Endosymbiosis, gene transfer and algal cell evolution Shinichiro Maruyama and John M Archibald
Shinichiro Maruyama and John M Archibald
Plastids, specifically chloroplasts, evolved from prokaryotic endosymbionts akin to modern cyanobacteria, as proposed by the endosymbiont hypothesis This theory suggests that these prokaryotes were initially engulfed by a phagotrophic eukaryote and retained rather than digested Over time, the relationship between host and endosymbiont evolved, leading to the transfer of genetic material and the eventual dependence of endosymbionts on their hosts, resulting in the formation of primary plastids Furthermore, some eukaryotes with primary plastids were later engulfed by other phagotrophic eukaryotes in a process known as secondary endosymbiosis, which contributed to the emergence of diverse algal lineages It is widely accepted that the origin of primary plastids occurred once in eukaryotic history, while secondary endosymbiosis is believed to have happened at least three times However, there is ongoing debate among researchers regarding these events, and despite the availability of numerous complete algal genome sequences, the relationships among modern algae plastids remain contentious in the study of eukaryotic evolution.
The endosymbiont hypothesis offers a comprehensive understanding of how eukaryotes developed the capability for oxygenic photosynthesis, yet the specific types of cells involved remain elusive due to the complexity and ancient nature of these endosymbiotic events Algal cells possess three genome-bearing organelles: the nucleus, plastid, and mitochondrion, indicating that their evolution is a blend of the histories of these distinct lineages Various scenarios exist for the emergence and evolution of primary plastid-bearing eukaryotes, highlighting the intricate relationships between plastid and algal nuclear phylogenies Considering secondary endosymbiosis complicates this further, introducing additional phylogenetic signals from the secondary endosymbiont's nucleus While genomic data aids in unraveling this complexity, it also reveals that even the most straightforward models of plastid origin and evolution may be overly simplistic.
A B C D plastid origin single multiple single multiple host lineages single clade single clade multi-clades multi-clades plastid bearers monophyletic ? ‘paraphyletic’ polyphyletic
Four competing scenarios explain the origin of primary plastids: (A) the widely accepted view that a derived eukaryote acquired primary plastids through a single event of primary endosymbiosis; (B) the hypothesis that multiple primary endosymbiotic events occurred within a single eukaryotic lineage; (C) the idea that after an initial primary endosymbiosis, some lineages retained plastids while others lost them; and (D) the possibility that separate lineages independently acquired primary endosymbionts, leading to the evolution of plastids, with some lineages experiencing secondary loss of plastids.
2nd rank classifi cation Organism Secondary plastids
Chloroplast sensu stricto green algae and land plants euglenophytes, chlorarachniophytes, the dinofl agellate Lepidodinium
Rhodoplast red algae cryptophytes, haptophytes, stramenopiles, dinofl agellates, chromerids, apicomplexans 2 Cyanelle (Muroplast) 1 glaucophytes
1 See text for discussion on the terminology
2 The red versus green origin of ‘ apicoplasts ’ in apicomplexans is still contentious (Cai et al 2003; Funes et al 2004; Lau et al 2009; Obornik et al 2009; Janouskovec et al 2010)
Endosymbiosis, gene transfer and algal cell evolution 23
Endosymbiotic gene transfer (EGT) has significantly influenced the genetic composition of algal nuclear genomes, as most plastid proteomes in photosynthetic eukaryotes are encoded by nuclear genes However, distinguishing between nuclear genes from organelles, transient endosymbionts, or ingested prey remains challenging yet crucial This chapter explores algal cell evolution through phylogenetics and comparative genomics, highlighting advancements made possible by extensive genomic data Despite these advancements, ongoing debates persist regarding the inference of past endosymbiotic events.
The primary endosymbiotic origin of plastid s
Extensive phylogenetic, biochemical, and morphological evidence indicates that modern cyanobacteria and plastids share a common ancestor However, significant differences exist, particularly in genome size; most plastid genomes are only 10-20% the size of the smallest sequenced cyanobacterial genomes, such as Prochlorococcus sp strain MED4 and the uncultured marine cyanobacterium UCYN-A This substantial difference in size and gene content implies that a major reduction in plastid genome size occurred in the ancestors of primary plastid-bearing eukaryotes.
The loss of genes from plastid genomes is a structured process rather than random Research by Race et al (1999) revealed that among the thousands of genes in cyanobacterial genomes, only 46 protein-coding genes are consistently retained across all sequenced plastid genomes, primarily those related to photosynthesis and ribosomal proteins Non-photosynthetic plastids exhibit even greater gene reduction but still maintain essential genes such as tRNA and ribosomal proteins (Krause 2008) Interestingly, some non-photosynthetic plastids retain genes associated with photosynthesis, like the rbcL gene in Euglena longa and the RuBisCO gene cluster in Cryptomonas paramecium (Gockel and Hachtel 2000; Donaher et al 2009) Furthermore, RuBisCO has been implicated in lipid biosynthesis in non-photosynthetic plastids found in land plant seeds (Schwender et al.).
2004), its function in the secondarily non-photosynthetic plastids of unicellular algae remains unclear (Krause 2008)
Which cyanobacterial lineage is most closely related to the plastid progenitor ?
Genome reduction and endosymbiotic gene transfer (EGT) complicate our understanding of plastid origins, despite offering insights into endosymbiosis A significant amount of genetic information crucial for reconstructing organelle phylogeny has likely been transferred from plastids to the nucleus, making it challenging to accurately identify which nuclear genes originated from the cyanobacterial ancestor of plastids Researchers often depend on nuclear genes with strong cyanobacterial signatures in their phylogenetic analyses, but caution is essential to avoid misinterpreting plastid phylogeny with genes that are not derived from the organelle.
Current studies have yet to determine which lineage of existing cyanobacteria most closely resembles the ancestor of plastids Phylogenetic analyses utilizing the 16S ribosomal RNA gene suggest that plastid sequences are either "near the root of the cyanobacterial line of descent" or "most closely related to N2-fixing unicellular cyanobacteria." In contrast, phylogenomic analyses indicate a basal branching of plastids, rather than a direct affiliation with any specific cyanobacterial lineage Recent research, including gene content surveys, proposes that the plastid progenitor may have been akin to heterocyst-forming nitrogen-fixing filamentous cyanobacteria, such as Anabaena and Nostoc However, a limitation of these studies is the small number of taxa available for whole-genome comparisons, leaving the conclusions still inconclusive.
To gain a clearer understanding of the relationship between plastids and cyanobacteria, it is essential to increase the number of sequenced plastid genomes, which currently represent only a limited selection of algal species Expanding sampling within major algal clades and including unidentified taxa at the base of the cyanobacterial tree will significantly enhance research efforts However, existing analyses reveal that plastid genomes exhibit mixed ancestry, complicating the determination of plastid origins in contemporary plants and algae.
How heterogeneous was the ancestral plastid genome ?
