The Chemistry Lying Deep Within a Volvox Colony

Robinson Libman, Meryem Louni, Ryan McGibbon, Ali Najjar

Abstract

Despite their apparent simplicity as microorganisms, Volvox are extremely complex living systems whose secrets have yet to be fully uncovered. This paper dives deep within Volvox to tackle the key chemical phenomena responsible for the basic functioning of the colony. The chemical processes involved in Volvox reproduction are based on protein-protein interactions and a sex inducer glycoprotein, enabling them to switch between asexual or sexual reproduction depending on their environment. The colony also optimizes its energy storage regardless of the level of oxygen in its surroundings via its hydrogen-based metabolism. Moreover, the extracellular matrix guarantees protection and flexibility of the colony thanks to its hydroxyproline-rich glycoproteins and their entanglement. And of course, like many other , Volvox generates energy through . This paper's study of the ways Volvox employs to define its structure and satisfy its needs once again demonstrates its uniqueness in the natural world and has revealed further design solutions to the challenges that arise within its aqueous environment.

Introduction

The green algae species in the Volvox genus are among the simplest multicellular organisms out there. With their very simple cell differentiation, consisting of only two types of cells, somatic and reproductive (gonidia), as well as the fact that they have relatively recently diverged from their single-celled counterpart, Chlamydomonas reinhardtii, 200 million years ago (Umen & Herron, 2021), they have become an ideal organism for studying various functions of eukaryotic cells, as well as the transition from unicellular to multicellular organisms.

Volvox, along with all other living organisms, are fascinating topics of study. The structure and design of these organisms is more intelligent than often assumed, and their application of scientific properties beautiful to observe. This essay will focus on Volvox‘s interaction with the chemical world and its ability to make use of chemical compounds to achieve its goals. Volvox must carefully regulate its reproductive cycle and how it does so will be covered in the first section of this paper. Following this, its metabolic functions will be discussed. Then, the interesting chemical properties and structure of the subzones of its extracellular matrix will be highlighted. Finally, Volvox's method for producing energy through photosynthesis will be explained. Studying the chemical structure of Volvox's extracellular matrix, and the chemistry behind their reproductive, photosynthetic, or metabolic systems can help researchers gain a deeper understanding of how these functions work in higher organisms without having to worry about more complex mechanisms and compounds that would otherwise interfere with their studies. This is not to say that Volvox are not complex, as they exhibit very intricate and functions, which shall be addressed in this paper.

How to be resilient and unique: the chemistry of Volvox's reproductive cycles

: Volvox's primary reproductive cycle

Asexual reproduction is the primary manner used by Volvox to reproduce. It occurs under favorable conditions, as a combination of several steps, each of them requiring a certain amount of incoming light. Firstly, a few cells on the posterior side of the colony become reproductive, meaning that they lose their flagella, and that their size increases up to ten times. These cells, now called gonidia, are pushed towards the Volvox's interior. Next, they undergo embryogenesis and inversion. Each embryo then differentiates into a new miniature juvenile spheroid. The final step of asexual reproduction consists of the daughter cells liberation through deterioration of the somatic cells that constitute the parent colony. This is due to the fact that somatic cells experience a substantial decrease in viability approximately 100 hours after embryogenesis, and die around 192 hours after this event (Matt & Umen, 2016). 

How can somatic cells “know” when to die?

As mentioned previously, it is crucial for the germ cells that the somatic cells which encapsulate them deteriorate. Otherwise, they could not be released into their environment, and the species' reproduction would be prevented. This process is called somatic cell senescence and could be caused by either environmental or intrinsic factors. In 1982, Pommerville and Kochert tested this on Volvox by altering several external factors such as temperature, light period, or culture medium. However, in these cases, survivorship curves all showed rapid declines in somatic cell viability, but at different times. This is inconsistent with the cell cycle inherent to Volvox, where each phase follows precise timing. As a result, one can infer that programmed senescence is not regulated by environmental factors in Volvox. Next, the researchers measured some intrinsic parameters such as the amount of total soluble cell , as Fig. 1 demonstrates. The latter showed a sharp decline 120 hours after embryogenesis. This result indicates that a protein synthesis inhibitor must be produced within the colony. Therefore, researchers incubated Volvox somatic cells with cycloheximide, a protein synthesis inhibitor, and realized these cells consistently showed a decline of viability sooner than those without the drug. It means that cycloheximide acted as a proteolytic enzyme, which either directly blocked protein synthesis or indirectly activated a protease, an enzyme which catalyzes proteolysis (destruction of a protein) (Pommerville & Kochert, 1982). Although the identity of the protein synthesis inhibitor has not yet been determined, it has been evidenced that this chemical intrinsic factor is the unique parameter that explains the programmed senescence and of somatic cells in Volvox.


