Variability in ITS1 and ITS2 sequences of historic herbaria and extant (fresh) Phalaris species (Poaceae).

BACKGROUND: Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882-2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species. RESULTS: We obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin. CONCLUSIONS: While not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way.

Nicotiana tabacum pollen-pistil interactions show unexpected spatial and temporal differences in pollen tube growth among genotypes.

KEY MESSAGE: This research revealed diverse PTG rates among intraspecific pollen-pistil interactions that showed variable dependency on the stigma and mature TT. Pollen-pistil interactions regulate pollen tube growth (PTG) rates and are determinants of fertilization and seed set. This research focuses on the diversity of intraspecific PTG rates and the spatial and temporal regulation of PTG among Nicotiana tabacum genotypes. Nonrandom mating within self-compatible species has been noted, but little is known on the mechanisms involved. To begin research on nonrandom mating, we took advantage of the model reproductive system of N. tabacum and used seventeen diverse N. tabacum genotypes in a complete pollination diallel to measure the diversity of intraspecific pollen-pistil interactions. The 289 intraspecific interactions showed surprisingly large differences in PTG rates. The interaction between specific males and females resulted in 18 specific combining abilities that were significantly different, indicating the importance of the specific genotype interaction in regulating intraspecific PTG. No single female or male genotype exerted overall control of PTG rates, as determined by a general combining ability analysis. Slow and fast pollen-pistil interactions showed spatial differences in growth rates along the style. Slower interactions had a slower initial PTG rate while fast interactions had faster consistent rates of growth indicating spatial regulation of PTG in the pistil. Removal of the stigma or the mature transmitting tissue (TT) showed the tissue-specific component of PTG regulation. Stigma removal resulted in slower or no change in PTG rate depending on the pollen and pistil genotypes. Removal of the TT, which necessitated removal of the stigma, showed no change, slower or unexpectedly, increased growth rates relative to growth rates without a stigma. These data show the diverse nature of pollen-pistil interactions in N. tabacum genotypes providing a system to further investigate the regulation of PTG.

Structure and function of class III pistil-specific extensin-like protein in interspecific reproductive barriers.

BACKGROUND: The transmitting tissue of the style is the pathway for pollen tube growth to the ovules and has components that function in recognizing and discriminating appropriate pollen genotypes. In Nicotiana tabacum, the class III pistil extensin-like (PELPIII) arabinogalactan protein is essential for the inhibition of N. obtusifolia pollen tube growth. The transmitting tissue-specific (TTS) arabinogalactan protein amino acid sequence and expression pattern is similar to PELPIII, but it facilitates self-pollinated N. tabacum. The TTS and PELPIII arabinogalactan protein can be divided into the less conserved N-terminal (NTD) and the more conserved C-terminal (CTD) domains. This research tested whether the NTD is the key domain in determining PELPIII function in the inhibition of interspecific pollen tube growth. Three variant PELPIII gene constructs were produced where the PELPIII NTD was exchanged with the TTS NTD and a single amino acid change (cysteine to alanine) was introduced into the PELPIII NTD. The PELPIII variants of N. tabacum were tested for activity by measuring the inhibition N. obtusifolia pollen tube growth by using them to complement a 3'UTR RNAi transgenic line with reduced PELPIII mRNA. RESULTS: The RNAi N. tabacum line had reduced PELPIII mRNA accumulation and reduced inhibition of N. obtusifolia pollen tube growth, but had no effect on self-pollen tube growth or pollen tube growth of 12 other Nicotiana species. The NTD of PELPIII with either the PELPIII or TTS CTDs complemented the loss PELPIII activity in the RNAi transgenic line as measured by inhibition of N. obtusifolia pollen tube growth. The TTS NTD with the PELPIII CTD and a variant PELPIII with a cysteine to alanine mutation in its NTD failed to complement the loss of PELPIII activity and did not inhibit N. obtusifolia pollen tube growth. CONCLUSION: The NTD is a key determinant in PELPIII's function in regulating interspecific pollen tube growth and is a first step toward understanding the mechanism of how PELPIII NTD regulates pollen tube growth.