A non -cyanobacterial RuBisCO operon in red algae
Inferring the evolution of plastids is challenging because their genomes are not merely simplified versions of cyanobacterial genomes A significant example of plastid genome heterogeneity is the discovery that red algal and red algal-derived plastids possess distinct RuBisCO large (rbcL) and small (rbcS) subunit genes, which are more closely related to those found in proteobacteria rather than cyanobacteria, green plants, or glaucophyte plastids This complexity is heightened by the fact that rbcS genes are considerably shorter than rbcL and are located in the nuclear genomes of green plants and glaucophytes, unlike the rbcLS operon found in red algal plastid genomes.
Endosymbiosis and gene transfer play crucial roles in the evolution of algal cells, particularly concerning the 25 distinct proteobacterial lineages The exact relationship of red algal rbcLS genes to these lineages remains uncertain To clarify, we will refer to the red algal rbcLS genes as 'non-cyanobacterial' type genes.
The rbcL gene serves as a widely utilized phylogenetic marker, having been sequenced across various lineages Its incongruence with other plastid genes, such as 16S and 23S rRNA, underscores the complex and heterogeneous nature of plastid genomes To explain the observed rbcL phylogeny, two extreme scenarios have been proposed: one involves lateral gene transfer (LGT) from a proteobacterial lineage to the plastid genome of red algae, while the other suggests ancestral gene duplication before the divergence of primary plastid-bearing eukaryotes, followed by subsequent gene losses.
The ongoing debate regarding the presence of non-cyanobacterial rbcL genes in green algae, plants, and glaucophytes remains unresolved Currently, no eukaryotes have been identified that contain both cyanobacterial and non-cyanobacterial rbcLS genes Maier et al investigated the cbbX gene family, which forms operons with rbcLS in various red algal and prokaryotic plastids They discovered that in the cryptophyte alga Guillardia theta, cbbX genes exist in both the red alga-derived plastid and the nucleomorph, sharing orthology with prokaryotic genes that possess non-cyanobacterial RuBisCO Fujita et al later confirmed that the cbbX genes from the red algal-type nuclear and plastid sources are orthologous and likely function as transcription regulators in the red alga Cyanidioschyzon merolae Maier et al also suggested that the ancestor of plastids may have possessed both cyanobacterial and non-cyanobacterial rbcL-S-cbbX operons, with subsequent lineages (green plants, red algae, and glaucophytes) losing one operon each This hypothesis aligns with the 'duplication and differential loss' model for rbcLS operon evolution However, the phylogenetic origin of the Oryza sativa cbbX homolog remains undetermined, leaving the origins of non-cyanobacterial rbcL-S-cbbX operons uncertain in relation to the common ancestor of primary plastids.
‘ Chimeric ’ origin s of the menaquinone/phylloquinone biosynthesis gene cluster
Gross et al (2008) conducted phylogenetic analyses on the plastid and nuclear men gene clusters, shedding light on the evolution of plastid genomes The men cluster is highly conserved across prokaryotes and eukaryotes, encoding enzymes essential for the biosynthesis of vitamin K, including menaquinone and phylloquinone Their research revealed that this gene cluster is exclusive to phototrophs among eukaryotes Notably, in the acidothermophilic red algal group Cyanidiales, most men genes are located in plastid genomes, whereas green plants and diatoms possess nuclear genes, including the menF-D-C-H fusion gene known as PHYLLO.
Chapter 2 reveals that many nuclear genes associated with plastid-localized proteins in Cyanidiales are closely related to non-cyanobacterial prokaryotic sequences Significant differences in phylogenetic affiliations between red algal and green plant genes were identified, with Cyanidiales exhibiting a unique combination of plastid-encoded MenA and MenE types, alongside a nuclear-encoded MenG In contrast, green plants possess cyanobacterial-type MenA and MenG, while MenE originates from deltaproteobacteria Gross et al (2008) argue that these complex phylogenetic patterns indicate evolutionary 'chimerism' in the ancestral plastid genome, suggesting that this ancestor may have incorporated extracellular DNA, potentially facilitated by viruses like cyanophages.
Cytological and cytochemical aspects in selected carrageenophytes (Gigartinales, Rhodophyta) Leonel Pereira
Red algae (Rhodophyta) are a diverse group of aquatic plants, ranging from unicellular to multicellular forms, known for their photosynthetic capabilities They exhibit various morphologies and simple anatomical structures, along with a variety of life cycles Approximately 98% of red algae species are found in marine environments, while only 2% inhabit freshwater, with a few rare species existing in terrestrial or sub-aerial habitats.
Red algae, classified under the phylum Rhodophyta, are true plants in a phylogenetic sense, sharing a common ancestor with green algae and higher plants This group is distinct from other eukaryotic algae due to several unique characteristics.
(i) Total absence of centrioles and any fl agellate phase
(ii) Presence of chlorophyll s a and d , and accessory pigments (light-harvest) called phycobilin s ( phycoerythrin and phycocyanin )
(iii) Plastid s with unstacked thylakoid s, and no external endoplasmic reticulum (iv) Absence of parenchyma and presence of pit-connection s between cells (i.e incomplete cytokinesis )
(v) Floridean starch as storage product
Red algae are traditionally categorized into three main groups based on their morphology: the first group consists of unicellular red algae that reproduce solely through binary cell division; the second group includes multicellular red algae characterized by the absence or incipient presence of a carpogonial branch, known as Bangiophyceae sensu lato; and the third group features multicellular red algae with well-developed carpogonial branches, referred to as Florideophyceae.
The Florideophyceae family features a specialized female filament known as the carpogonial branch, where the female gamete, or carpogonium, is distinguished by the presence of a trichogyne—an elongated structure that facilitates the reception of male gametes (spermatium) Following fertilization, the zygote undergoes in situ germination, leading to the formation of carpospores or the development of a parasitic generation of female gametophyte, which produces carpospores within the cystocarp.
The cystocarp consists of the carposporophyte along with the surrounding protective sterile haploid tissue from the female gametophyte, known as the pericarp Within this structure, carpospores emerge and give rise to a subsequent free-living stage called the tetrasporophyte.
In red algae, the tetrasporophyte phase can exhibit either isomorphic or heteromorphic alternation of generations, producing tetrasporangia through meiosis that release tetraspore s The meiotic division within the tetrasporangium is consistently one of three types: cruciate, tetrahedral, or zonate Each released tetraspore develops into a haploid gametophyte, which can be either male or female (Gurgel and Lopez 2007).
In general Gigartinales present triphasic isomorphic or heteromorphic, diplo-haplotic (haploid gametophyte, diploid carposporophyte and diploid tetrasporophyte) or diphasic diplo-haplotic lifecycles (Maggs 1990; Brown et al 2004; Thornber 2006)
A large number of genera of high economic interest ( carrageenophyte s), are mem- bers of this order and most of them are phylogenetically related (Freshwater et al 1994; Fredericq et al 1996)
Algal cell walls primarily consist of cellulose fibrils, with some containing xylan fibrils, embedded in a hydrocolloid matrix In red algae, this hydrocolloid matrix is made up of sulfated polysaccharides, which are categorized into two main types: agar and carrageenan.