Fig. 1 Evolution of the amount of soluble proteins in Volvox carteri‘s cells. This quantity increases rapidly during the progeny's development that lasts until 120 hours after embryogenesis. Then, it steeply decreases during the senescence of somatic cells, which leads to the release of daughter cells in the environment (Pommerville & Kochert, 1982).

To conclude, asexual reproduction in Volvox relies on three key processes: embryonic cleavage, morphogenesis, and cellular differentiation. While physical theories helped explain the underlying mechanism behind some of them in the previous paper, they also left several questions unanswered. Therefore, the aim of this part is to complete the started using an approach centered on chemistry.

An intriguing embryonic cleavage

Embryogenesis is characterized by the rapid and synchronous division of each gonidium. The important number of divisions led to calling this type of cell cycle multiple fission, also known as palintomy. One interesting property of multiple fission is its prolonged G1 phase, which allows cells to enlarge in size. This feature is of utter importance for cellular differentiation and will be explained later in this section. The G1 phase allows cells to prepare to replicate their DNA in the S phase, and since this phenomenon is systematic, one can infer that it is not due to environmental stress or DNA damage, some common reasons for G1 phase prolongation. Moreover, it has been shown that proteins called cyclin-dependent kinases extend the growth period prior to replication in Chlamydomonas, which share the same cell cycle as Volvox. More specifically, cyclin A – CDKA and cyclin B – CDKB are involved in a negative loop feedback that regulates the time gonidia spend in the G1 phase (Atkins & Cross, 2018). Concretely, CDK's become active once they bind to cyclins to form CDK-Cyclin complexes. Then, the latter complex phosphorylates specific proteins involved in cell cycle progression, which affects the conditions required for the G1 to S phase checkpoint to open (Peeper et al., 1993).

Proteins as initial motors for the colony's inversion

 Once embryos are formed, the vegetative (asexual) reproductive cycle of Volvox is marked by morphogenesis. It is characterized by the inversion of spherical embryos that turn themselves inside out, to achieve their adult configuration. Both changes in cell shape and movement of intercellular bridges are necessary to explain Volvox's inversion. Previously, the analysis focused on the mechanical forces that led to an “elastic snap through” which enabled the sphere to invert. Nevertheless, while it provides a solid base to understand the mechanism, a chemical approach is required to complete the gaps. For instance, it has been observed that cytoplasmic bridges are localized at the midpoints of cells that have not yet passed through the region of maximum curvature, whereas they are found at the chloroplast ends of the cells once this point has been reached (Viamontes et al., 1979). Therefore, bridge relocation leads to bringing the cells into contact at their narrowest points as described in Fig. 2, but there is still mystery concerning the initial source of energy, the motor that drives the whole process. Thankfully, studies have identified an inversion-specific gene called invA present in Volvox by tagging it with a transposon named Jordan, named after its jumping abilities. Also, mutants in which invA has been inactivated by a Jordan insertion could not undergo relocation of their cytoplasmic bridges, which prevented inversion. More specifically, invA is a type of kinesin, a well-known motor protein, and is located in cytoplasmic bridges (Kirk & Nishii, 2001). Therefore, it is activated during inversion, and attempts to walk along the microtubule adjacent to a given bridge. However, the protein is anchored in the bridge and the force it generates to walk is converted into a bending moment that induces the curvature required to invert the cell sheet (Baluska et al., 2006). As a result of this change in cell shape, intercellular bridges occupy a different position.

