Leaf-FISH : microscale imaging of bacterial taxa on phyllosphere

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 8 (2018): 2669, doi:10.3389/fmicb.2017.02669.Molecular methods for microbial community characterization have uncovered environmental and plant-associated factors shaping phyllosphere communities. Variables undetectable using bulk methods can play an important role in shaping plant-microbe interactions. Microscale analysis of bacterial dynamics in the phyllosphere requires imaging techniques specially adapted to the high autoflouresence and 3-D structure of the leaf surface. We present an easily-transferable method (Leaf-FISH) to generate high-resolution tridimensional images of leaf surfaces that allows simultaneous visualization of multiple bacterial taxa in a structurally informed context, using taxon-specific fluorescently labeled oligonucleotide probes. Using a combination of leaf pretreatments coupled with spectral imaging confocal microscopy, we demonstrate the successful imaging bacterial taxa at the genus level on cuticular and subcuticular leaf areas. Our results confirm that different bacterial species, including closely related isolates, colonize distinct microhabitats in the leaf. We demonstrate that highly related Methylobacterium species have distinct colonization patterns that could not be predicted by shared physiological traits, such as carbon source requirements or phytohormone production. High-resolution characterization of microbial colonization patterns is critical for an accurate understanding of microbe-microbe and microbe-plant interactions, and for the development of foliar bacteria as plant-protective agents.Funding was provided by the J. Unger Vetleson Foundation to SS

Shared up-regulation and contrasting down-regulation of gene expression distinguish desiccation-tolerant from intolerant green algae

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Peredo, E. L., & Cardon, Z. G. Shared up-regulation and contrasting down-regulation of gene expression distinguish desiccation-tolerant from intolerant green algae. Proceedings of the National Academy of Sciences of the United States of America, 117(29), 1(2020): 7438-17445, doi:10.1073/pnas.1906904117.Among green plants, desiccation tolerance is common in seeds and spores but rare in leaves and other vegetative green tissues. Over the last two decades, genes have been identified whose expression is induced by desiccation in diverse, desiccation-tolerant (DT) taxa, including, e.g., late embryogenesis abundant proteins (LEA) and reactive oxygen species scavengers. This up-regulation is observed in DT resurrection plants, mosses, and green algae most closely related to these Embryophytes. Here we test whether this same suite of protective genes is up-regulated during desiccation in even more distantly related DT green algae, and, importantly, whether that up-regulation is unique to DT algae or also occurs in a desiccation-intolerant relative. We used three closely related aquatic and desert-derived green microalgae in the family Scenedesmaceae and capitalized on extraordinary desiccation tolerance in two of the species, contrasting with desiccation intolerance in the third. We found that during desiccation, all three species increased expression of common protective genes. The feature distinguishing gene expression in DT algae, however, was extensive down-regulation of gene expression associated with diverse metabolic processes during the desiccation time course, suggesting a switch from active growth to energy-saving metabolism. This widespread downshift did not occur in the desiccation-intolerant taxon. These results show that desiccation-induced up-regulation of expression of protective genes may be necessary but is not sufficient to confer desiccation tolerance. The data also suggest that desiccation tolerance may require induced protective mechanisms operating in concert with massive down-regulation of gene expression controlling numerous other aspects of metabolism.Dr. Louise Lewis (University of Connecticut) provided F. rotunda and A. deserticola. Suzanne Thomas and Jordan Stark provided expert technical assistance. This work was supported by the NSF, Division of Integrative Organismal Systems (1355085 to Z.G.C.), and an anonymous donor (to Z.G.C.)

