Cellular and Developmental Biology of Plants

Brief descriptions of some of our current research topics

The targeted migration of the ECM protein pherophorin-S reveals the extensive dynamics of the extracellular matrix in Volvox carteri

Hydroxyproline-rich glycoproteins (HRGPs) constitute a major group of proteins of the extracellular matrix (ECM). The multicellular green alga Volvox carteri is a suitable model organism in which to study the evolutionary transition to multicellularity, including the basic principles and characteristics of an ECM. In Volvox, the ECM is dominated by a single HRGP family: the pherophorins. Our inventory amounts to 117 pherophorin-related genes in V. carteri. We focused on a pherophorin with an unexpected characteristic: pherophorin-S is a soluble, non-cross-linked ECM protein. Using transformants expressing a YFP-tagged pherophorin-S we observed the synthesis and secretion of pherophorin-S by somatic cells in vivo, and we then traced the protein during its conspicuous migration to the ECM around prehatching juveniles and its localized concentration there. Our results provide insights into how an ECM zone surrounding the progeny is remotely affected by distantly located parental somatic cells. In view of the properties and migration of pherophorin-S, we conclude that pherophorin-S is likely to act as an ECM plasticizer to allow for dynamic ECM remodeling.

Reference: von der Heyde, B. & Hallmann, A. (2020) Plant J. 103, 2301-2317.

The basal body protein Babo1 illuminates cell division in Volvox carteri

In photosynthetic organisms many processes are light dependent and sensing of light requires light-sensitive proteins. The supposed eyespot photoreceptor protein Babo1 (formerly Vop1) has previously been classified as an opsin due to the capacity for binding retinal. Here, we analyze Babo1 and provide evidence that it is no opsin. Due to the localization at the basal bodies, the former Vop1 and Cop1/2 proteins were renamed V.c. Babo1 and C.r. Babo1. We reveal a large family of more than 60 Babo1-related proteins from a wide range of species. The detailed subcellular localization of fluorescence-tagged Babo1 shows that it accumulates at the basal apparatus. More precisely, it is located predominantly at the basal bodies and to a lesser extent at the four strands of rootlet microtubules. We trace Babo1 during basal body separation and cell division. Dynamic structural rearrangements of Babo1 particularly occur right before the first cell division. In four-celled embryos Babo1 was exclusively found at the oldest basal bodies of the embryo and on the corresponding d-roots. The unequal distribution of Babo1 in four-celled embryos could be an integral part of a geometrical system in early embryogenesis, which establishes the anterior-posterior polarity and influences the spatial arrangement of all embryonic structures and characteristics. Due to its retinal-binding capacity, Babo1 could also be responsible for the unequal distribution of retinoids, knowing that such concentration gradients of retinoids can be essential for the correct patterning during embryogenesis of more complex organisms. Thus, our findings push the Babo1 research in another direction.

Reference: von der Heyde, E. L. & Hallmann, A. (2020) Plant J. 102, 276-298.

The grand challenges in algae biotechnology

In our book „Grand Challenges in Algae Biotechnology” researchers and practitioners working in the field present the major promises of algae biotechnology and they critically discuss the challenges arising from applications. Based on this assessment, the authors explore the great scientific, industrial and economic potential opened up by algae biotechnology. The first part of the book presents recent developments in key enabling technologies, which are the driving force to unleash the enormous potential of algae biotechnology. The second part of the book focuses on how practical applications of algae biotechnology may provide new solutions to some of the grand challenges of the 21st century.

Algae offer great potential to support the building of a bio-based economy and they can contribute new solutions to some of the grand challenges of the 21st century. Despite significant progress, algae biotechnology is yet far from fulfilling its potential. How to unleash this enormous potential is the challenge that the own field is facing. New cultivation technologies and bioprocess engineering allow for optimization of the operation strategy of state-of the art industrial-scale production systems and they reduce the production costs. Parallel to this, new molecular technologies for genetic and metabolic engineering of (micro)algae develop quickly. The optimization of existing biochemical pathways or the introduction of pathway components makes high-yield production of specific metabolites possible. Novel screening technologies including high-throughput technologies enables testing of extremely large numbers of samples and, thus, allow for large scale modelling of biomolecular processes, which would have not been possible in the past. Moreover, profitable production can demand for integrated biorefining, which combines consecutive processes and various feedstocks to produce both transportation fuel, electric energy and valuable chemicals.

