Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells

Background Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. Methodology/Principal Findings In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. Conclusions/Significance Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin\u27s role in tip growing plant cells

P. patens genomic and transcriptomic analyses

The model organism Physcomitrium patens, formerly Physcomitrella patens is a moss in the Funariaceae family. Due to P. patens ability to generate easily transgenic plants via homologous recombination, the interest of scientists worldwide was attracted. P. patens was the world's first completely sequenced non-seed plant genome (V1). Constant improvements of the genome assembly and the associated gene annotations resulted in the current P. patens pseudo-chromosomal genome version (V3). This genome version is the basis of all analyses performed in this thesis. Since P. patens became a U.S. Department of Energy Joint Genome Institute (DOE JGI) plant flagship genome 1 and a member of the JGI Gene Atlas project 2, hundreds of P. patens RNA-seq samples were generated. During my time as a PhD student, I analysed the JGI Gene Atlas RNA-seq samples and several dozen other RNA-seq samples from different projects. These RNA-seq samples contained data from five different P. patens ecotypes/accessions (Gransden, Kaskaskia, Reute, Villersexel, and Wisconsin).To efficiently analyse this data, I developed a powerful RNA-seq pipeline to perform differentially expressed gene (DEG) calling. The performance of the RNA-seq pipeline was tested by comparing its results to commercial software solutions and multiple RNA-seq samples from different species. My newly generated gene expression results, together with previous published expression data from a variety of other projects, were stored at our novel online tool PEATmoss. Furthermore, my gene version lookup tables were implemented in a database structure. This, allows PEATmoss users to find gene models of different gene annotation versions and to use them in PEATmoss. With an updated version of my RNA-seq pipeline, I identified and analysed sequence variations in P. patens accessions. A clear clustering by individual accessions could be shown. I could demonstrate, that due to decades of vegetative propagation in laboratories, somatic mutations have accumulated in Gransden laboratory plants. In addition, we used restriction fragment length polymorphism (RFLP) to offer a simple method for quick identification of unknown P. patens plants. 1 https://jgi.doe.gov/our-science/science-programs/plant-genomics/plant-flagship-genomes/ 2 https://jgi.doe.gov/doe-jgi-plant-flagship-gene-atlas

Model Organisms in Plant Genetics

Model plants are required for research when targeted plant species are difficult to study or when research material is unavailable. Importantly, knowledge gained from model plants can be generally translated to other related plant species because many key cellular and molecular processes are conserved and regulated by ‘blueprint’ genes inherited from a common ancestor. Model Organisms in Plant Genetics addresses characteristics of model plants such as Arabidopsis, moss, soybean, maize, and cotton, highlighting their advantages and limitations as well as their importance in studies of plant development, plant genome polyploidization, adaptive selection, evolution, and domestication, as well as their importance in crop improvement

