The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.

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SUMMARYThe charophyte green algae (CGA, Streptophyta, Viridiplantae) occupy a key phylogenetic position as the immediate ancestors of land plants but, paradoxically, are less well-studied than the other major plant lineages. This is particularly true in the context of functional genomic studies, where the lack of an efficient protocol for their stable genetic transformation has been a major obstacle. Observations of extant CGA species suggest the existence of some of the evolutionary adaptations that had to occur for land colonization; however, to date, there has been no robust experimental platform to address this genetically. We present a protocol for high-throughput Agrobacterium tumefaciens-mediated transformation of Penium margaritaceum, a unicellular CGA species. The versatility of Penium as a model for studying various aspects of plant cell biology and development was illustrated through non-invasive visualization of protein localization and dynamics in living cells. In addition, the utility of RNA interference (RNAi) for reverse genetic studies was demonstrated by targeting genes associated with cell wall modification (pectin methylesterase) and biosynthesis (cellulose synthase). This provided evidence supporting current models of cell wall assembly and inter-polymer interactions that were based on studies of land plants, but in this case using direct observation in vivo. This new functional genomics platform has broad potential applications, including studies of plant organismal biology and the evolutionary innovations required for transition from aquatic to terrestrial habitats.

Application of the dintroaniline compound, oryzalin, which inhibits microtubule formation, to the unicellular green alga Penium margaritaceum caused major perturbations to its cell morphology, such as swelling at the wall expansion zone in the central isthmus region. Cell wall structure was also notably altered, including a thinning of the inner cellulosic wall layer and a major disruption of the homogalacturonan (HG)-rich outer wall layer lattice. Polysaccharide microarray analysis indicated that the oryzalin treatment resulted in an increase in HG abundance in treated cells but a decrease in other cell wall components, specifically the pectin rhamnogalacturonan I (RG-I) and arabinogalactan proteins (AGPs). The ring of microtubules that characterizes the cortical area of the cell isthmus zone was significantly disrupted by oryzalin, as was the extensive peripheral network of actin microfilaments. It is proposed that the disruption of the microtubule network altered cellulose production, the main load-bearing component of the cell wall, which in turn affected the incorporation of HG in the two outer wall layers, suggesting coordinated mechanisms of wall polymer deposition.

The growth of a plant cell encompasses a complex set of subcellular components interacting in a highly coordinated fashion. Ultimately, these activities create specific cell wall structural domains that regulate the prime force of expansion, internally generated turgor pressure. The precise organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying mechanics of the cell wall in a single plant cell.

Like all taxa, populations of aquatic insects may respond to climate change by evolving new physiologies or behaviors, shifting their ranges, exhibiting physiological and behavioral plasticity, or by going extinct. We evaluated the importance of plasticity by measuring changes in growth, survival, and respiratory phenotypes of salmonfly nymphs (the stonefly Pteronarcys californica) in response to experimental combinations of dissolved oxygen and temperature. Overall, smaller individuals grew more rapidly during the six-week experimental period, and oxygen and temperature interacted to affect growth in complex ways. Survival was lower for the warm treatment, though only four mortalities occurred (91.6 vs 100%). Nymphs acclimated to warmer temperatures did not have higher critical thermal maxima (CTMAX), but those acclimated to hypoxia had CTMAX values (in normoxia) higher by approximately 1 °C. These results suggest possible adaptive plasticity of systems for taking up or delivering oxygen. We examined these possibilities by measuring the oxygen-sensitivity of metabolic rates and the morphologies of tracheal gill tufts located ventrally on thoracic and abdominal segments. Mass-specific metabolic rates of individuals acclimated to warmer temperatures were higher in acute hypoxia but lower in normoxia, regardless of their recent history of oxygen exposure during acclimation. The morphology of gill filaments, however, changed in ways that appeared to depress rates of oxygen delivery in functional hypoxia. Our combined results from multiple performance metrics indicate that rising temperatures and hypoxia may interact to magnify the risks to aquatic insects, but that physiological plasticity in respiratory phenotypes may offset some of these risks.

The giant, agglutinated foraminiferan Bathysiphon Sars, previously Triassic–Recent, occurs in much older sedimentary rock (Early Ordovician, late early Tremadocian) of Avalonia. The genus extends back to c. 485 Ma based on its discovery in platform mudstone of the Chesley Drive Group in Cape Breton Island, Nova Scotia. Elongate (up to 60 mm), epibenthic Bathysiphon tubes occur in wave-rippled, green-grey mudstone with a low diversity, probably dysoxic fauna. The mudstone is coeval with and lithologically similar to the Shineton Formation in Shropshire and the Welsh Borderlands. Scanning microscopy of the Bathysiphon walls shows imbricated mica grains that parallel the long axis of the tests. The lumen has a mélange of packed sediment grains, some of which are spherical structures of siliciclastic mud studded with tetrahedral pyrite crystals. A felt-like, agglutinated test, a lumen packed with spherical structures (probable stercomata) and the domal ends of some specimens are consistent with modern Bathysiphon. This report is the first time that cytoplasmic activity and stercomata formation have been used to refer fossil protists to a modern group. Bathysiphon differs from the Cambrian foraminiferan Platysolenites Pander, which has an open lumen without stercomata, but support a comparable, sediment deposit-feeding niche. Bathysiphon is truly a ‘living fossil’, with a mode of test construction, cytoplasmic activity that formed stercomata and a niche unchanged for almost 500 million years. Foraminiferans have not been found prior to the Cambrian Period, and the Early Cambrian appearance of agglutinated foraminiferans is part of the radiation of Phanerozoic communities.

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