Reference: Hallmann, A. & Rampelotto, P. H. (2020). Grand challenges in algae biotechnology. Springer, Cham, Switzerland.

Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases

BACKGROUND: The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two-the channelrhodopsins-were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics. RESULTS: Here we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called "two-component cyclase opsins" (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery. CONCLUSIONS: Cr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.

Reference: Tian, Y., Gao, S., von der Heyde, E. L., Hallmann, A. & Nagel, G. (2018). BMC Biol. 16, 144..

Whole transcriptome RNA-Seq analysis reveals extensive cell type-specific compartmentalization in Volvox carteri

BACKGROUND: One of evolution's most important achievements is the development and radiation of multicellular organisms with different types of cells. Complex multicellularity has evolved several times in eukaryotes; yet, in most lineages, an investigation of its molecular background is considerably challenging since the transition occurred too far in the past and, in addition, these lineages evolved a large number of cell types. However, for volvocine green algae, such as Volvox carteri, multicellularity is a relatively recent innovation. Furthermore, V. carteri shows a complete division of labor between only two cell types - small, flagellated somatic cells and large, immotile reproductive cells. Thus, V. carteri provides a unique opportunity to study multicellularity and cellular differentiation at the molecular level. RESULTS: This study provides a whole transcriptome RNA-Seq analysis of separated cell types of the multicellular green alga V. carteri f. nagariensis to reveal cell type-specific components and functions. To this end, 246 million quality filtered reads were mapped to the genome and valid expression data were obtained for 93% of the 14,247 gene loci. In the subsequent search for protein domains with assigned molecular function, we identified 9435 previously classified domains in 44% of all gene loci. Furthermore, in 43% of all gene loci we identified 15,254 domains that are involved in biological processes. All identified domains were investigated regarding cell type-specific expression. Moreover, we provide further insight into the expression pattern of previously described gene families (e.g., pherophorin, extracellular matrix metalloprotease, and VARL families). Our results demonstrate an extensive compartmentalization of the transcriptome between cell types: More than half of all genes show a clear difference in expression between somatic and reproductive cells. CONCLUSIONS: This study constitutes the first transcriptome-wide RNA-Seq analysis of separated cell types of V. carteri focusing on gene expression. The high degree of differential expression indicates a strong differentiation of cell types despite the fact that V. carteri diverged relatively recently from its unicellular relatives. Our expression dataset and the bioinformatic analyses provide the opportunity to further investigate and understand the mechanisms of cell type-specific expression and its transcriptional regulation.

Reference: Klein, B., Wibberg, D., Hallmann, A. (2017). BMC Biol. 15, 111.

Algal photobiology: A rich source of unusual light sensitive proteins for synthetic biology and optogenetics

The light absorption system in eukaryotic (micro)algae includes highly sensitive photoreceptors, which change their conformation in response to different light qualities on a subsecond time scale and induce physiological and behavioral responses. Some of the light sensitive modules are already in use to engineer and design photoswitchable tools for control of cellular and physiological activities in living organisms with various degrees of complexity. Thus, identification of new light sensitive modules will not only extend the source material for the generation of optogenetic tools but also foster the development of new light-based strategies in cell signaling research. Apart from searching for new proteins with suitable light-sensitive modules, smaller variants of existing light-sensitive modules would be helpful to simplify the construction of hybrid genes and facilitate the generation of mutated and chimerized modules. Advances in genome and transcriptome sequencing as well as functional analysis of photoreceptors and their interaction partners will help to discover new light sensitive modules.

Reference: Kianianmomeni, A. & Hallmann, A. (2016). Methods Mol Biol. 1408, 37-54.

Distinct shape-shifting regimes of bowl-shaped cell sheets - embryonic inversion in the multicellular green alga Pleodorina