Red algal hydrocolloids, known as phycocolloids, are polysaccharides with gel-forming abilities, classified into two main types: agars and carrageenans Both types share a sugar skeleton composed of 1,3-linked β-D-galactopyranose, with agars containing 1,4-linked 3,6-anhydro-α-L-galactopyranose and carrageenans containing 1,4-linked 3,6-anhydro-α-D-galactopyranose Natural phycocolloids are complex mixtures of neutral and charged polysaccharides, and their gel quality is determined by rheological properties such as gel strength, density, and gelling points, influenced by the chemical composition and ratios of different polysaccharides Certain red algae are categorized as agarophytes or carrageenophytes based on their predominant polysaccharide production, with notable families including Gracilariales, Gelidiales, and various families within Gigartinales.
Cytological and spectroscopic analysis techniques
Ground, dried algal samples were analyzed for their natural phycocolloids composition using FTIR-ATR and FT-Raman techniques, following the methodologies outlined by Pereira (2006) and Pereira et al (2009) FTIR spectra of both native and alkali-modified carrageenan were obtained on an IFS 55 spectrometer with a Golden Gate single reflection diamond ATR system, eliminating the need for sample preparation Each spectrum reported in this study represents the average of two counts, consisting of 128 scans at a resolution of 2 cm−1 Additionally, room temperature FT-Raman spectra were recorded using a RFS-100 Bruker FT-spectrometer equipped with a Nd:YAG laser for excitation.
1064 nm Each spectrum was the average of two repeated measurements, with 150 scans at a resolution of 2 cm −1
Cytological and cytochemical aspects in selected carrageenophytes 83
H-NMR spectra were acquired using a Bruker AMX600 spectrometer at 500.13 MHz and 65 °C, with 64 scans and a 5 s interpulse delay, as the T1 values for the anomeric protons of κ- and ι-carrageenan are under 1.5 s The carrageenan sample (5 mg mL−1) was prepared by dissolving it in D2O at 80 °C, supplemented with 1 mM TSP and 20 mM Na2HPO4, and subjected to three 1-hour sonication sessions in a Branson 2510 sonicator Chemical shifts were referenced to the internal TSP standard (δ = −0.017 ppm), following IUPAC recommendations by van de Velde et al (2004) The assignments of the 1H-NMR spectra were based on chemical shift data from van de Velde et al (2002, 2004) and Pereira and van de Velde (2011).
Two methods were utilized for the localization of cellulose (β-glucan): first, fresh sections were observed using fluorescence microscopy with ultraviolet light (345-365 nm) and Calcofluor-white staining at a concentration of 0.04% (Gretz et al 1997); second, identical sections were examined under a polarization microscope (Nikon Optiphot) with cross polarization filters to investigate crystalline or para-crystalline structures (Gretz et al 1997).
Two techniques were employed for the identification and localization of sulphated polysaccharides, specifically carrageenans The first method involved staining tissue sections with toluidine blue (0.05% in 0.1 M acetate buffer, pH 4.4) for light microscopy, which allows for the detection of acid polysaccharides through metachromasia (Gretz et al 1997) The second method utilized energy dispersive X-ray analysis (EDX) to assess variations in sulphur concentration, conducted using a Hitachi H900 electron microscope paired with an X-ray spectrometer and detector (Russ 1974; McCandless et al 1999).
Various cytochemical stains are utilized for specific identifications in microscopy, including the Thiéry test for localizing floridean starch in electron microscopy, Periodic Acid Schiff (PAS) for detecting polysaccharides like floridean starch, and Black Sudan B for identifying lipids in light microscopy.
Morphological, anatomical and cytological aspects
Ahnfeltiopsis devoniensis and Gymnogongrus crenulatus ( Phyllophoraceae )
The thallus of G crenulatus features cartilaginous, flattened dark red fronds that emerge from a short cylindrical stipe, anchored to the substrate by a small disc-shaped holdfast measuring 10 mm in diameter These fronds exhibit a repeated dichotomous branching pattern with rounded tips, which may occasionally appear bleached (Ardré 1977; Gayral and Cosson 1986; Cabioch et al 1995).
G crenulatus exhibits a diphasic lifecycle that lacks a carposporophyte Its monoecious gametophytes develop a post-fertilization structure known as a tetrasporoblast or carpotetrasporophyte, originating from auxiliary cells This process results in wart-like excrescences on the gametophyte that produce tetrasporangial nemathecia Meiosis is believed to occur during the tetrasporogenesis phase.
Evolution of vacuolar targeting in algae Burkhard Becker and Kerstin Hoef-Emden
Burkhard Becker and Kerstin Hoef-Emden
Vacuoles are essential, single membrane-bound compartments within the cytoplasm of cells, playing crucial roles in various cellular functions In angiosperms, the large central vacuole is vital for cell viability, providing structural support, storage, waste disposal, and contributing to cell protection and growth In contrast, vacuoles in algae vary significantly in size; unicellular algae typically have small vacuoles, while coenocytic and multicellular algae may possess a single large vacuole akin to that of embryophytes It is widely believed that vacuoles in algae fulfill similar functions as those in land plants.
Vacuoles are integral components of a cell's endomembrane system, interconnected with other compartments through vesicular transport (Surpin and Raikhel, 2004) The majority of vacuolar proteins are produced by ribosomes attached to the rough endoplasmic reticulum and are translocated into the ER during the co-translational process (Vitale and Raikhel).
Coated vesicles are responsible for transporting vacuolar proteins from the endoplasmic reticulum (ER) through the Golgi apparatus to the vacuole (Vitale and Raikhel, 1999) In angiosperms, there is evidence of direct trafficking of vacuolar proteins from the ER to vacuoles, bypassing the Golgi apparatus (Vitale and Galili, 2001) The presence of two distinct types of vacuoles in the same plant cell complicates this process: the lytic vacuole (LV), which is acidic and lysosome-like, and the protein storage vacuole (PSV), which is less acidic and accumulates reserve materials (Park et al., 2004) However, the existence of both vacuole types in all angiosperm cells remains a topic of debate (Frigerio et al., 2008).
Soluble proteins intended for the vacuole possess vacuolar sorting signals (VSS) that bind to vacuolar sorting receptors (VSR) in the trans-Golgi network (TGN) These proteins are then transported in clathrin-coated vesicles to the prevacuolar compartment, where the cargo protein and VSR complex dissociates, allowing VSRs to return to the Golgi complex Eukaryotes have identified three distinct VSRs, with mammalian cells primarily utilizing the mannose-6-phosphate receptor for lysosomal protein sorting Additionally, some lysosomal hydrolases are directed to the lysosome via sortilin, which is homologous to the yeast vps10p receptor The yeast vps10p represents a second type of eukaryotic VSR, while an unrelated third type has been noted in angiosperms.