Shaping plant microtubule networks via overlap formation

Microtubules are long filaments made up from protein building blocks and ubiquitously employed by eukaryotic cells for a wide range of often essential cellular processes. To perform these functions, microtubules are virtually always organized into higher order networks. Microtubule networks in cells of land plants are fundamental for guiding growth processes and for bringing about their unique mode of cell division. The latter is facilitated by the so‑called phragmoplast network, consisting of two opposing sets of microtubules that foster in their centre the formation and radial outgrowth of a disc-shaped membrane compartment (termed the cell plate) that ultimately divides the two daughter cells. The mechanisms driving the spatial organisation of such networks are of outstanding interest because plant cells do not rely on major microtubule organizers as in most other organisms. Instead, plant cells use a wide range of dispersed interactions among individual microtubules to shape functional microtubule networks. Chapter 1 introduces encounters between microtubules of opposite polarity and consequent bundling as potentially powerful handles to organize microtubules into networks. These encounters generate an area of antiparallel microtubule overlap and such overlaps are a striking feature of the phragmoplast microtubule network. For long it is recognized that the short overlaps formed among the two opposing sets of phragmoplast microtubules and the membranous structures of the cell plate fall within the same plane. In chapter 2 we hypothesize that the limited length of these overlaps is required for the confined accumulation of cell plate membranes. To investigate this, we start out by co-visualizing overlaps and cell-plate membrane material in living cells of the moss Physcomitrella patens, an emerging model plant system with a convenient genetic toolset and tissues readily observable through microscopy. We reaffirm an early association between overlaps and membranes and further explored this association by experimentally altering overlap length. Incited by length control mechanisms of overlaps in animal cells, we identify two kinesin-4 motor proteins that jointly limit the length of phragmoplast microtubule overlaps in moss. Using cells lacking these kinesin-4s we then show that over-elongation of microtubule overlaps leads to a broadening of initial cell plate membrane depositions and a delayed progression of radial cell plate outgrowth. The cross walls ultimately formed by the wider membrane depositions were found to be thick and irregularly shaped. We thus demonstrate that kinesin-4-dependent overlap shortening in the phragmoplast defines the site of cell plate synthesis for the proper scaffolding of a new cell wall segment separating two daughter cells. In chapter 3 we further investigate molecular mechanisms that could explain how linkage between a microtubule overlap and membrane assembly activity is realized. We focus on the exocyst tethering complex, one of the membrane tethering complexes involved in cell plate formation in flowering plants. We survey the localization of several moss exocyst subunits during cell division and find that one (Sec6) localizes to microtubule overlaps already before the onset of cell plate biogenesis. Experiments in which overlap length is altered and overlap formation is suppressed reveal that these structures play an important role in positioning Sec6 during cell division. The ability of moss Sec6 to interact with an evolutionary conserved factor in cell plate membrane fusion called KEULE is demonstrated, signifying a potential functional link between membrane tethering and fusion activities during cell plate formation. The precise role of Sec6 positioning by overlaps is as yet unclear, but in the light of the importance of overlaps for spatial control of cytokinesis will prove to be an intriguing direction for future research efforts. In chapter 4 we gain further mechanistic insight in kinesin-4 mediated overlap length control and governance of division apparatus length as a whole. We focus on microtubule growth in overlaps regulated by kinesin-4, the poleward transport of microtubule polymers (termed flux), and the interplay between these processes. First, a method based on localized photo-activation is established for the quantitative assessment of microtubule flux. We demonstrate that initially flux in the metaphase spindle occurs synchronized and at high rates, to be replaced by a heterogeneous and on average much slower microtubule flux in the phragmoplast. Since polymerisation of microtubules could provide direct fuel for flux, we postulate that the rate of microtubule growth at sites of overlap could determine flux rates. To test this, we experimentally enhance polymerisation rates through knock-out of kinesin-4 proteins. This approach is validated by experiments demonstrating that they can supress microtubule outgrowth at overlaps in an in vivo setting. Upon kinesin-4 removal, flux rates are enhanced signifying coupling to rates of polymerization. We also find that lack of kinesin-4s leads elongation of the entire division apparatus and that this length change is proportional to the temporal activity patterns of the two kinesin-4s. Based on these findings we propose a mechanism for length regulation through a balance of microtubule growth in the overlap zone, retrograde microtubule translocation and putatively microtubule breakdown at the poles. Microtubule turnover in this system is high in the metaphase spindle (~1.5 μm/min), which, partly through kinesin-4 action, is succeeded by a more slowly turning over system in the form of the phragmoplast. While in general the involvement of antiparallel microtubule overlaps in spatial organization of bipolar microtubule configurations is evident, how they could help shape other geometries is largely unknown. Chapter 5 starts out with the observation that within the unipolarized microtubule array of tip growing moss cells during interphase, there is occasional formation of overlaps at dispersed sites in the network. Tip growth is a mode of growth allowing rapid colonization of the environment and is achieved through highly polarized secretion, whereby the microtubule network reportedly steers the grows axis. We identify one kinesin-4 motor (Kin4-Ia) recruited to the observed overlaps within this network and use knock-out of Kin4-Ia to assess its role in tip growth. This reveals that absence of Kin4-Ia leads to a less adaptable axis of tip growth, prompting further investigation of Kin4-Ia behaviour at interphase overlaps. We find that this kinesin-4 is recruited with a slight delay to overlaps after their formation and inhibits plus end polymerization of overlap microtubules, thereby limiting overlap length. We then uncover that this activity helps to keep the network polarized towards the tip and prevent the overall organization from becoming hyperaligned with the cell axis. We propose that the latter observation might explain the decrease in growth axis adaptability. Overall, this thesis demonstrates that in plant microtubule networks of varying architecture, the formation of antiparallel overlaps provides a defined network feature for the recruitment of other microtubule-based process. Together, overlaps and activities coordinated from there, are potent organizers of functional plant microtubule arrays. The potential wider implications of these findings, their relationship to membrane-bound cytokinetic processes, and their evolutionary context are briefly discussed in Chapter 6.</p