Polymorphism and structure of style-specific arabinogalactan proteins as determinants of pollen tube growth in Nicotiana.

BACKGROUND: Pollen tube growth and fertilization are key processes in angiosperm sexual reproduction. The transmitting tract (TT) of Nicotiana tabacum controls pollen tube growth in part by secreting pistil extensin-like protein III (PELPIII), transmitting-tract-specific (TTS) protein and 120 kDa glycoprotein (120 K) into the stylar extracellular matrix. The three arabinogalactan proteins (AGP) are referred to as stylar AGPs and are the focus of this research. The transmitting tract regulates pollen tube growth, promoting fertilization or rejecting pollen tubes. RESULTS: The N-terminal domain (NTD) of the stylar AGPs is proline rich and polymorphic among Nicotiana spp. The NTD was predicted to be mainly an intrinsically disordered region (IDR), making it a candidate for protein-protein interactions. The NTD is also the location for the majority of the predicted O-glycosylation sites that were variable among Nicotiana spp. The C-terminal domain (CTD) contains an Ole e 1-like domain, that was predicted to form beta-sheets that are similar in position and length among Nicotiana spp. and among stylar AGPs. The TTS protein had the greatest amino acid and predicted O-glycosylation conservation among Nicotiana spp. relative to the PELPIII and 120 K. The PELPIII, TTS and 120 K genes undergo negative selection, with dn/ds ratios of 0.59, 0.29 and 0.38 respectively. The dn/ds ratio for individual species ranged from 0.4 to 0.9 and from 0.1 to 0.8, for PELPIII and TTS genes, respectively. These data indicate that PELPIII and TTS genes are under different selective pressures. A newly discovered AGP gene, Nicotiana tabacum Proline Rich Protein (NtPRP), was found with a similar intron-exon configuration and protein structure resembling other stylar AGPs, particularly TTS. CONCLUSIONS: Further studies of the NtPRP gene are necessary to elucidate its biological role. Due to its high similarity to the TTS gene, NtPRP may be involved in pollen tube guidance and growth. In contrast to TTS, both PELPIII and 120 K genes are more diverse indicating a possible role in speciation or mating preference of Nicotiana spp. We hypothesize that the stylar AGPs and NtPRP share a common origin from a single gene that duplicated and diversified into four distinct genes involved in pollen-style interactions.

Increases in synthetic cannabinoids-related harms: Results from a longitudinal web-based content analysis.

BACKGROUND: Synthetic Cannabinoid Receptor Agonists (SCRA), also known as "K2" or "Spice," have drawn considerable attention due to their potential of abuse and harmful consequences. More research is needed to understand user experiences of SCRA-related effects. We use semi-automated information processing techniques through eDrugTrends platform to examine SCRA-related effects and their variations through a longitudinal content analysis of web-forum data. METHOD: English language posts from three drug-focused web-forums were extracted and analyzed between January 1st 2008 and September 30th 2015. Search terms are based on the Drug Use Ontology (DAO) created for this study (189 SCRA-related and 501 effect-related terms). EDrugTrends NLP-based text processing tools were used to extract posts mentioning SCRA and their effects. Generalized linear regression was used to fit restricted cubic spline functions of time to test whether the proportion of drug-related posts that mention SCRA (and no other drug) and the proportion of these "SCRA-only" posts that mention SCRA effects have changed over time, with an adjustment for multiple testing. RESULTS: 19,052 SCRA-related posts (Bluelight (n=2782), Forum A (n=3882), and Forum B (n=12,388)) posted by 2543 international users were extracted. The most frequently mentioned effects were "getting high" (44.0%), "hallucinations" (10.8%), and "anxiety" (10.2%). The frequency of SCRA-only posts declined steadily over the study period. The proportions of SCRA-only posts mentioning positive effects (e.g., "High" and "Euphoria") steadily decreased, while the proportions of SCRA-only posts mentioning negative effects (e.g., "Anxiety," 'Nausea," "Overdose") increased over the same period. CONCLUSION: This study's findings indicate that the proportion of negative effects mentioned in web forum posts and linked to SCRA has increased over time, suggesting that recent generations of SCRA generate more harms. This is also one of the first studies to conduct automated content analysis of web forum data related to illicit drug use.