Recent studies have focused on protein trafficking to vacuoles in angiosperms, highlighting the identification of three distinct vacuolar sorting signals (VSSs) The first type features a sequence-specific VSS at the N- or C-terminus, such as the N-terminal NPIR (Asn-Pro-Ile-Arg) The second type is characterized by a C-terminal VSS with variable length, often rich in hydrophobic amino acids Additionally, some proteins necessitate extended segments from various parts of the polypeptide for effective vacuolar targeting.
The first identified VSR in plants was the pea trans-membrane protein BP-80, which led to the discovery of a small protein family in Arabidopsis thaliana, comprising seven members known as AtVSR 1-7 This protein family appears to be exclusive to plants, with all known angiosperm members sharing specific characteristics These include a large N-terminal luminal domain of approximately 400 amino acids that features a protease-associated domain at its N-terminus, several epidermal growth factor (EGF) signature sequences in the C-terminal part, a single trans-membrane region, and a short cytoplasmic tail containing a YXXI tyrosine-motif The PA-domain and EGF repeats are believed to play a crucial role in ligand binding.
2000) and the tyrosine-motif is apparently involved in sorting the receptor ligand complex into clathrin-coated vesicles formed at the TGN (Happel et al 2004)
A second type of vacuolar sorting receptor, named RMR, has been identified, belonging to a unique protein family with a ReMembR-H2 domain, and is likely involved in transport to storage vacuoles in plants This receptor appears to be restricted to Tracheophyta Research by Masclaux et al (2005) categorized seven VSRs from A thaliana into three phylogenetic clades, suggesting distinct functions related to lytic, storage vacuole, and endocytic pathways However, recent studies indicate that while some functional specialization exists among VSRs, many perform redundant roles The precise functions and in vivo cargo of various VSRs and RMR isoforms remain unclear, necessitating further investigation.
Recent research has revealed a lack of knowledge regarding protein trafficking to vacuoles in eukaryotic algae A study conducted by Becker and Hoef-Emden (2009) explored completed algal genomes for AtVSR homologues to understand the evolution of vacuolar sorting in algae The findings indicated that all investigated viridiplants possess at least one plant-type VSR, while red algae and likely glaucophytes do not have AtVSR homologues Moreover, several chromalveolate species appear to have acquired a putative VSR from green algae through horizontal or endocytotic gene transfer This study revisits the topic by including additional species, such as Ectocarpus siliculosus, Albugo laibachii, Emiliania huxleyi, Fragilariopsis cylindrus, Phytophthora infestans, Picea sitchensis, Selaginella moellendorffii, and Coccomyxa sp C169 genomes, as well as Klebsormidium subtile and Coloechaete scutata ESTs in the analysis.
Evolution of vacuolar targeting in algae 107
Recent genome releases have shown slight modifications in protein sequences, particularly different adaptin motifs in Tetrahymena thermophila, Phaeodactylum tricornutum VSR2, Chlorella sp NC64A, and Micromonas pusilla, which were incorporated into our analyses The updated Phytophthora sojae genome now features a VSR protein comparable in length to those found in other chromalveolates.
Identifi cation of putative vacuolar sorting receptors in protists
To explore vacuolar targeting in algae, we conducted a screening of public databases for the presence of plant-type and vps10p type VSRs in protists using the BLASTP program with a conservative cut-off of e-20 The findings, detailed in Table 5.1, reveal that plant-type VSRs are primarily found in viridiplants, stramenopiles, and ciliates, while putative vps10p homologues appear in opisthokonts, alveolates, and chlorophytes Notably, some eukaryotic groups lack both VSR types, suggesting they may rely on alternative, unrelated VSRs for vacuolar protein targeting This article will focus on the structure and evolution of plant-type VSRs in algae.
Table 5.1 Distribution of vps10p and plant-type vacuolar sorting receptors among eukaryotes Phyla are grouped into the currently discussed 5 eukaryotic supergroups vps10p plant-type VSR
Short ESTs may exhibit limited similarity confined to a single domain, and no analogous proteins were identified through BLASTP or tBLASTN analysis, utilizing an e-value cut-off of e-20 with the protein EST database at NCBI.
Significant similarities to Arabidopsis thaliana VSRs were found in various protists, including green algae, ciliates, diatoms, brown algae, pelagophytes, and oomycetes, but not in haptophytes, rhodophytes, or glaucophytes Several protists exhibited multiple putative homologues to AtVSRs, with two putative VSRs identified in the genomes of Micromonas sp., Chlorella sp., Cocco-myxa sp., Albugo laibachii, and Thalassiosira pseudonana BLASTP searches revealed three putative VSRs in both Phaeodactylum tricornutum and Fragilariopsis cylindrus Additionally, a highly modified fourth protein sequence from Phaeodactylum tricornutum and a derived sequence from Aureococcus anophageferrens were not suitable for phylogenetic analyses A newly released sequence from the Tetrahymena thermophila genome showed limited similarity to viridiplant VSRs and was excluded from further analysis Notably, the genome of Paramecium tetraurelia contained nine putative VSRs, indicating a small protein family presence.
P tetraurelia , only sequences for the best three BLASTP hits ( Table 5.2 and Table 5.3 ) were retrieved for Paramecium tetraurelia and used for phylogenetic analyses and protein structure predictions
Table 5.2 VSR-homologues in protists, bryophytes and pteridophytes
Arabidopsis thaliana VSR query VSR 1 – VSR 7
Chlorella sp NC64A jgi|ChlNC64A_1|144184| jgi|ChlNC64A_1|144100|
Coccomyxa sp C169 jgi|Coc_C169_1|49231| jgi|Coc_C169_1|26335|
Fragilariopis cylindrus jgi|Fracy1|171314| jgi|Fracy1|269990| jgi|Fracy1|244424|
Micromonas pusilla jgi|MicpuC2|43569| jgi|MicpuC2|56945|
Table continued on next page
Evolution of vacuolar targeting in algae 109
Arabidopsis thaliana VSR query VSR 1 – VSR 7
Micromonas sp RCC299 jgi|MicpuN2|58183| jgi|MicpuN2|66647|
Oryza sativa gi|115469398| gi|115481614| gi|115487010
Paramecium tetraurelia 1 gi|146184097| gi|145501005| gi|145504074|
Phaeodactylum tricornutum jgi|Phatr2|38456| jgi|Phatr2|32074| jgi|Phatr2|32073| jgi|Phatr2|36020|
Physcomitrella patens ref|XP_001759820.1| ref|XP_001777810.1| ref|XP_001776090.1| ref|XP_001765481.1| ref|XP_001751750.1| ref|XP_001785165.1| ref|XP_001759928.1| gb|AAG60258.1|
Selaginella moellendorffi i gi|302770398| gi|302788188| gi|302768689| gi|302821453|
Thalassiosira pseudonana jgi|Thaps3|42545| jgi|Thaps3|25062|
Rhodophyta (NCBI ESTs, and Galdieria sulphuraria and Cyanidioschyzon merolae genomes)
No protein similar to AtVSRs found
Glaucocystophyceae (NCBI ESTs) Haptophyta ( Emiliania huxleyi )
The Paramecium genome features numerous hypothetical proteins that exhibit similarities to plant virus resistance proteins (VSRs) For clarity and to represent the protein family, only three members are utilized in the phylogenetic analysis.