BACKGROUND: The multicellular volvocine alga Pleodorina is intermediate in organismal complexity between its unicellular relative, Chlamydomonas, and its multicellular relative, Volvox, which shows complete division of labor between different cell types. The volvocine green microalgae form a group of genera closely related to the genus Volvox within the order Volvocales (Chlorophyta). Embryos of multicellular volvocine algae consist of a cellular monolayer that, depending on the species, is either bowl-shaped or comprises a sphere. During embryogenesis, multicellular volvocine embryos turn their cellular monolayer right-side out to expose their flagella. This process is called 'inversion' and serves as simple model for epithelial folding in metazoa. While the development of spherical Volvox embryos has been the subject of detailed studies, the inversion process of bowl-shaped embryos is less well understood. Therefore, it has been unclear how the inversion of a sphere might have evolved from less complicated processes. RESULTS: In this study we characterized the inversion of initially bowl-shaped embryos of the 64- to 128-celled volvocine species Pleodorina californica. We focused on the movement patterns of the cell sheet, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. The development of living embryos was recorded using time-lapse light microscopy. Moreover, fixed and sectioned embryos throughout inversion and at successive stages of development were analyzed by light and transmission electron microscopy. We generated three-dimensional models of the identified cell shapes including the localization of CBs. CONCLUSIONS: In contrast to descriptions concerning volvocine embryos with lower cell numbers, the embryonic cells of P. californica undergo non-simultaneous and non-uniform cell shape changes. In P. californica, cell wedging in combination with a relocation of the CBs to the basal cell tips explains the curling of the cell sheet during inversion. In volvocine genera with lower organismal complexity, the cell shape changes and relocation of CBs are less pronounced in comparison to P. californica, while they are more pronounced in all members of the genus Volvox. This finding supports an increasing significance of the temporal and spatial regulation of cell shape changes and CB relocations with both increasing cell number and organismal complexity during evolution of differentiated multicellularity.

Reference: Höhn, S. & Hallmann, A. (2016). BMC Dev. Biol. 16, 35.

Spotlighted brains: Optogenetic activation and silencing of neurons

Optogenetics is revolutionizing cell biology and neuroscience research by allowing precise biochemical control of neuronal activity through light-activated channels. Light-induced ion transporters have been used extensively for cellular activation, and now light-gated inhibitory channels have been discovered. These represent a key new tool to elucidate the molecular mechanisms underlying neurological and neuropsychiatric disorders.

Reference: Kianianmomeni, A. & Hallmann, A. (2015). Trends Biochem. Sci. 40, 624-627.

Algal photoreceptors: in vivo functions and potential applications

Many algae, particularly microalgae, possess a sophisticated light-sensing system including photoreceptors and light-modulated signaling pathways to sense environmental information and secure the survival in a rapidly changing environment. Over the last couple of years, the multifaceted world of algal photobiology has enriched our understanding of the light absorption mechanisms and in vivo function of photoreceptors. Moreover, specific light-sensitive modules have already paved the way for the development of optogenetic tools to generate light switches for precise and spatial control of signaling pathways in individual cells and even in complex biological systems.

Reference: Kianianmomeni, A. & Hallmann, A. (2014). Planta 239, 1-26.

The genome of Volvox, the “fierce roller”: Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri

The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138-megabase genome of V. carteri and compared its ~14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal–specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.

Reference: Prochnik, S. E., Umen, J., Nedelcu, A., Hallmann, A. et al. (2010). Science 329, 223-226.

There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox globator

Epithelial folding is a common morphogenetic process during the development of multicellular organisms. In metazoans, the biological and biomechanical processes that underlie such three-dimensional (3D) developmental events are usually complex and difficult to investigate. Spheroidal green algae of the genus Volvox are uniquely suited as model systems for studying the basic principles of epithelial folding. Volvox embryos begin life inside out and then must turn their spherical cell monolayer outside in to achieve their adult conguration; this process is called "inversion." There are two fundamentally different sequences of inversion processes in Volvocaceae: type A and type B. Type A inversion is well studied, but not much is known about type B inversion. How does the embryo of a typical type B inverter, V. globator, turn itself inside out? We investigated the type B inversion of V. globator embryos and focused on the major movement patterns of the cellular monolayer, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. Isolated intact, sectioned and fragmented embryos were analyzed throughout the inversion process using light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques. We generated 3D models of the identified cell shapes, including the localizations of CBs. We show how concerted cell-shape changes and concerted changes in the position of cells relative to the CB system cause cell layer movements and turn the spherical cell monolayer inside out. The type B inversion of V. globator is compared to the type A inversion in V. carteri. Concerted, spatially and temporally coordinated changes in cellular shapes in conjunction with concerted migration of cells relative to the CB system are the causes of type B inversion in V. globator. Despite significant similarities between type A and type B inverters, differences exist in almost all details of the inversion process, suggesting analogous inversion processes that arose through parallel evolution. Based on our results and due to the cellular biomechanical implications of the involved tensile and compressive forces, we developed a global mechanistic scenario that predicts epithelial folding during embryonic inversion in V. globator.