The plant-specific protein FEHLSTART controls male meiotic entry, initializing meiotic synchronization in Arabidopsis.

Meiosis marks the transition from the sporophyte to the gametophyte generation in the life cycle of flowering plants, and creates genetic variations through homologous recombination. In most flowering plants, meiosis is highly synchronized within each anther, which is significant for efficient fertilization. To date, little is known about the molecular mechanisms of entry into meiosis and exit from it, and only a few genes in Arabidopsis have been characterized with a role in regulating meiotic progression. In this study, we report the functional characterization of a plant-specific basic helix-loop-helix (bHLH) protein, FEHLSTART (FST), a defect in which leads to premature meiotic entry and asynchronous meiosis, and results in decreased seed yield. Investigation of the time course of meiosis showed that the onset of leptotene, the first stage of prophase I, frequently occurred earlier in fst-1 than in the wild type. Asynchronous meiosis followed, which could manifest in the disruption of regular spindle structures and symmetric cell divisions in fst-1 mutants during the meiosis I/II transition. In accordance with frequently accelerated meiotic entry, whole-transcriptome analysis of fst-1 anthers undergoing meiosis revealed that 19 circadian rhythm genes were affected and 47 pollen-related genes were prematurely expressed at a higher level. Taken together, we propose that FST is required for normal meiotic entry and the establishment of meiotic synchrony.

The transmitting tissue of Nicotiana tabacum is not essential to pollen tube growth, and its ablation can reverse prezygotic interspecific barriers.

The Nicotiana tabacum transmitting tissue is a highly specialized file of metabolically active cells that is the pathway for pollen tubes from the stigma to the ovules where fertilization occurs. It is thought to be essential to pollen tube growth because of the nutrients and guidance it provides to the pollen tubes. It also regulates gametophytic self-incompatibility in the style. To test the function of the transmitting tissue in pollen tube growth and to determine its role in regulating prezygotic interspecific incompatibility, genetic ablation was used to eliminate the mature transmitting tissue, producing a hollow style. Despite the absence of the mature transmitting tissue and greatly reduced transmitting-tissue-specific gene expression, self-pollen tubes had growth to the end of the style. Pollen tubes grew at a slower rate in the transmitting-tissue-ablated line during the first 24 h post-pollination. However, pollen tubes grew to a similar length 40 h post-pollination with and without a transmitting tissue. Ablation of the N. tabacum transmitting tissue significantly altered interspecific pollen tube growth. These results implicate the N. tabacum transmitting tissue in facilitating or inhibiting interspecific pollen tube growth in a species-dependent manner and in controlling prezygotic reproductive barriers.

PELPIII: the class III pistil-specific extensin-like Nicotiana tabacum proteins are essential for interspecific incompatibility.

Pre-zygotic interspecific incompatibility (II) involves an active inhibition mechanism between the pollen of one species and the pistil of another. As a barrier to fertilization, II effectively prevents hybridization and maintains species identity. Transgenic ablation of the mature transmitting tract (TT) in Nicotiana tabacum resulted in the loss of inhibition of pollen tube growth in Nicotiana obtusifolia (synonym Nicotiana trigonophylla) and Nicotiana repanda. The role of the TT in the II interaction between N. tabacum and N. obtusifolia was characterized by evaluating N. obtusifolia pollen tube growth in normal and TT-ablated N. tabacum styles at various post-pollination times and developmental stages. The II activity of the TT slowed and then arrested N. obtusifolia pollen tube growth, and was developmentally synchronized. We hypothesize that proteins produced by the mature TT and secreted into the extracellular matrix inhibit interspecific pollen tubes. When extracts from the mature TT of N. tabacum were injected into the TT-ablated style prior to pollination, the growth of incompatible pollen tubes of N. obtusifolia and N. repanda was inhibited. The class III pistil-specific extensin-like protein (PELPIII) was consistently associated with specific inhibition of pollen tubes, and its requirement for II was confirmed through use of plants with antisense suppression of PELPIII. Inhibition of N. obtusifolia and N. repanda pollen tube growth required accumulation of PELPIII in the TT of N. tabacum, supporting PELPIII function in pre-zygotic II.