Fig. 2 Representation of the cellular mechanism of inversion in Volvox cells. On the left, the colony has not inverted yet and the cytoplasmic system (red line) links the cells (green outlines) at their widest point, right below the nucleus. Once morphogenesis starts as depicted on the right, cells move inward past the cytoplasmic bridge system, which makes them link one to another at their narrowest point. This bending force then spreads to the rest of the colony, allowing it to turn inside out completely (Baluska et al., 2006).

To conclude, the presence of a type of kinesin is required for morphogenesis to start. However, the process is divided into two halves: bending of the cell sheet and formation of a cross-shaped opening (phialopore), followed by the inversion of the posterior hemisphere. Hence, some uncertainty still accompanies this second step. Nishii and Ogihara evidenced in 1999 that inhibiting actomyosin only stopped inversion towards its end. Moreover, they discovered that the consequence of it was that the posterior hemisphere was too large and unable to fit through the phialopore. From here, one can deduce that actomyosin activity allows for the contraction of this hemisphere of the colony. Hence, its width is temporarily reduced by chemically induced contraction to ensure its passage through the phialopore.

The size of a cell influences its fate: the chemical mechanism behind cellular differentiation

Once embryogenesis is completed, a wild-type juvenile spheroid is composed of approximately 16 large cells and 2000 small cells. Over time, large cells differentiate in gonidia and take on reproductive functions, while small cells specialize into somatic cells. While this statement is observed to be true, it is interesting to examine why the size of a cell influences its fate, and whether this is the only determining factor. In fact, not all species of Volvox use the same mechanisms for germ-soma specialization. For instance, while only cell size influences Volvox carteri cell specialization, both size and position within the embryo influence the fate of a juvenile spheroid for Volvox obversus (Ransick, 1991). As far as size is concerned, the scientific community still acknowledges that the relationship between this property and its use by the colony during cellular differentiation remains unclear (Matt & Umen, 2016). Nevertheless, an interesting assumption deserves to be mentioned, as it may be confirmed in the coming years. First, large cells are characterized by very different surface-to-volume ratios than those of small cells. In other words, while their genetic information is similar, their respective amounts of cellular material such as chloroplasts and mitochondria are likely to vary substantially. Consequently, their compositions in terms of metabolic by-products may differ, which has been proven to affect gene expression (Goodenough et al., 2014). This theory is supported by the fact that gonidia and somatic cells synthesize different proteins (Kirk & Kirk, 1983), meaning that they do not express the same genes. As expected, in 2001, Kirk showed rigorously that germ-soma differentiation is controlled by genetic factors. For instance, the mutant regA “forces” cells to become gonidia, even though they have already specialized as somatic cells, as seen in Fig. 3.

To sum up, one promising hypothesis to explain the mechanism by which Volvox cells differentiate after embryogenesis is the following: size influences chemical composition of a cell, which has an impact on its inherent genetic expression. Nowadays, the last part of the theory is certain, as researchers have found evidence that some genes were responsible for assigning roles to cells in the colony. Nevertheless, one question remains unanswered: How do small and large cells express different genes?

Fig. 3 Micrographs of a wild-type (WT) Volvox adult colony (left), versus a regAadult mutant colony (right). The wild-type is mainly composed of numerous small somatic cells, which encapsulate a couple of larger germ cells. However, the expression of the regAgene in the mutant forces all somatic cells to re-differentiate as larger gonidia. This proves that cellular differentiation is eventually controlled by genetic expression (Matt & Umen, 2016).

Volvox's ability to switch from asexual to sexual reproduction

While the vegetative cycle is the primary manner used by Volvox to reproduce, it is not well suited to extreme conditions, such as droughts, heat shocks, or nutrient deficiencies (Umen & Herron, 2021). This type of reproduction is characterized by the addition of a sex inducer before embryogenesis, which generates two types of Volvox cells; some of them are females and carry eggs and others are males and carry sperm-packets. Then, sperm packets are released and enter female cells to fertilize their eggs, which generates diploid zygospores. The major advantage of sexual reproduction is that these diploid zygotes have a thick cell wall and remain viable for several years in a dormant state. This design solution developed by Volvox allows the species to overcome harsh conditions and start reproducing again when the situation is more favorable. Once their environment has improved, and that light as well as nutrients are abundant, the zygospores undergo germination and meiosis to produce a haploid progeny, which then enters the vegetative cycle again by default (Umen, 2020).

