Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity(1,2), manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping(3-6). Results from this model organism have impacted not only plant science, but also mammalian biology(7). This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic(1), Transcriptomic(8), and Proteomic(9) information for this species is freely available, whereas the previously reported methods(6,10) to genetically transform Ostreococcus are known to few laboratories worldwide. In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species

The Coumarin Glucoside, Esculin, Reveals Rapid Changes in Phloem-Transport Velocity in Response to Environmental Cues

The study of phloem transport and its vital roles in long-distance communication and carbon allocation have been hampered by a lack of suitable tools that allow high-throughput, real-time studies. Esculin, a fluorescent coumarin glucoside, is recognized by Suc transporters, including AtSUC2, which loads it into the phloem for translocation to sink tissues. These properties make it an ideal tool for use in live-imaging experiments, where it acts as a surrogate for Suc. Here, we show that esculin is translocated with a similar efficiency to Suc and, because of its ease of application and detection, demonstrate that it is an ideal tool for in vivo studies of phloem transport. We used esculin to determine the effect of different environmental cues on the velocity of phloem transport. We provide evidence that fluctuations in cotyledon Suc levels influence phloem velocity rapidly, supporting the pressure-flow model of phloem transport. Under acute changes in light levels, the phloem velocity mirrored changes in the expression of AtSUC2. This observation suggests that under certain environmental conditions, transcriptional regulation may affect the abundance of AtSUC2 and thus regulate the phloem transport velocity

Persistence ability of soil seed banks in burnt landscapes

Seed banks play a critical role in the persistence of plant populations in variable environments. In the context of fire, seed banks are a particularly important source of seed for species which are killed by fire and rely on seed stored in the soil to regenerate and persist in the landscape following such disturbance events. Fire severity can vary, depending on a number of factors including: intensity, duration of the fire, fuel loads, fuel and vegetation type, topography, climatic condition, soil texture and moisture, soil organic content, time since last fire and area burnt. Essentially fires that consume a large amount of fuel can result in greater heat penetration of the soil profile, greater soil temperatures and longer residence times during the passage of fire. This is particularly important for species with a soil-stored seed bank as heating of soil may promote or inhibit the germination of seeds in the soil and affect the regeneration of species following fires of different severities. I examined the soil seed banks of five different plant communities in the Gibraltar Range and Washpool National Parks, New South Wales, Australia. The five communities ranged from those dominated by mesic elements with low fire frequency (Wet Sclerophyll Forest and Rainforest), to those with a strong sclerophyll component and a relatively higher expected fire frequency (Dry Sclerophyll Forest, Rocky Outcrop and Wet Health). This thesis consists of five data chapters, each written in the style of a scientific paper

Quantitative analysis of regulatory flexibility under changing environmental conditions

The circadian clock controls 24-h rhythms in many biological processes, allowing appropriate timing of biological rhythms relative to dawn and dusk. Known clock circuits include multiple, interlocked feedback loops. Theory suggested that multiple loops contribute the flexibility for molecular rhythms to track multiple phases of the external cycle. Clear dawn- and dusk-tracking rhythms illustrate the flexibility of timing in Ipomoea nil. Molecular clock components in Arabidopsis thaliana showed complex, photoperiod-dependent regulation, which was analysed by comparison with three contrasting models. A simple, quantitative measure, Dusk Sensitivity, was introduced to compare the behaviour of clock models with varying loop complexity. Evening-expressed clock genes showed photoperiod-dependent dusk sensitivity, as predicted by the three-loop model, whereas the one- and two-loop models tracked dawn and dusk, respectively. Output genes for starch degradation achieved dusk-tracking expression through light regulation, rather than a dusk-tracking rhythm. Model analysis predicted which biochemical processes could be manipulated to extend dusk tracking. Our results reveal how an operating principle of biological regulators applies specifically to the plant circadian clock

Virus resistance is not costly in a marine alga evolving under multiple environmental stressors