Reference: Höhn, S. & Hallmann, A. (2011). BMC Biol 9, 89.

A gender-specific retinoblastoma-related protein implies a role for the retinoblastoma protein family in sexual development

The green alga Volvox carteri is one of the simplest multicellular organisms with only two cell types, somatic and reproductive, making it suitable as a model for studying cell division, multicellularity, and cellular differentiation. We cloned and characterized the RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) from the green alga Volvox carteri. Likewise, other key elements of the retinoblastoma tumor suppressor pathway like E2F1 and DP1 have been identified in Volvox carteri. RBR1 expression increases substantially during embryogenesis and in response to the sex-inducer glycoprotein, but it decreases significantly under heat stress. While RBR1 is expressed in gonidia (asexual reproductive cells) and embryos, the largest proportion of RBR1 mRNA is found in parental somatic cells. The presence of 4 splice variants and 15 potential cyclin-dependent kinase phosphorylation sites suggests that RBR1 is subject to control at the posttranscriptional and posttranslational levels. Surprisingly, RBR1 is a gender-specific gene, mapping exclusively to the female mating-type locus. A procedure for stable nuclear transformation of males was established to generate RBR1-expressing males. These transformants exhibit enlarged reproductive cells, altered growth characteristics, and a prolonged embryogenesis. The results suggest that a functionally related analog of RBR1 exists in males. The reason for the divergent evolution of RBRs in females and males appears to be based on sexual development: males and females respond to the same sex-inducer with different cleavage programs and substantial differences in cellular differentiation. Thus, the gender-specific presence of RBR1 provides evidence for an additional, novel role for retinoblastoma family proteins in sexual development.

References: Kianianmomeni, A., Nematollahi, G. & Hallmann, A. (2008). Plant Cell 20, 2399-2419. __Hallmann, A. (2009). Commun. Integr. Biol. 2, 396-399. __Hallmann, A. (2009). Commun. Integr. Biol. 2, 538-544.

How 5000 independent rowers coordinate their strokes in order to row into the sunlight: Phototaxis in the multicellular green alga Volvox

The evolution of multicellular motile organisms from unicellular ancestors required the utilization of previously evolved tactic behavior in a multicellular context. Volvocine green algae are uniquely suited for studying tactic responses during the transition to multicellularity because they range in complexity from unicellular to multicellular genera. Phototactic responses are essential for these flagellates because they need to orientate themselves to receive sufficient light for photosynthesis, but how does a multicellular organism accomplish phototaxis without any known direct communication among cells? Several aspects of the photoresponse have previously been analyzed in volvocine algae, particularly in the unicellular alga Chlamydomonas. Recently, we analyzed the phototactic behavior in the spheroidal, multicellular volvocine green alga Volvox rousseletii (Volvocales, Chlorophyta). In response to light stimuli, not only did the flagella waveform and beat frequency change, but the effective stroke was reversed. Moreover, there was a photoresponse gradient from the anterior to the posterior pole of the spheroid, and only cells of the anterior hemisphere showed an effective response. The latter caused a reverse of the fluid flow that was confined to the anterior hemisphere. The responsiveness to light is consistent with an anterior-to-posterior size gradient of eyespots. At the posterior pole, the eyespots are tiny or absent, making the corresponding cells appear to be blind. Pulsed light stimulation of an immobilized spheroid was used to simulate the light fluctuation experienced by a rotating spheroid during phototaxis. The results demonstrated that in free-swimming spheroids, only those cells of the anterior hemisphere that face toward the light source reverse the beating direction in the presence of illumination; this behavior results in phototactic turning. Moreover, positive phototaxis is facilitated by gravitational forces. Under our conditions, V. rousseletii spheroids showed no negative phototaxis. On the basis of our results, we developed a mechanistic model that predicts the phototactic behavior in V. rousseletii. The model involves photoresponses, periodically changing light conditions, morphological polarity, rotation of the spheroid, two modes of flagellar beating, and the impact of gravity. Our results also indicate how recently evolved multicellular organisms adapted the phototactic capabilities of their unicellular ancestors to multicellular life.

Reference: Ueki, N., Matsunaga, S., Inouye, I. & Hallmann, A. (2010). BMC Biol. 8, 103.