While Volvox may be able to switch from one reproductive cycle to another in the blink of an eye, this mechanism is in fact highly complex and requires a biochemical approach to understand the role of the sex-inducing molecule. Even though Volvox are descendants of Chlamydomonas, their sex-inducing mechanism is completely different. Sexual reproduction is solely triggered by nitrogen deficiency in Chlamydomonas, but it occurs as a result of the of a glycoprotein in Volvox (Umen, 2011). Vegetative gonidia are either male or female, even though this property has no impact during the asexual cycle. However, upon exposure to this molecule, they show different cleavage patterns which lead to modified embryos, as Fig. 4 shows. After maturation, both types contain opposed sexual germ cells (Hallmann et al., 1998).

Fig. 4 Comparison of the effect of the sex-inducing molecule on both male and female cells in Volvox. Its first impact is that both types of cells undergo different embryonic cleavages. Once all steps of sexual development have been completed, males (b) are composed of sperm packets and somatic cells in similar amounts, while females (a) contain much more somatic cells than eggs. (Hallmann et al., 1998).

The glycoprotein results from the expression of a specific gene in Volvox cells. This gene is silent for all kinds of asexual cells but is activated by either drastic environmental changes, or the presence of the gene's product itself. The second possibility allows for the rapid switch of all cells to the new reproductive cycle, which is confirmed by the fact that a concentration of 10-17 mol/L is sufficient to guarantee an effective transition from an asexual to sexual cycle across the whole colony (Tschochner et al., 1987). Surprisingly, the gene is only activated in male gonidia, or by somatic cells in very specific conditions due to a heat shock. In terms of characterization, glycoproteins are biomolecules made of a protein attached to several carbohydrate chains. They are made of various amino acids assembled in a polypeptide chain, with five different types of carbohydrates binding to them, as detailed in Fig. 5. The primary specificity of these molecules are their unusual chemical stability, which is explained by their high degree of glycosylation. This notion refers to the amount of attached carbohydrates compared to the original protein size, and is evaluated at 40% in this case (Gilles et al., 1981).

Fig. 5 List of the carbohydrates attached to the polypetide chain of the sex-inducing glycoprotein in Volvox, and their relative abundance (Tschochner et al., 1987).

Furthermore, the effect of this sex inducer highlights the essential role of the in Volvox's transition to sexual reproduction. The ECM is considered as a dynamic structure that constantly adapts to its surroundings. Indeed, exposure to this inducer triggers the synthesis of a tyrosine sulfate glycoprotein (SG70) by the ECM. It acts as a carrier since it collects the positively charged sex-inducing pheromones, and transports them through the negatively charged ECM of the gonidia cell's membrane, to activate the gene responsible for switching reproductive cycles (Hallmann, 2003).

To conclude, asexual reproduction prevails in Volvox under favorable conditions. It involves the formation of daughter colonies originating from one parent to obtain clones. This mechanism can be completed in a couple of days without requiring high inputs of energy and allows Volvox to form numerous daughter cells rapidly. Nevertheless, asexual reproduction does not permit genetic mixing, which is crucial for the species to evolve and remain adapted to its changing environment. Therefore, Volvox reproduces sexually at least once a year, even though it implies higher energy expenses. While the reproductive rate is reduced due to the time and energy it takes to differentiate female and male cells, fertilize eggs, and develop the zygote, sexual reproduction has the tremendous advantage of increasing the adaptability of the species through mutations. Finally, zygotes are more resilient than all cells present in the asexual cycle and can remain dormant until conditions are more clement. In other words, in Volvox, sexual reproduction guarantees survival, whereas the asexual cycle allows for efficiency in the offspring's formation.