T able 5.3 In-silico characterization of putati v e viridiplant and chromalv eolate VSRs Organism Protein length (aa)
SP prediction 1 TMD prediction 2 à -adaptin binding motif 3
The study explores various signatures and domains in different species, including Paramecium tetraurelia and Tetrahymena thermophila, highlighting their unique characteristics such as Asx-hydroxyl and EGF signatures The research also examines the presence of specific domains in Phaeophytes like Ectocarpus siliculosus and diatoms such as Fragilariopsis cylindrus and Phaeodactylum tricornutum, noting variations in their sequences and functional attributes Additionally, the analysis includes Oomycetes, specifically Albugo laibachii, emphasizing the diversity of biochemical signatures across these organisms.
Evolution of vacuolar targeting in algae 111
T able 5.3 (Continued) Table continued on next page
SP prediction 1 TMD prediction 2 à -adaptin binding motif 3
The study analyzes various signatures and domains across different species, including Asx-hydroxyl 4 EGF1, 4 EGF2, and 4 EGF-Ca signatures, along with the PA domain It highlights the significant findings related to Albugo laibachii, Phytophthora sojae, Phytophthora ramorum, and Phytophthora infestans, detailing their respective sequences and identifiers Additionally, the research encompasses Viridiplants, specifically the Chlorophyta group, featuring species such as Chlorella spec NC64A and Chlamydomonas reinhardtii, while also examining Coccomyxa sp and Micromonas pusilla, revealing critical insights into their genetic structures and evolutionary relationships.
T able 5.3 (Continued) Organism Protein length (aa)
SP prediction 1 TMD prediction 2 à -adaptin binding motif 3
Cytokinesis of brown algae Christos Katsaros, Chikako Nagasato, Makoto Terauchi and Taizo Motomura
Makoto Terauchi and Taizo Motomura
Brown algae (Phaeophyceae) are complex photosynthetic organisms that have garnered significant interest from phycologists due to their economic value and unique evolutionary traits They are one of the few eukaryotic lineages to have independently evolved complex multicellularity, distinct from green plants, with which they share a distant relationship A notable advancement in research is the sequencing of the 214 Mbp genome of Ectocarpus siliculosus, a filamentous seaweed and model organism closely related to kelps This multicellularity is associated with a diverse set of signal transduction genes, particularly a family of receptor kinases, which parallels similar developments in the evolution of multicellularity in animals and green plants.
Recent molecular studies have highlighted the significance of brown algae, drawing attention to their unique structural features, particularly in the context of mitosis and cytokinesis Notably, brown algae do not exhibit unicellular or simple unbranched forms; instead, the simplest morphology is represented by branched uniseriate filaments, such as Ectocarpus Given the vast diversity of brown algae, ranging from the simple filaments of Ectocarpus to the complex giant kelps like Macrocystis and Alaria, the critical role of cell division in their development is well recognized.
The study of cell division in brown algae began with light microscopy in the late 19th and early 20th centuries, with significant contributions from researchers such as Swingle, Strasburger, and Yamanouchi The advent of electron microscopy in the 1970s and 1980s revealed new insights into mitosis and cytokinesis in these organisms Pioneering ultrastructural studies identified two distinct patterns of cytokinesis: one involving furrowing of the plasma membrane, observed in species like Pylaiella littoralis and Fucus vesiculosus, and another characterized by the outgrowth of a partition membrane, as noted in Ascophyllum nodosum This ongoing debate highlights the complexity of cytokinesis in brown algae.
Chapter 7 highlighted unresolved questions about the cytokinesis mechanism in brown algae, prompting subsequent research on cell division across various brown algal species.
Brown algae exhibit unique cellular characteristics that make them valuable models for studying cytoskeleton organization and cell division Notably, they lack cortical microtubules, preprophase microtubule bands, phragmoplasts, and phycoplasts, while possessing centrosomes and a cortical actin filament cytoskeleton Unlike animals and yeasts, brown algae do not display an actomyosin contractile ring The application of immunofluorescence techniques has significantly enhanced our understanding of the organization of microtubules and actin filaments in brown algal cytokinesis Combined immunofluorescence and transmission electron microscopy studies have provided insights into the cytokinetic mechanisms of brown algae, although a key question remains regarding vesicle delivery to the equatorial plane in the absence of phragmoplast or phycoplast microtubules Despite minor variations in cytokinesis among different brown algal cell types, a general framework has been proposed, highlighting four key aspects: the involvement of microtubules and actin filaments in cytokinesis, the absence of phycoplasts and phragmoplasts, a lack of a clear furrowing mechanism, and the formation of the cytokinetic diaphragm through the fusion of flat cisternae and Golgi-derived vesicles.
Cytoskeleton of brown algae and its role in cytokinesis and cell wall morphogenesis
Cytoskeletal elements, including microtubules (MTs) and actin filaments (AFs), play crucial roles in mitosis and cytokinesis in higher plants and various algae In animal cells and yeasts, cytokinesis begins at the cell's periphery, where a contractile actomyosin ring pulls the plasma membrane inward to form a furrow In contrast, higher plant somatic cells utilize a unique cytoskeletal structure known as the phragmoplast, which comprises overlapping MTs and AFs that are oriented transversely to the division plane This structure facilitates the delivery of Golgi-derived vesicles, forming a cell plate that expands outward and ultimately fuses with the existing cell walls Additionally, the cytoskeleton of brown algal cells differs significantly from that of land plants, as it is characterized by the persistent presence of a centrosome, or microtubule organizing center (MTOC), throughout the entire cell cycle.
Brown algal cells present a unique model for studying microtubule (MT) organization during the cell cycle due to the absence of cortical MTs, MT preprophase bands, and phragmoplasts, alongside the presence of centrosomes The centrosome consists of a pair of centrioles and pericentriolar materials, highlighting its structural complexity Additionally, gamma-tubulin plays a crucial role in microtubule nucleation, while centrin is consistently found in these cells, indicating their importance in MT dynamics.
Cytokinesis of brown algae 145 on centrosomes in brown algae (Katsaros and Galatis 1992; Karyophyllis et al 2005)
In brown algae, centrosomes serve as the primary microtubule-organizing centers (MTOCs) throughout the entire cell cycle, as illustrated in Figure 7.1 The only microtubules present in brown algal cells radiate from these centrosomes, with the exception of a single instance where microtubules were identified at cortical sites (Corellou et al 2005).