Viruses are important evolutionary drivers of host ecology and evolution. The marine picoplankton Ostreococcus tauri has three known resistance types that arise in response to infection with the Phycodnavirus OtV5: susceptible cells (S) that lyse following viral entry and replication; resistant cells (R) that are refractory to viral entry; and resistant producers (RP) that do not all lyse but maintain some viruses within the population. To test for evolutionary costs of maintaining antiviral resistance, we examined whether O. tauri populations composed of each resistance type differed in their evolutionary responses to several environmental drivers (lower light, lower salt, lower phosphate and a changing environment) in the absence of viruses for approximately 200 generations. We did not detect a cost of resistance as measured by life-history traits (population growth rate, cell size and cell chlorophyll content) and competitive ability. Specifically, all R and RP populations remained resistant to OtV5 lysis for the entire 200-generation experiment, whereas lysis occurred in all S populations, suggesting that resistance is not costly to maintain even when direct selection for resistance was removed, or that there could be a genetic constraint preventing return to a susceptible resistance type. Following evolution, all S population densities dropped when inoculated with OtV5, but not to zero, indicating that lysis was incomplete, and that some cells may have gained a resistance mutation over the evolution experiment. These findings suggest that maintaining resistance in the absence of viruses was not costly

A Palette of Minimally Tagged Sucrose Analogues for Real‐Time Raman Imaging of Intracellular Plant Metabolism

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy

Post-fire recovery of woody plants in the New England Tableland Bioregion

The resprouting response of plant species to fire is a key life history trait that has profound effects on post-fire population dynamics and community composition. This study documents the post-fire response (resprouting and maturation times) of woody species in six contrasting formations in the New England Tableland Bioregion of eastern Australia. Rainforest had the highest proportion of resprouting woody taxa and rocky outcrops had the lowest. Surprisingly, no significant difference in the median maturation length was found among habitats, but the communities varied in the range of maturation times. Within these communities, seedlings of species killed by fire, mature faster than seedlings of species that resprout. The slowest maturing species were those that have canopy held seed banks and were killed by fire, and these were used as indicator species to examine fire immaturity risk. Finally, we examine whether current fire management immaturity thresholds appear to be appropriate for these communities and find they need to be amended

Putting the Squeeze on Plasmodesmata:A Role for Reticulons in Primary Plasmodesmata Formation

Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER during cell-plate formation. Here we show that members of the reticulon (RTNLB) family of ER-tubulating proteins may play a role in formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant co-localisation at PD with the viral movement protein of tobacco mosaic virus while super-resolution imaging (3D-SIM) of primary PD revealed the central desmotubule to be labelled by RTNLB6. FRAP studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its centre where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation

Data assimilation constrains new connections and components in a complex, eukaryotic circadian clock model

Circadian clocks generate 24-h rhythms that are entrained by the day/night cycle. Clock circuits include several light inputs and interlocked feedback loops, with complex dynamics. Multiple biological components can contribute to each part of the circuit in higher organisms. Mechanistic models with morning, evening and central feedback loops have provided a heuristic framework for the clock in plants, but were based on transcriptional control. Here, we model observed, post-transcriptional and post-translational regulation and constrain many parameter values based on experimental data. The model's feedback circuit is revised and now includes PSEUDO-RESPONSE REGULATOR 7 (PRR7) and ZEITLUPE. The revised model matches data in varying environments and mutants, and gains robustness to parameter variation. Our results suggest that the activation of important morning-expressed genes follows their release from a night inhibitor (NI). Experiments inspired by the new model support the predicted NI function and show that the PRR5 gene contributes to the NI. The multiple PRR genes of Arabidopsis uncouple events in the late night from light-driven responses in the day, increasing the flexibility of rhythmic regulation

Sticky mucilages and exudates of plants: putative microenvironmental design elements with biotechnological value

Plants produce a wide array of secretions both above- and belowground. Known as mucilages or exudates, they are secreted by seeds, roots, leaves and stems and fulfil a variety of functions including adhesion, protection, nutrient acquisition or infection. Mucilages are generally polysaccharide-rich and often occur in the form of viscelastic gels and in many cases have adhesive properties. In some cases, progress is being made in understanding the structure-function relations of mucilages such as for the secretions that allow growing ivy to attach to substrates and the biosynthesis and secretion of the mucilage compounds of the Arabidopsis seed coat. Work is just beginning in understanding root mucilage and the proposed adhesive polymers involved in the formation of rhizosheaths at root surfaces and for the secretions involved in host plant infection by parasitic plants. In this article, we summarize knowledge on plant exudates and mucilages within the concept of their functions in microenvironmental design, focusing especially on their bioadhesive functions and the molecules responsible for them. We draw attention to areas of future knowledge need, including the microstructure of mucilages and their compositional and regulatory dynamics. This article is protected by copyright. All rights reserved