Metabolic functions of Volvox

Metabolic functions include all chemical reactions that occur throughout an organism, within each cell, that provide it with energy (Sánchez & Raja, 2022). In order to survive, all living organisms make use of their environment by taking the necessary nutrients and substances that will help them with movement, growth, development, and reproduction. These processes are catalyzed by enzymes, which are proteins that have specialized functions in anabolism (building up molecules for energy storage) and catabolism (breaking down molecules to obtain energy) (Sánchez & Raja, 2022). As a green alga, Volvox is a photoautotroph, meaning that it uses energy in the form of light to synthesize its organic molecules. This also means that the growth of cells is photosynthesis limited (Kirk, 2003). The specifics of photosynthesis in Volvox will be discussed in a following section. This section will focus on the hydrogen metabolism of Volvox, as well as lipid composition and metabolism.

Hydrogen metabolism in Volvox carteri

Hydrogen gas is critical component in the metabolism of a wide variety of microorganisms (Cornish et al., 2015). In a study by Cornish et al., it was found that Volvox carteri evolves H (a chemical reaction that yields H2) both when supplied with an abiotic (non-living) electron donor and under physiological conditions. The study analyzes the genome of Volvox and highlights the two genes that encode [FeFe]-hydrogenases (HYDA1 and HYDA2) (Cornish et al., 2015). Before presenting the results, some background information is needed.

The production of H2 in biological organisms is primarily catalyzed by two classes of enzymes: hydrogenases and nitrogenases. Hydrogenases also contribute to hydrogen uptake. In autotrophic microorganisms, such as green algae like Volvox, hydrogen plays a complex role in their life cycle, as it is linked to both photosynthetic and fermentative processes (Cornish et al., 2015). Green algae both evolve (produce) and consume hydrogen gas using [FeFe]-hydrogenases, which are metalloproteins that can catalyze proton reduction and H2 oxidation (Cornish et al., 2015). The electrons needed for the production of hydrogen gas can either be obtained from photosynthetic water-splitting or by the fermentation of carbon sources.

In many green algae, hydrogenases are encoded by two genes: HYDA1 and HYDA2. These genes are paralogs that have very similar sequences. However, evidence suggests that HYDA1 might contribute more to H2 production in the light (Cornish et al., 2015). In order to assemble the catalytic site, maturation proteins are necessary, and the corresponding genes (HYDEF and HYDG in green algae) are very present in the genome of Volvox. Moreover, [FeFe]-hydrogenases are irreversibly inactivated when oxygen gas is present, and expression of the HYD genes responsible for maturation proteins is induced under anaerobiosis (life in the absence of free oxygen). This oxygen gas sensitivity limits production of H2 during oxygenic photosynthesis. In Chlamydomonas reinhardtii, the unicellular ancestor of Volvox carteri, this limitation is overcome by channeling electrons to synthesize H2. One method is sulfur deprivation, limiting O2 production by photosystem II while letting electrons obtained from photosystem I to be used to produce H2. Another method is to allow carbon stores generated by photosynthesis to be used to produce H2. As the two related volvocine algae have very similar genomes, Volvox carteri shares many metabolic processes with C. reinhardtii (Cornish et al., 2015).

In order to assess hydrogen gas production in V. carteri both in vivo and in vitro, a culture of cells was acclimated to anaerobiosis for 4 hours in degassed media. Then, these cells were transferred to anaerobic assay vials either containing only anaerobic media or supplemented with an abiotic electron donor (Na2S2O4) and an electron mediator, methyl viologen dichloride (MV), which increases the rate of electron transfer (Cornish et al., 2015). The same process was done for aerobic cells (oxygen-exposed) under similar conditions. The accumulation of hydrogen gas in the headspace (volume above each vial) was measured as shown in Fig. 6 (Cornish et al., 2015). 

Fig. 6 Hydrogen gas evolution in V. carteri cells per mg of chlorophyll per second. Whole cell cultures were acclimated to aerobiosis or anaerobiosis and then either supplied with an abiotic electron donor system (supplemented) or not (unsupplemented) during H2 measurement (Cornish et al., 2015).

The results showed that for anaerobic cells that were supplemented with an abiotic electron donor system, H2 production could be measured within 45 minutes and continued at a fast rate. As for unsupplemented cells, they did not accumulate a significant amount of H2 even after 48 hours. For aerobic cells, H2 production was not appreciable even when supplemented with an abiotic electron donor system. Therefore, the data indicates that under these conditions, Volvox needs both anaerobic acclimation and enough reducing equivalents to produce hydrogen gas (Cornish et al., 2015).