Light-gated ion channels that control photomovement responses: The Channelrhodopsins

Channelrhodopsins are light-gated ion channels involved in the photoresponses of microalgae. Here we describe the characterization of two channelrhodopsins, VChR1 and VChR2, from the multicellular green alga Volvox carteri. Both are encoded by nuclear single copy genes and are highly expressed in the small biflagellated somatic cells but not in the asexual reproductive cells (gonidia). Expression of both VChRs increases after cell cleavage and peaks after completion of embryogenesis when the biosynthesis of the extracellular matrix begins. Likewise, expression of both transcripts increases after addition of the sex-inducer protein, but VChR2 is induced much more than VChR1. The expression of VChR1 is specifically promoted by extended dark periods, and heat stress reduces predominantly VChR1 expression. Expression of both VChRs increased under low light conditions, whereas cold stress and wounding reduced expression. Both VChRs were spectroscopically studied in their purified recombinant forms. VChR2 is similar to the ChR2 counterpart from Chlamydomonas reinhardtii with respect to its absorption maximum (460 nm) and photocycle dynamics. In contrast, VChR1 absorbs maximally at 540 nm at low pH (D540), shifting to 500 nm at high pH (D500). Flash photolysis experiments showed that after light excitation, the D540 dark state bleaches and at least two photoproducts, P600 and P500, are sequentially populated during the photocycle. We hypothesize that VChR2 is a general photoreceptor, which is responsible for the avoidance of blue light and might play a key role in sexual development, whereas VChR1 is the main phototaxis photoreceptor under vegetative conditions, as it is more specifically adapted to environmental conditions and the developmental stages of Volvox.

Reference: Kianianmomeni, A., Stehfest, K., Nematollahi, G., Hegemann, P. & Hallmann, A. (2009). Plant Physiol. 151, 347-366.

VCRPs, small cysteine-rich proteins that might be involved in extracellular signaling

The sex-inducer of the spherical green alga Volvox carteri is one of the most potent biological effector molecules known: it is released into the medium by sexual males and triggers the switch to the sexual cleavage program in the reproductive cells of vegetatively grown males and females even at concentrations as low as 10 (-16) M. In an adult Volvox alga, all cells are embedded in an extensive extracellular matrix (ECM), which constitutes >99% of the volume of the spheroid. There exist no cytoplasmic connections between the cells in an adult alga, so any signal transduction between different cells or from the organism’s environment to a reproductive cell must involve the ECM. A small cysteine-rich extracellular protein, VCRP, was identified in Volvox and shown to be quickly synthesized by somatic cells in response to the sex-inducer. Due to its characteristics, VCRP was speculatedto be an extracellular second messenger from somatic cells to reproductive cells. There is also a related protein, VCRP2, which exhibits 56% amino acid sequence identity with VCRP. Two possible scenarios for signal transduction from the sex-inducer to the reproductive cell are discussed.
In the future, we want to investigated this signal transduction in more detail to learn more about the corresponding molecular mechanisms.

References: Hallmann, A. (2008). Plant Signal. Behav. 3, 124-127. __Hallmann, A. (2007). Planta 226, 719-727.

The Chlamydomonas genome reveals the evolution of key animal and plant functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella.
Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Reference: Merchant, S. S. et al. (2007). Science 318, 245-250.

Stable nuclear transformation of volvocine algae

Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. For a detailed analysis of  this molecular evolution, transformation techniques are required, which allow for genetic manipulation of these algae. Until recently, transformation procedures have only been established for the volvocine algae Chlamydomonas reinhardtii and Volvox carteri. Now we achieved the stable nuclear transformation of Gonium pectorale. Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. Stable nuclear transformation of G. pectorale was achieved using a heterologous dominant antibiotic resistance gene, the aminoglycoside 3'-phosphotransferase VIII gene (aphVIII) of Streptomyces rimosus, as a selectable marker. Heterologous 3'- and 5'-untranslated flanking sequences, including promoters, were from Chlamydomonas reinhardtii or from Volvox carteri. After particle gun bombardment of wild type Gonium cells with plasmid-coated gold particles, transformants were recovered. The transformants were able to grow in the presence of the antibiotic paromomycin and produced a detectable level of the AphVIII protein. The plasmids integrated into the genome, and stable integration was verified after propagation for over 1400 colony generations. Co-transformants were recovered with a frequency of ~30-50% when cells were co-bombarded with aphVIII-based selectable marker plasmids along with unselectable plasmids containing heterologous genes. The transcription of the co-transformed, unselectable genes was confirmed. After heterologous expression of the luciferase gene from the marine copepod Gaussia princeps, which was previously engineered to match the codon usage in C. reinhardtii, Gonium transformants show luciferase activity through light emission in bioluminescence assays. Conclusions: Flanking sequences that include promoters from C. reinhardtii and from V. carteri work in G. pectorale and allow the functional expression of heterologous genes, such as the selectable marker gene aphVIII of S. rimosus or the co-transformed, codon-optimized G. princeps luciferase gene, which turned out to be a suitable reporter gene in Gonium. The availability of a method for transformation of Gonium makes genetic engineering of this species possible and allows for detailed studies in molecular evolution using the unicellular Chlamydomonas, the 16-celled Gonium, and the multicellular Volvox.