A novel pollen tube growth assay utilizing a transmitting tract-ablated Nicotiana tabacum style.

Sexual plant reproduction requires multiple pollen-pistil interactions from the stigma (pollen adhesion, hydration, and germination) to the ovary (fertilization). Understanding the factors that regulate pollen tube growth is critical to understanding the processes essential to sexual reproduction. Many pollen tube growth assays (PTGAs) have shorter and slower pollen tube growth when compared to pollen tube growth through the style. The identification and study of factors that regulate pollen tube growth have been impeded by a lack of an efficient and reproducible PTGA. The objective of this research is to develop a robust assay for Nicotiana tabacum pollen tube growth in an environment that supports sustained and normal growth yet is amenable to testing the effects of specific factors. In this paper, we introduce a novel PTGA, which uses pistils from N. tabacum that lack a mature transmitting tract (TT) due to tissue-specific ablation. The TT-ablated style supports normal pollen tube growth and the hollow structure of the style allows modification of the growth environment by direct injection of test material. This PTGA is robust and allows for rapid and accurate measurement of pollen tube length and pollen tube morphology, supporting pollen tube growth from 20 to 35°C and at pH ranging from 4.8 to 7.6. Use of the ablated style for a PTGA is a novel method for the culture of pollen tubes with sustained growth in vivo while permitting the application of treatments to the growing pollen tubes.

Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes.

BACKGROUND: Meiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes. RESULTS: A high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes. CONCLUSION: These findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.

A glycine-rich protein that facilitates exine formation during tomato pollen development.

Formation of the unique and highly diverse outer cell wall, or exine, of pollen is essential for normal pollen function and survival. However, little is known about the many contributing proteins and processes involved in the formation of this wall. The tomato gene LeGRP92 encodes for a glycine-rich protein produced specifically in the tapetum. LeGRP92 is found as four major forms that accumulate differentially in protein extracts from stamens at different developmental stages. The three largest molecular weight forms accumulated during early microspore development, while the smallest molecular weight form of LeGRP92 was present in protein extracts from stamens from early microsporogenesis through anther dehiscence, and was the only form present in dehisced pollen. Light microscopy immunolocalization experiments detected LeGRP92 at only two stages, late tetrad and early free microspore. However, we observed accumulation of the LeGRP92 at the early tetrad stage of development by removing the callose wall from tetrads, which allowed LeGRP92 detection. Transmission electron microscopy confirmed the LeGRP92 accumulation from microspore mother cells, tetrads through anther dehiscence. It was observed in the callose surrounding the microspore mother cells and tetrads, the exine of microspores and mature pollen, and orbicules. Plants expressing antisense RNA had reduced levels of LeGRP92 mRNA and protein, which correlated to pollen with altered exine formation and reduced pollen viability and germination. These data suggest that the LeGRP92 has a role in facilitating sporopollenin deposition and uniform exine formation and pollen viability.

Production of male- and female-sterile plants through reproductive tissue ablation.

Male and female sterilities have many useful applications in horticultural crops, including reducing the invasive potential of new ornamentals, elimination of pollen allergens and redirecting resources from seeds to vegetative growth. In this study, we tested a male- and female-sterility (MS; FS) gene construct in Nicotiana tabacum to evaluate its effectiveness and effect on phenotype. Three T1 Nicotiana tabacum lines expressing the MS (p108:barnase) and FS (sp41:barnase) genes (MS/FS lines) and a control Nicotiana tabacum line (WT GUS) were measured for plant height, leaf length and width, corolla length, number of nodes on the main stem and stem diameter. No significant differences were found in these growth measurements between MS/FS lines and WT GUS. No pollen was observed on any of the lines carrying the MS and FS genes, indicating that the male sterility was complete. Seed set was greatly reduced or completely eliminated in plants with the MS and FS genes, after heavy pollinations of mature flowers with WT GUS pollen. However, pollinations of immature flowers resulted in very low seed set. This may be due to the nature of the promoter controlling expression of the FS gene as it had the highest expression levels at anthesis. The combination of male- and female-sterility genes was effective in eliminating seed set in all the lines examined and has direct application for reducing invasiveness of ornamental plants.