Najas flexilis (Hydrocharitaceae) in Alaska : a reassessment

Author Posting. © New England Botanical Club, 2015. This article is posted here by permission of New England Botanical Club for personal use, not for redistribution. The definitive version was published in Rhorora 117 (2015): 354-370, doi:10.3119/15-03.Fifteen Najas flexilis collections were made in Alaska during the summer of 2012, with 13 of the stations representing either new or formerly undocumented localities for this imperiled Alaskan species. These field collections characterize the Alaskan habitats of N. flexilis as shallow water sites (<1.5 m) with sand-dominated substrates (71% of sites) and have documented an additional 28 species associates (a 300% increase). However, the additional collections have not extended the elevational, latitudinal, or longitudinal extent of N. flexilis from the limits indicated by previous Alaskan collections. Najas flexilis remains rare in Alaska as evidenced by a low specimen recovery rate (10%) from potentially suitable sites, and a total of only 12 geographically distinct localities known across the entire state. The new collections have furnished valuable study material for morphological and genetic analyses, which have confirmed the identity of Alaskan populations as N. flexilis, rather than N. canadensis, a recently identified, cryptic, allotetraploid derivative. A synthesis of information indicates that N. flexilis is indigenous to Alaska, where it originated via past (versus recent) migrations from other North American rather than Old World populations.Portions of this work were funded by National Science Foundation grant DEB-0841658 to D.H.L

A model suite of green algae within the Scenedesmaceae for investigating contrasting desiccation tolerance and morphology

Author Posting. © The Company of Biologists, 2018. This article is posted here by permission of The Company of Biologists for personal use, not for redistribution. The definitive version was published in Journal of Cell Science 131 (2018): jcs212233, doi:10.1242/jcs.212233.Microscopic green algae inhabiting desert microbiotic crusts are remarkably diverse phylogenetically, and many desert lineages have independently evolved from aquatic ancestors. Here we worked with five desert and aquatic species within the family Scenedesmaceae to examine mechanisms that underlie desiccation tolerance and release of unicellular versus multicellular progeny. Live cell staining and time-lapse confocal imaging coupled with transmission electron microscopy established that the desert and aquatic species all divide by multiple (rather than binary) fission, although progeny were unicellular in three species and multicellular (joined in a sheet-like coenobium) in two. During division, Golgi complexes were localized near nuclei, and all species exhibited dynamic rotation of the daughter cell mass within the mother cell wall at cytokinesis. Differential desiccation tolerance across the five species, assessed from photosynthetic efficiency during desiccation/rehydration cycles, was accompanied by differential accumulation of intracellular reactive oxygen species (ROS) detected using a dye sensitive to intracellular ROS. Further comparative investigation will aim to understand the genetic, ultrastructural and physiological characteristics supporting unicellular versus multicellular coenobial morphology, and the ability of representatives in the Scenedesmaceae to colonize ecologically diverse, even extreme, habitats.This work was supported by the National Science Foundation, Division of Integrative Organismal Systems [1355085 to Z.G.C.], an anonymous donor [to Z.G.C.], the Marine Biological Laboratory [to M.B.] and the Environmental and Molecular Sciences Laboratory (EMSL) [48938 to Z.G.C.], a Department of Energy, Office of Science User Facility sponsored by the Office of Biological and Environmental Research, located at Pacific Northwest National Laboratory.2019-04-1

Extraction of high-quality, high-molecular-weight DNA depends heavily on cell homogenization methods in green microalgae

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Stark, J. R., Cardon, Z. G., & Peredo, E. L. Extraction of high-quality, high-molecular-weight DNA depends heavily on cell homogenization methods in green microalgae. Applications in Plant Sciences, 8(3), (2020): e11333, doi:10.1002/aps3.11333.Premise New sequencing technologies have facilitated genomic studies in green microalgae; however, extracting high‐quality DNA is often a bottleneck for long‐read sequencing. Methods and Results Here, we present a low‐cost, highly transferrable method for the extraction of high‐molecular‐weight (HMW), high‐purity DNA from microalgae. We first determined the effect of sample preparation on DNA quality using three homogenization methods: manual grinding using a mini‐pestle, automatic grinding using a vortex adapter, and grinding in liquid nitrogen. We demonstrated the versatility of grinding in liquid nitrogen followed by a modified cetyltrimethylammonium bromide (CTAB) extraction across a suite of aquatic‐ and desert‐evolved algal taxa. Finally, we tested the protocol's robustness by doubling the input material to increase yield, producing per sample up to 20 μg of high‐purity DNA longer than 21.2 kbp. Conclusions All homogenization methods produced DNA within acceptable parameters for purity, but only liquid nitrogen grinding resulted in HMW DNA. The optimization of cell lysis while minimizing DNA shearing is therefore crucial for the isolation of DNA for long‐read genomic sequencing because template DNA length strongly affects read output and length.The authors thank Dr. Louise Lewis (University of Connecticut) for providing Flechtneria rotunda and Acutodesmus deserticola, and Suzanne Thomas for expert technical assistance. This work was supported by the National Science Foundation, Division of Integrative Organismal Systems (1355085 to Z.G.C.) and an anonymous donor (to Z.G.C.)