Immunofluorescence microscopy images reveal the cellular structure of Dictyota dichotoma and Silvetia babingtonii, utilizing anti-tubulin, anti-actin, and anti-γ-tubulin antibodies, with nuclei stained by DAPI In the apical region of D dichotoma, microtubules (MTs) appear in green, highlighting their radiating pattern from two centrosomes in each large apical cell, sub-apical cells, and cortical cells, while the nuclei are depicted in blue.
(B) MTs radiate from two centrosomes Two γ -tubulin dots can be detected in each one
(C) MTs (green) and AFs (red) during early cyotokinesis MTs radiate from centrosomes of two nuclei (blue) in telophase, and AFs begin to accumulate beween them
(D) MTs (green) and AFs (red) during cyotokinesis of S babingtonii Actin plate (orange) can be detected at the cytokinetic plane, where MTs from both centrosomes intermingle
This study investigated the re-organization of microtubule (MT) arrays during the development of Fucus zygotes and embryos through in vivo observation after fluorescent tubulin microinjection Microtubules play a crucial role in cytokinesis, particularly in higher plants where the phragmoplast, an MT assembly, facilitates cell plate formation from Golgi-derived vesicles, thereby separating daughter cells In brown algae, microtubules from centrosomes associate with each nucleus to establish the cytokinetic plane, where the actin disc forms The involvement of MTs in cytokinesis is further supported by the observation that treatment with anti-MT agents, as well as taxol, inhibits this process Notably, in taxol-treated cells, MTs on the cytokinetic plane were observed, which is not seen in untreated cells, indicating a distinct alteration in the MT system during cytokinesis.
Fluorescence microscopy has revealed the presence of actin filaments (AFs) in brown algae, utilizing rhodamine-phalloidin labeling or specific antibodies to detect actin molecules This research has been documented in various studies, including those by Brawley and Robinson (1985), Kropf et al (1989), Bouget et al (1996), Alessa and Kropf (1999), Karyophyllis et al (2000a), Hable et al (2003), and Bisgrove and Kropf.
The distribution of actin filaments (AFs) varies throughout the cell cycle, with a well-organized cortical AF system forming at the cytoplasmic surface during interphase, similar to the cortical microtubule (MT) system in higher plant cells (Karyophyllis et al 2000a) Research utilizing fluorescence microscopy, transmission electron microscopy (TEM), and actin inhibitors has demonstrated that this cortical AF system plays a crucial role in cell wall morphogenesis by orienting cellulose microfibrils (Karyophyllis et al 2000b) Additionally, AFs are found to colocalize with spindle MTs from metaphase to early telophase (Karyophyllis et al 2000a; Nagasato and Motomura 2009) Prior to cytokinesis, an actin plate forms at the cytokinetic plane, which does not constrict like the actin contractile ring observed in animal cells and persists during the creation of the new cell partition In the zygotes of Silvetia compressa, this actin plate appears at the cell's center and expands toward the plasma membrane (Bisgrove and Kropf 2004), while in Fucus distichus, this expansion coincides with the growth of the new cell partition membrane (Belanger and Quatrano 2000).
Recent advances on the study of the cytokinesis of brown algae
Since the 1970s, two distinct patterns of cytokinesis in brown algae have been identified through conventional chemical fixation and transmission electron microscopy (TEM) The first pattern involves centripetal furrowing of the plasma membrane, while the second is characterized by centrifugal outgrowth of a partition membrane This furrowing was a key feature of brown algal cytokinesis during that period However, chemical fixation proved inadequate for observing cytokinesis due to its inability to preserve the rapid dynamic changes of delicate membranous structures In the 2000s, advancements in TEM using cryo-fixation and freeze substitution provided improved insights into these processes.
Recent studies on cytokinesis in various brown algae species, including Scytosiphon lomentaria, Dictyota dichotoma, Halopteris congesta, Sphacelaria rigidula, and Silvetia babingtonii, have revealed significant findings Notably, in the vegetative cells of Sphacelaria rigidula, cytokinesis predominantly occurs through furrowing of the plasma membrane In contrast, Dictyota dichotoma exhibits a diaphragm formation that lacks a clear centrifugal or centripetal pattern Additionally, the cytokinesis process in the zygotes of brown algae shows distinct characteristics compared to vegetative cells.
In S lomentaria and Silvetia babingtonii, cytokinesis primarily occurs through the expansion of the cell partition membrane (Nagasato and Motomura, 2002a; Nagasato et al., 2010) This conclusion is further corroborated by studies on fucoid zygotes utilizing FM4-64, a dye that assesses membrane trafficking related to endocytosis and exocytosis (Belanger and Quatrano, 2000; Bisgrove and Kropf, 2004).
Development of antheridial fi laments and spermatozoid
Qiaojun Jin and Karl H Hasenstein
The green alga Chara is considered the closest relative of land plants, exhibiting advanced features like oogamous sexual reproduction and phragmoplastic cell division, while still retaining ancestral traits such as zygotic meiosis Notably, Chara shares the formation of motile spermatozoids with bryophytes and ferns, produced in the antheridium, a bright orange, multi-cellular structure approximately 0.5 mm in diameter The antheridium's outer layer consists of interlocked shield cells containing antheridial filaments, which develop from the second capitulum and give rise to individual spermatozoids Upon maturity, the shield cells rupture, releasing the spermatozoids through liberation pores Each mature spermatozoid features two flagella originating from the lateral anterior end, minimal cytoplasm, and a unique structure that includes approximately 30 mitochondria and a dense, cylindrical nucleus, along with six starch-filled plastids arranged with associated mitochondria.
Spermatogenesis and the ultrastructure of spermatozoa are well understood; however, the release process remains largely unexplored The swimming patterns of spermatozoa indicate that microtubules (MT) and microfilaments are crucial for their development and motility Nonetheless, the specific roles of these cytoskeletal elements in the activation and release of spermatozoa are still unknown.
The cytoskeleton's functionality is often examined through the use of drugs that either disrupt or stabilize its components, hindering the dynamic rearrangement of cytoskeletal elements One such drug, the dinitroaniline herbicide Oryzalin, binds to tubulin dimers, leading to microtubule depolymerization and facilitating the study of microtubule-dependent cellular processes Additionally, Latrunculin, derived from the marine sponge Latrunculia magnifica, has been demonstrated to depolymerize F-actin both in vivo and in vitro by binding to actin monomers and altering their interface.
The oxime derivative 2,3-butanedione monoxime (BDM) serves as a myosin ATPase inhibitor in eukaryotic cells It uncompetitively inhibits both myosin and myofibril ATPase activity by enhancing the equilibrium constant of the cleavage step and delaying the release of inorganic phosphate This mechanism plays a crucial role in preventing polymerization processes, as highlighted by Walter et al (2000) and Herrmann et al.
