A Model Suite of Green Algae Within the Scenedesmaceae for Investigating Contrasting Desiccation Tolerance and Morphology Zoe G
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© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs212233. doi:10.1242/jcs.212233 RESEARCH ARTICLE A model suite of green algae within the Scenedesmaceae for investigating contrasting desiccation tolerance and morphology Zoe G. Cardon1,*, Elena L. Peredo1, Alice C. Dohnalkova2, Hannah L. Gershone3 and Magdalena Bezanilla4,5,‡ ABSTRACT desert-dwelling and aquatic representatives. These related species Microscopic green algae inhabiting desert microbiotic crusts are harbor rich, natural variation that has developed in the presence of remarkably diverse phylogenetically, and many desert lineages have selective pressures within aquatic and desert environments. independently evolved from aquatic ancestors. Here we worked with Lewis and Flechtner (2004) described three independently five desert and aquatic species within the family Scenedesmaceae to evolved lineages of desert-dwelling green algae that fell within Scenedesmus: S. rotundus S. deserticola examine mechanisms that underlie desiccation tolerance and release the genus , and S. bajacalifornicus of unicellular versus multicellular progeny. Live cell staining and time- , that were isolated from the Sevilleta desert, lapse confocal imaging coupled with transmission electron microscopy San Nicolas Island (off the coast of California) and Baja California, established that the desert and aquatic species all divide by multiple respectively. These organisms are particularly intriguing because (rather than binary) fission, although progeny were unicellular in three aquatic relatives within the Scenedesmaceae have been studied for species and multicellular ( joined in a sheet-like coenobium) in two. decades by a number of researchers, who focused on genetic and During division, Golgi complexes were localized near nuclei, and all physiological controls over cell division by multiple fission (e.g. š species exhibited dynamic rotation of the daughter cell mass within the Bi ová and Zachleder, 2014), abiotic and biotic environmental mother cell wall at cytokinesis. Differential desiccation tolerance across drivers of phenotypic plasticity (e.g. Trainor et al., 1976; Lampert the five species, assessed from photosynthetic efficiency during et al., 1994), the evolution of terrestriality within green plants (e.g. desiccation/rehydration cycles, was accompanied by differential Lewis and Flechtner, 2004; Lewis and Lewis, 2005; Gray et al., accumulation of intracellular reactive oxygen species (ROS) detected 2007; Cardon et al., 2008) and, more recently, biofuels development using a dye sensitive to intracellular ROS. Further comparative (e.g. Ho et al., 2014) and bioremediation (e.g. Suresh Kumar et al., investigation will aim to understand the genetic, ultrastructural and 2015). We are building on this broad foundation, studying the three physiological characteristics supporting unicellular versus multicellular desert-derived taxa described by Lewis and Flechtner (2004) Scenedesmus sensu lato coenobial morphology, and the ability of representatives in the together with two sister aquatic Meyen Scenedesmaceae to colonize ecologically diverse, even extreme, (s.l. Scenedesmoideae) (An et al., 1999; Lewis and Flechtner, 2004; habitats. Hegewald et al., 2010) as a new model suite of organisms. Although not yet genetically tractable, the common genetic background of KEY WORDS: ROS, Photosynthesis, Multiple fission, Scenedesmus, these five species in the Scenedesmaceae, coupled with their Enallax, Tetradesmus ecological and morphological distinctiveness, make them a promising group for comparative analyses from the variety of INTRODUCTION biological perspectives noted above. Microscopic green algae have long been known to inhabit Evolutionary relationships among our five target organisms are microbiotic crusts in arid regions world-wide (e.g. Friedmann firmly established (Hegewald et al., 2013; Sciuto et al., 2015; et al., 1967; Metting, 1981). Over the last two decades, researchers Wynne and Hallan, 2015). Desert-derived S. deserticola and combining morphological and molecular information have S. bajacalifornicus (Lewis and Flechtner, 2004) – recently renamed demonstrated that these green algae are remarkably diverse (e.g. Acutodesmus deserticola and Acutodesmus bajacalifornicus, Lewis and Lewis, 2005), belonging to at least six different green respectively (Hegewald et al., 2013) – fall within the same genus as algal classes (Cardon et al., 2008). Phylogenetic analyses further aquatic S. obliquus, which itself was recently renamed Tetradesmus revealed that, even within families and genera (e.g. Lewis and obliquus (Wynne and Hallan, 2015; see Materials