNAs, 2,033 miRNA variants (isomiRNAs) were identified as well. Those isomiRNAs with variation at the 3’-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs) were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers of a second round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.

Complete plastid genome sequence of goosegrass (Eleusine indica) and comparison with other Poaceae

Eleusine indica, also known as goosegrass, is a serious weed in at least 42 countries. In this paper we report the complete plastid genome sequence of goosegrass obtained by de novo assembly of paired-end and mate-paired reads generated by Illumina sequencing of total genomic DNA. The goosegrass plastome is a circular molecule of 135,151bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 20,919 bases. The large (LSC) and the small (SSC) single-copy regions span 80,667 bases and 12,646 bases, respectively. The plastome of goosegrass has 38.19% GC content and includes 108 unique genes, of which 76 are protein-coding, 28 are transfer RNA, and 4 are ribosomal RNA. The goosegrass plastome sequence was compared to eight other species of Poaceae. Although generally conserved with respect to Poaceae, this genomic resource will be useful for evolutionary studies within this weed species and the genus Eleusine.