In maize root tips, BDM treatment significantly altered the distribution of plant myosin VIII and a potential myosin II homologue, leading to the loss of endoplasmic and cortical microtubule assemblies while increasing their presence at plasmodesmata and pit fields This study examines the effects of Oryzalin, Latrunculin B, and BDM on the spermatozoid release process in Chara contraria A Braun ex Kützing, exploring the connection between the cytoskeleton and the release of spermatozoids.
Experimental set up and manipulations
Chara contraria (Characeae) was cultivated in 20 L glass tanks filled with local pond soil beneath a 3 cm sand layer, using tap water at room temperature (22-25 °C) and natural window light Mature antheridia were extracted from the thalli and placed in artificial pond water containing varying concentrations of LatB (0.01, 0.1, 1, or 5 µM), Oryzalin (10 or 50 µM), and BDM (1, 10, or 20 mM) on glass slides The antheridia were then opened, and the release of spermatozoids was monitored via video microscopy with an Olympus DP71 digital camera.
In a study conducted using a Nikon Eclipse E600FN microscope, compounds dissolved in methanol were tested against untreated controls immersed in 0.05% (v/v) methanol in culture medium Spermatozoid release was monitored over a period of approximately 30 minutes, or until the majority of spermatozoids had been released Each experiment was repeated a minimum of three times to ensure reliability of the results.
The study analyzed the timing and dynamics of spermatozoid release from antheridia, focusing on key metrics such as the duration from antheridia cutting to spermatozoid release, their movement prior to release, and the onset of motility Spermatozoid size was quantified using Image J software, defined by the length-to-diameter ratio (L/D) The release dynamics were modeled using a logistic function to express the percentage of spermatozoids released over time, with parameters indicating the release timing and rate Average release rates were derived from the minimized sums of squares of the distribution, and statistical analyses were conducted using Proc GLM with Tukey-Kramer adjustments for multiple comparisons.
Development of antheridial fi laments and spermatozoid release 163
Mature antheridia were cut into halves One half of each antheridium was placed into
For effective cell fixation, incubate samples with 1 à M LatB or 50 à M Oryzalin for one hour before fixing in 1.5% formaldehyde in PHEM buffer containing 5% DMSO for two hours at room temperature, following the method by Brown and Lemon (1995) The second half of the sample serves as a control After digesting the cell wall with a mixture of 1% cellulase Y-O and 0.1% pectolyase Y-23, both sourced from Koamicho Corporation, and containing 1% BSA for 30 minutes, stain the microtubules (MTs) using the monoclonal antibody YOL 1/34 from Accurate Chemical.
& Scientifi c Corp., Westbury, NY) followed by Alexa 488 -conjugated secondary anti-rat antibody (Molecular Probes (Invitrogen), CA, USA)
F-actin visualization was achieved by incubating samples in Alexa-phalloidin 488 for 5 hours following treatment with cellulase and pectolyase This was followed by washing with 5% glycerol in PHEMD buffer for 10 minutes and 0.1% Triton X-100 in PHEMD buffer for 30 minutes Nuclei were stained using 0.005% propidium iodide in phosphate-buffered saline (pH 7) for two minutes Images were captured using a confocal laser scanning microscope (MRC-1024, BioRad, Hercules, CA) with Lasersharp 2000 software (v 5.1), and each experiment was repeated at least three times for reliability.
Mature antheridial filaments were stained for 10 minutes using a 0.02% (w/v) calcofluor solution in artificial pond water Fluorescence imaging was conducted with an Olympus DP71 digital camera attached to a Nikon Eclipse E600FN microscope.
The antheridia at various developmental stages provide insights into the evolution of cytoskeletal organization within antheridial filaments Mitotic processes result in increasingly dense nuclear packaging before the nuclei transform into coiled spermatozoids Mature spermatozoids feature two lateral anterior flagella, minimal cytoplasm, and a distinct arrangement of nuclei and microtubules that remain straight during filamentous cell release, subsequently adopting a motile coiled shape as free-swimming spermatozoids The posterior region of the spermatozoid is noted for its thick microtubular composition.
The process of spermatozoid release
After the antheridia open, the coiled spermatozoids stay within the antheridial filaments for approximately 12 to 20 minutes The release process initiates with the flagella's movement inside the cell for about 1 to 2 minutes before the spermatozoids are expelled through the liberation pore.
The development of antheridial filaments in Chara contraria involves cell divisions that result in progressively smaller cytoplasmic volumes Although the diameter of the filaments remains constant, the volume of each cell decreases, leading to a larger proportion of nuclear material The symmetry of these cell divisions is highlighted by the alternating zones of bright microtubule staining Once the filaments have completed cell mitosis, they are devoid of cytoplasmic material.
Mature spermatozoids in antheridial filaments demonstrate microtubules aligned with their coiled nucleus, both before and after passing through the liberation pore Newly released spermatozoids are elongated, with microtubules concentrated at the tail end All images are presented at similar magnifications, with a scale bar of 10 μm.
Development of antheridial fi laments and spermatozoid release 165
Dinofl agellate bioluminescence – a key concept for studying
Kirsten Heimann, Paul L Klerks and
Bioluminescence in Pyrocystis lunula is influenced by the circadian movement of scintillons and chloroplasts, which are transported by the cytoskeleton Research utilizing cytoskeleton-specific drugs revealed that chloroplast movements could be visualized through chlorophyll autofluorescence and confocal laser scanning microscopy The drugs Latrunculin B and Oryzalin inhibited chloroplast translocation, while bioluminescence was suppressed four hours into the night phase by Latrunculin B, the myosin inhibitor 2,3 butanedione monoxime (BDM), and Oryzalin in a dose-dependent manner Although the actin stabilizer Jasplakinolide slightly enhanced bioluminescence, it countered the effects of Latrunculin B Interestingly, Colchicine, a microtubule depolymerizer, did not impact bioluminescence These findings indicate that both F-actin and microtubules play crucial roles in the movement of chloroplasts and scintillons, though additional research is required to clarify whether the observed effects on bioluminescence stem from disrupted translocations of these organelles.
Mechanisms of circadian-controlled establishment and down regulation of bioluminescence in dinofl agellate s
In photosynthetic bioluminescent dinoflagellates, such as Pyrocystis lunula, light emission is regulated by circadian rhythms, being down-regulated during the day and re-established at night This process involves the reciprocal movements of chloroplasts and scintillons, with chloroplasts positioned at the cell periphery during the day and centrally located at night While the distribution of scintillons has been challenging to clarify, recent studies confirm their peripheral position at night and central location during the day Furthermore, Western blot analysis reveals the presence of luciferase in both day and night phase cells of P lunula, indicating that this species does not rely on diurnal scintillon reassembly, unlike many other bioluminescent dinoflagellates.