The Plastid Genome of Najas flexilis: Adaptation to Submersed Environments Is Accompanied by the Complete Loss of the NDH Complex in an Aquatic Angiosperm

The re-colonization of aquatic habitats by angiosperms has presented a difficult challenge to plants whose long evolutionary history primarily reflects adaptations to terrestrial conditions. Many aquatics must complete vital stages of their life cycle on the water surface by means of floating or emergent leaves and flowers. Only a few species, mainly within the order Alismatales, are able to complete all aspects of their life cycle including pollination, entirely underwater. Water- pollinated Alismatales include seagrasses and water nymphs ( Najas ), the latter being the only freshwater genus in the family Hydrocharitaceae with subsurface water-pollination. We have determined the complete nucleotide sequence of the plastid genome of Najas flexilis. The plastid genome of N. flexilis is a circular AT-rich DNA molecule of 156 kb, which displays a quadripartite structure with two inverted repeats (IR) separating the large single copy (LSC) from the small single copy (SSC) regions. In N. flexilis, as in other Alismatales, the rps19 and trnH genes are localized in the LSC region instead of within the IR regions as in other monocots. However, the N. flexilis plastid genome presents some anomalous modifications. The size of the SSC region is only one third of that reported for closely related species. The number of genes in the plastid is considerably less. Both features are due to loss of the eleven ndh genes in the Najas flexilis plastid. In angiosperms, the absence of ndh genes has been related mainly to the loss of photosynthetic function in parasitic plants. The ndh genes encode the NAD(P)H dehydrogenase complex, believed essential in terrestrial environments, where it increases photosynthetic efficiency in variable light intensities. The modified structure of the N. flexilis plastid genome suggests that adaptation to submersed environments, where light is scarce, has involved the loss of the NDH complex in at least some photosynthetic angiosperms

Genetic stability of in vitro conserved germplasm of Humulus lupulus L.

The genetic and epigenetic stability of hop accessions cryopreserved for one year or cold stored for three years was evaluated using several molecular markers (RAPD, AFLP, and MSAP). Clear, repetitive patterns were obtained among accessions and between control and treated samples. Although no genetic changes were detected among the control plants grown in the greenhouse and in vitro plants regenerated from slow-cooling cryopreserved shoot tips or cold stored in vitro shoots, MSAP analysis detected methylation changes in 36% of the loci. Nevertheless, only 2.6 to 9.8% of the detected changes could be ascribed to the conservation procedure and most of them seemed to be generated as a result of the in vitro introduction. Due to the number of accessions analysed (51) we can cautiously deduce that the genetic behaviour described in this work after cryopreservation or cold-storage protocols is common to most hop genotypes and these storage procedures are suitable for standard use. However, it is important to keep in mind the epigenetic changes produced, particularly during any in vitro processes.vo

Evaluation of Microsatellite Detection Using Autoradiography and Capillary Electrophoresis in Hops

Two sequence tagged sites (STS) detection techniques were used in the identification of 25 hop cultivars. In cultivar identification, capillary electrophoresis is more efficient than autoradiography. First, it was more sensitive in detection of fragments that allowed observation of the instability in the locus 7a82 due to the small size differences of its alleles. Additionally, the fluorescent detection of fragments was a nonsubjective technique suitable of automation that increased the flexibility of work because no radioactive labeling or long vertical polyacrylamide electrophoresis was needed. Only two cultivars, Hersbrucker and Hallertauer, were indistinguishable, but further molecular analysis revealed that both were included in a separate cluster within the European hop cultivars. The statistical analysis of fluorescence and radioactive detection data including allelic frequencies, heterozygosity, and probability of identity are displayed in this study, which corroborates the following advantages of the capillary electrophoresis technique: high sensitivity, amenable to automation, and its nonsubjectivity. Therefore, fluorescent-labeled primer microsatellite detection by capillary electrophoresis is a powerful tool in cultivar identification for hop breeders, merchants, and brewers. Keywords: DNA typing, Fluorescence detection, Humulus lupulus, STS.Peer reviewe