and Methods for Flechtner, 2002, 2004; Fucíková et al., 2013), multiple desert discussion of taxonomic nomenclature in flux). Desert-derived S. lineages have evolved from aquatic ancestors independently, rotundus – recently renamed Fletchneria rotunda (Sciuto et al., resulting in suites of extant related green algal species that have 2015) – and aquatic Scenedesmus costatus – recently renamed Enallax costatus (Hegewald et al., 2013) – are consistently more 1Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 USA. basal within the family Scenedesmaceae, no matter which gene 2Environmental Molecular Sciences Laboratory, Pacific Northwest National region was used for phylogenetic analysis (Sciuto et al., 2015). Laboratory, Richland, WA 99352 USA. 3Biological Sciences and Environmental Studies Departments, Mt. Holyoke College, South Hadley, MA 01075 USA. Here, we test several hypotheses regarding cellular and 4Department of Biology, University of Massachusetts, Amherst, MA 01003 USA. physiological traits in this target suite of five ecologically and 5Whitman Center, Marine Biological Laboratory, Woods Hole, MA 02543 USA. ‡ morphologically contrasting desert and aquatic taxa. Lewis and Present address: Department of Biological Sciences, Dartmouth College, A. bajacalifornicus A. deserticola F. Hanover, NH 03755 USA. Flechtner (2004) noted that , and rotunda are unicellular, but intracellular membrane characteristics *Author for correspondence ([email protected]) and details of division have not been previously explored. T. obliquus E. costatus Z.G.C., 0000-0001-8725-7842 and are coenobial: each mother cell produces daughter cells that are permanently physically joined side by side in a Received 16 October 2017; Accepted 15 February 2018 sheet-like, multicellular coenobium. Because of their contrasting Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212233. doi:10.1242/jcs.212233 morphology, these five taxa provide intriguing potential as a system membranes were stained with the lipophilic dye FM4-64; and to study determinants of multicellularity within a common genetic chloroplasts exhibited chlorophyll autofluorescence. We found that, background. in all species, the nucleus of each mother cell divided first into We first tested the hypothesis that all five taxa divide by multiple multiple nuclei, followed by chloroplast division and, finally, fission, as expected considering many other Scenedesmus sp. (Bišová cytokinesis within the mother cell wall. Fig. 1 shows representative and Zachleder, 2014). During multiple fission, a single mother cell images for two stages of division. T. obliquus has undergone commonly gives rise to more than two daughter cells during asexual nuclear but not chloroplast division, and cell membranes have not reproduction. The nucleus and chloroplast in the mother cell divide yet formed around daughter cells. For the other species, Fig. 1 into multiple copies, and cytokinesis compartmentalizes new shows daughter cells held within the mother cell wall – having daughter cells prior to release when the mother cell wall ruptures. completed nuclear and chloroplast division, and cytokinesis – but Bišová and Zachleder (2014) reviewed a large body of literature not yet released from the mother cell wall. In all species, nuclear focused on multiple fission in Chlamydomonas and Scenedesmus division occurred in the presence of one large chloroplast with an quadricauda, noting that, in Chlamydomonas, each of the multiple intact pyrenoid (e.g. T. obliquus, Fig. 1, pyrenoids indicated by daughter cells is independent and unicellular. In S. quadricauda, white arrows; chlorophyll autofluorescence channel). The pyrenoid multiple daughter cells are physically joined in a coenobium is a subcellular structure within the chloroplast where ribulose 1,5- (although cytokinesis is complete). We examined whether the cells bisphosphate carboxylase/oxygenase (RuBisCO) is concentrated; in our five target species divided by multiple fission by using pyrenoids are commonly part of the CO2-concentrating mechanism overnight, time-lapse imaging of cell division and release of in green algae and appear electron dense in transmission electron unicellular or coenobial daughter cells in gas-permeable microscopy (TEM). After nuclear division, the chloroplast divided, polymethylsiloxane (PDMS) microfluidic chambers (Bascom and the daughter cells were packaged by membranes delineating et al., 2016). We then explored intracellular organellar and daughters (e.g. FM4-64 channel, all species except T. obliquus, membrane patterns during division in more depth, using confocal Fig. 1). The pyrenoid reformed after chloroplast division and was microscopy for all five species and transmission electron microscopy visible in daughter cells still enclosed within the mother cell wall for