Case 1982) suggest that circadian control of mechanically induced bioluminescence by diurnal translocation of scintillons may be common in the genus Pyrocystis (Sweeney
1981, Colepicolo et al 1993, Seo and Fritz 2000)
Circadian bioluminescence in Lingulodinium polyedrum relies on the diurnal disassembly and reassembly of scintillons, contrasting with the complex movements observed in the genus Pyrocystis The formation of pre-scintillons occurs near the Golgi apparatus in the cell center, and they are transported to the cell periphery at night Mechanical bioluminescence in dinoflagellates necessitates scintillon association with the vacuole, as outlined in the "proton trigger" model, where a mechanical stimulus generates action potentials at the tonoplast This depolarization leads to the influx of protons from the acidic vacuole, acidifying the scintillon lumen The resulting acidification activates luciferase, an enzyme that oxidizes luciferin, producing light Therefore, the cytoskeleton may play a dual role in facilitating diurnal scintillon translocation and anchoring scintillons to the vacuolar membrane.
Involvement and mechanism of the cytoskeleton in organelle movement
Filamentous actin (F-actin) plays a crucial role in organelle trafficking across various cell types, including animal, plant, and fungal cells Research has demonstrated that actin filaments are integral to the endocytotic vesicle trafficking process Key studies have utilized cytoskeletal drugs such as Latrunculin B, Oryzalin, and Colchicine to investigate the role of cytoskeletal components in the movement of chloroplasts.
In 1999, Foissner and Wasteneys highlighted the specificity of certain drugs, such as Latrunculin B, which binds to monomeric G-actin, and Colchicine and Oryzalin, which target tubulin dimers These interactions shift the equilibrium from polymerized to monomeric components, resulting in the disintegration of key cytoskeletal structures (Spector et al 1983, Morejohn et al 1987a, Morejohn et al 1987b, Spector et al.).
F-actin and microtubules are known to facilitate the transport of membranous organelles, but their role in the translocation of non-membranous particles remains unclear Recent research conducted on Xenopus oocytes has demonstrated that F-actin plays a significant role in the movement of non-membranous subnuclear organelles.
Our study utilized cytoskeletal drugs alongside confocal microscopy and bioluminescence measurements to investigate the cytoskeleton's role in the diurnal movement of scintillons and chloroplasts in the marine dinoflagellate Pyrocystis lunula The findings indicate that both F-actin and microtubules are crucial for circadian chloroplast movements, highlighting their involvement in the translocation of scintillons and their attachment to the tonoplast.
Experimental design and set up
Cell cultivation and preparation for drug treatments and measurement of bioluminescence
Pyrocystis lunula (Sch ü tt) was sourced from Lumitox ® Gulf L.C in Slidell, LA, USA, and cultured in a Percival ® environmental growth chamber at a temperature of 20 °C The organisms were maintained under a 12:12 hour light-dark cycle in synthetic dinoflagellate medium, with an irradiance of 65 µmol photons m−2 s−1, following the methodology outlined by Heimann et al (2002).
To achieve consistent organelle distribution, cultures were utilized at the end of the exponential growth phase (4 weeks) for all experiments The impact of cytoskeletal drugs on diurnal chloroplast translocations was assessed through chlorophyll autofluorescence and confocal laser scanning microscopy (MRC-1024 BioRad) Additionally, the influence of cytoskeletal drugs on bioluminescence was evaluated for mechanically inducible bioluminescence, following the methodologies outlined in Heimann et al (2002).
Briefl y, cells were homogenously suspended by stirring on a magnetic stirrer for
Fifteen minutes to two hours before the night phase began, cell concentrations of the cultures were determined using a Sedgewick Rafter counting chamber The cultures were then diluted to a concentration of 100 cells per mL with artificial seawater for the assays.
In this study, 3 mL volumes of a cell suspension at a density of 100 cells mL−1 were incubated with various cytoskeletal drugs, including Latrunculin B, Jasplakinolide, 2,3 butanedione monoxime, Oryzalin, and Colchicine, before transitioning from day to night photoperiods Stock solutions were prepared with DMSO, except for BDM, which used sterile artificial seawater, maintaining DMSO concentration below 1% Bioluminescence measurements were taken 4 hours post-transition in a light-controlled environment to prevent interference with the cells' night phase A custom-built bioluminometer measured cumulative bioluminescence over one minute To ensure the cytoskeletal drugs did not produce general physiological effects, total bioluminescence was stimulated with 0.5% acetic acid for both treated and control groups Recovery experiments were conducted to confirm that observed effects were not due to cytotoxicity The impact of the drugs on bioluminescence was expressed as a percentage relative to controls, and data analysis was performed using one-way ANOVA with Statistica 7.
Cytoskeletal drug treatment and quantifi cation of chloroplast movements
This study investigates the impact of cytoskeletal drugs on diurnal chloroplast translocation by incubating cells in 10 µM Latrunculin B for 2 hours or 50 µM Oryzalin for 1 hour, prior to transitions between day and night The effective concentrations of these cytoskeletal drugs were determined through bioluminescence assays Micrographs were captured 4 hours after the photoperiod transitions, with a total of 50 cells analyzed for each treatment Additionally, differential interference contrast microscopy was employed to document cell morphology following Oryzalin treatment.
The study investigated the impact of Latrunculin B on the actin cytoskeleton by fixing cells with a methanol:acetic acid solution (3:1, v:v) at -20 ºC for 15 minutes After fixation, the cells were washed three times with PHEM/DMSO buffer and subsequently labeled with FITC-phalloidin, following the manufacturer's instructions The resulting fluorescence was captured using confocal laser scanning microscopy (LaserSharp 2000, v 5.1).
Circadian distribution and locations of scintillon s and chloroplast s
In Pyrocystis lunula, chloroplasts are evenly distributed in the cytosol during the day and aggregate at the cell center at night under a 12:12 h light:dark photoperiod Bioluminescence is down-regulated within 20 minutes when transitioning from night to day, while full bioluminescence restoration takes 2 hours after the day to night transition During the night, bioluminescence remains stable for 2 hours If light is absent at the start of the day, bioluminescence drops to 50% of the normal night emission and remains constant for 1 hour Conversely, exposure to light at the onset of the day results in a complete down-regulation of bioluminescence within 20 minutes These findings indicate that bioluminescent cells can effectively respond to changes in illumination within 12:12 light:dark cycles.
Table 9.1 Summary of cytoskeletal drug treatment regimes for bioluminescence experiments in Pyrocystis lunula
Colchicine 1 m M 2 h a Cells of P lunula were treated with cytoskeletal drugs for the times indicated prior to photoperiod transition from day to night
Figures 9.1 – 9.4 Diurnal morpholo- gies and bioluminescent profi les of Pyrocystis lunula
Nomarski micrographs illustrate the differences between day and night phase cells, highlighting the distribution of chloroplasts In the day phase cell, chloroplasts are extended throughout the cytosol, while in the night phase cell, they are centrally located Both figures feature a scale bar representing 20 µm.