Reference: Lerche, K. & Hallmann, A. (2009). BMC Biotechnol. 9, 64.

Algal transgenics and biotechnology

Transgenesis in algae is a complex and fast-growing technology. Selectable marker genes, promoters, reporter genes, transformation techniques, and other genetic tools and methods are already available for various species and currently ~25 species are accessible to genetic transformation. Fortunately, large-scale sequencing projects are also planned, in progress, or completed for several of these species; the most advanced genome projects are those for the red alga Cyanidioschyzon merolae, the diatom Thalassiosira pseudonana, and the three green algae Chlamydomonas reinhardtii, Volvox carteri and Ostreococcus tauri. The vast amount of genomic and EST data coming from these and a number of other algae has the potential to dramatically enlarge not only the algae’s molecular toolbox. A powerful driving force in algal transgenics is the prospect of using genetically modified algae as bioreactors. In general, today’s non-transgenic, commercial algal biotechnology produces food additives, cosmetics, animal feed additives, pigments, polysaccharides, fatty acids, and biomass. But recent progress in algal transgenics promises a much broader field of application: molecular farming, the production of proteins or metabolites that are valuable to medicine or industry, seems to be feasible with transgenic algal systems. Indeed, the ability of transgenic algae to produce recombinant antibodies, vaccines, insecticidal proteins, or bio-hydrogen has already been demonstrated.
Genetic modifications that enhance physiological properties of algal strains and optimization of algal production systems should further improve the potential of this auspicious technology in the future.

Reference: Hallmann, A. (2007). Transgenic Plant J. 1, 81-98.

Quantitative analysis of cell-type specific gene expression

The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there is an ongoing genome project in Volvox carteri and a completed genome project in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluated the potential of real-time RT-PCR for quantifying gene transcript levels. We analyzed a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contained previously known genes with unknown localization of cellular expression, 28 novel genes which were described for the first time, and a few known, cell-type specific genes as a control. The respective gene products were, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provided expression data for all these genes. The results showed that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox. Our expression data also provided a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes.
In the future, this approach will be extended to a much larger number of genes and to developmental or metabolic mutants.

Reference: Nematollahi, G., Kianianmomeni, A. & Hallmann, A. (2006). BMC Genomics 7, 321.

Translational control of a key gene, regA, controlling cell differentiation

The complete division of labour between the reproductive and somatic cells of the green alga Volvox carteri is controlled by three types of genes. One of these is the regA gene, which controls terminal differentiation of the somatic cells. We examined translational control elements located in the 5' UTR of regA, particularly the eight upstream start codons (AUGs) that have to be bypassed by the translation machinery before regA can be translated. The results of our systematic mutational, structural and functional analysis of the 5' UTR led us to conclude that a ribosome-shunting mechanism - rather than leaky scanning, ribosomal reinitiation, or internal ribosome entry site (IRES)-mediated initiation - controls the translation of regA mRNA. This mechanism, which involves dissociation of the 40S initiation complex from the message, followed by reattachment downstream, in order to bypass a secondary structure block in the mRNA, was validated by deleting the predicted 'landing site' (which prevented regA expression) and inserting a stable 64 nucleotide hairpin just upstream of this site (which did not prevent regA expression). This is the first report suggesting that translation of an mRNA in a green eukaryote is controlled by ribosome shunting.
In the future, we want to investigate whether other key genes in development are also controlled by ribosome shunting.

Reference: Babinger, K., Hallmann, A. & Schmitt, R. (2006). Development 133, 4045-4051.