An anther-specific cysteine-rich protein of tomato localized to the tapetum and microspores.

The tapetum is a nutritive tissue of the stamen that is essential for normal microspore development. While numerous tapetal-specific genes have been identified, little information is available on the localization and function of the proteins produced by these genes. The tapetally produced protein 5B-CRP is cysteine-rich, has a secretory signal sequence and lacks an endoplasmic reticulum retention sequence. The 5B-CRP mRNA is expressed specifically within the tapetum and accumulates from premeiosis to tetrad release. Antibodies generated against an Escherichia coli fusion protein only recognized 5B-CRP in the reduced state. The 5B-CRP was detected as a 6 kDa protein in extracts of stamens from microspore meiosis through anthesis and was also observed in extracts from dehisced pollen. In situ, 5B-CRP was localized in stamens to the tapetum and the developing microspores, from the tetrad through early free microspore stages. Based on similarity to proteins with known functions, 5B-CRP may inhibit proteasome activity within the stamen locule.

Functional analysis of a beta-1,3-glucanase gene (Tag1) with anther-specific RNA and protein accumulation using antisense RNA inhibition.

A critical stage in pollen development is the dissolution of tetrads into free microspores. Tetrads are surrounded by a wall composed primarily of beta-1,3-glucan. At the completion of meiosis, tetrads are released into the anther locule after hydrolysis of the callose by a beta-1,3-glucanase complex. The cDNA corresponding to a beta-1,3-glucanase cloned from tobacco (Tag 1) represents a gene that is highly similar to other beta-1,3-glucanases and is expressed exclusively in anthers from the tetrad to free microspore stage of pollen development. Tag 1 protein was overexpressed in E. coli, accumulating in insoluble inclusion bodies. Polyclonal antibodies against Tag 1 recombinant protein identify a single 33 kD protein accumulating only in anthers at tetrad and free microspore stages where beta-1,3-glucanase activity is present. Transgenic plants expressing Tag 1 antisense RNA were produced. Although Tag 1 RNA and protein levels were greatly reduced, tetrad dissolution and pollen development were normal. These data indicate that under the conditions these tobacco plants were grown, wild type levels of Tag 1 protein are not necessary for male fertility.

Vernalization, photoperiod and GA3 interact to affect flowering of Japanese radish (Raphanus sativus Chinese Radish Jumbo Scarlet).

Raphanus sativus L. Chinese Radish Jumbo Scarlet has characteristics that make it an excellent plant model for vernalization studies. This study further characterizes flower induction of R. sativus Chinese Radish Jumbo Scarlet. Seed were imbibed in distilled water containing 0, 10-5 M or 10-3 M GA3 for 24 h and were then exposed to 6 +/- 0.5 degrees C (vernalized) for 0, 2, 4, 6, or 8 days. Seedlings were then grown under a short- (8 h) or long-day photoperiod (8 h with or without a 4-h night interruption; 2200-0200 h). Of unvernalized plants grown under long- and short-day conditions, 45 and 3% flowered, respectively. Saturation of the vernalization response occurred after a 4- or 8-day vernalization treatment when plants were placed under long- or short-days, respectively. Basal leaf number and days to anthesis decreased when seeds were cooled for 2 or 4 days and were imbibed with 10-3 M GA3 compared to distilled water only. These data indicate that R. sativus Chinese Jumbo Scarlet has principally an obligate vernalization requirement when grown under short-days. GA3 application only facilitated flowering when the length of the vernalization treatment was marginal. Taken together, these data support the use of this plant as a model plant for identifying vernalization responses under short-day conditions.