Volume 1, Chapter 5-1: Ecophysiology of Development: Hormones

https://digitalcommons.mtu.edu/bryo-ecol-subchapters/1025/thumbnail.jp

Comparative transcriptone analysis in the moss Physcomitrella patens and the genetic basis of key reproductive innovations

During land plant evolution plants underwent extensive transformations. For the successful adaptation to the terrestrial environment the vegetative tissues were modified to cope with low water availability while new reproductive organs and strategies emerged to ensure plant dispersal and survival. Importantly, plant life cycles were dramatically modified, evolving from gametophyte dominant in early land plants such as Bryophytes to sporophyte dominance as it is observed in flowering plants.(...

HAG1 and SWI3A/B control of male germ line development in P. patens suggests conservation of epigenetic reproductive control across land plants

Bryophytes as models to study the male germ line: loss-of-function mutants of epigenetic regulators HAG1 and SWI3a/b demonstrate conserved function in sexual reproduction

Plant protein prenylation in environmental responses and protein production.

Lipid post-translational modifications enhance a protein’s interaction with membranes and other proteins. In this dissertation, I studied a recently identified Arabidopsis gene, PROTEIN PRENYLTRANSFERASE ALPHA SUBUNIT-LIKE (PPAL), by mapping a second-site mutation that rescues the original mutation phenotype and could be an additional factor involved in sugar homeostasis/sensing. I found that mutant phenotype associated with suppressor line was too weak and variable to consistently score, which resulted in not finding any linked markers. I also generated crosses between ppal-1 and ABA biosynthesis and signal transduction mutants to find any possible connection between these pathways and PPAL. By using several physiological screens, I concluded that PPAL’s response is dependent on ABA. A cell wall composition analysis of ppal-1xaba3-1 showed a severe drop in the lignin content of these plants, which shows promise for biofuels and biomaterial applications and indicates possible crosstalk among ABA biosynthesis, sugar homeostasis, and lignin biosynthesis. I found that PPAL functions in the ABA signal-transduction pathway. To investigate the possible cause for distorted segregation of ppal-1 in this study, reciprocal crosses were generated between ppal-1 and Col-0, and F1 genotyping again revealed only Col-0 band and no ppal-1 insertion was found, indicating a possible gametophytic lethality. I also used a mutant of the moss Phycomitrella patens to establish a novel heterologous protein expression system. This mutant, ggb, reverts the plants into undifferentiated, unicellular plants resembling green algae. ggb was used successfully to express three fungal lignolytic enzymes: aryl alcohol dehydrogenase, glyoxal oxidase and laccase. Western blots of total protein from transgenic lines showed the expected protein bands. I found the laccase activity in extracellular fluid from transgenic lines by development of blue-green color with ABTS; however, similar color was also found in an untransformed ggb line. In this dissertation, a bioreactor was developed to test the activity of recombinant proteins. I showed the advantages of ggb as a heterologous protein expression system, including its amenability to easy protein isolation compared to other plant-based protein expression systems

Stomata and sporophytes of the model moss physcomitrium patens

Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana, moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release