In nature, green algae can ferment carbon stores to drive H2 production, but initially no appreciable hydrogen gas accumulation was observed in anaerobically acclimated V. carteri cells in the absence of an abiotic electron donor system. This was most likely due to insufficient fixed carbon levels in the original growth conditions (Cornish et al., 2015). To remediate this issue, cell cultures of V. carteri were supplied with sodium carbonate and incubated in the light for 72 hours. They were then acclimated to anaerobiosis for 4 hours and tested for H2 production under dark anaerobic conditions. The cells produced significant amounts of hydrogen gas under anaerobic, dark conditions, as demonstrated in Fig. 7. Therefore, the data shows that Volvox can also producehydrogen gas under physiological conditions (Cornish et al., 2015).

Fig. 7 H2 production of V. carteri cells with sodium bicarbonate per mg of chlorophyll. Both anaerobic cells and negative control H production are shown. The negative control represents unsupplemented cells (without an electron donor system) incubated in anaerobic media (Cornish et al., 2015).

Lipid composition and metabolism in Volvox carteri

In a 1980 research paper, Mosely & Thompson Jr. characterized the membrane structural lipids of somatic cells and gonidia isolated from Volvox carteri spheroids. The main polar lipid components of these two cell types include sulfoquinovosyl diglyceride, mono- and digalactosyl diglyceride, phosphatidylglycerol, phosphatidylethanolamine, and 1(3),2-diacylglyceryl-(3)-O-4'-(N,N,N,-trimethyl)homoserine (DGTH) (Moseley & Thompson Jr., 1980).

The lipid content was analyzed for Volvox spheroids in stage I, which was defined as the period of development starting with the release of daughter spheroids and ending with the first division of the gonidia in the daughter spheroid (Moseley & Thompson Jr., 1980). The obtained value was dependent upon gonidia size, as they were rapidly growing. Fig. 8 below compiles the molar ratios of the major lipid classes relative to phospholipids (Moseley & Thompson Jr., 1980).


Fig. 8 Molar ratios of some principal Volvox lipids in spheroid colonies, gonidia, and somatic cells relative to PL. Abbreviations: phospholipid (PL), chlorophyll (Chl) lipid galactose (GL), sulfolipid (SL), chlorophyll a (Chl a), chlorophyll b (Chl b), monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) (Moseley & Thompson Jr., 1980).

Then, portions of the three lipid fractions from gonidia and somatic cells were converted to fatty acid methyl esters and then analyzed by gas–liquid chromatography (GLC). The results are tabulated in Fig. 9 below. All the aliquots (fractions) contained high levels of palmitic acid (Moseley & Thompson Jr., 1980). From this table, it can be observed that the degree of unsaturation in the membrane lipids is higher in gonidia than in somatic cells. This can result in more fluid membranes in gonidia (Moseley & Thompson Jr., 1980).

Fig. 9 Fatty acid distribution in Volvox lipid classes. Abbreviations: neutral lipids (NL), lipid galactose (GL), phospholipids (PL), 1(3),2-diacylglyceryl-(3)-O-4'-(N,N,N,-trimethyl) homoserine (DGTH) (Moseley & Thompson Jr., 1980).

After these tests, radioisotope-labelling experiments were performed in order to examine lipid metabolic pathways in Volvox. |14C|palmitate was incorporated into light synchronized Volvox colonies as a radiotracer (Moseley & Thompson Jr., 1980). Palmitate, also known as palmitic acid, is a straight 16-carbon, saturated long-chain fatty acid (National Library of Medicine, 2023). The radioactivity in membrane lipids of both cell types (gonidia and somatic cells) was primarily associated with DGTH. After being incorporated into complex lipids, the |14C|palmitic acid had little metabolic changes. |14C|palmitate was therefore incorporated specifically into a lipid fraction that was not available for elongation, desaturation, or exchange into different complex lipids (Moseley & Thompson Jr., 1980). This is hypothesized to be due to cells being unable to convert externally supplied long chain fatty acids such as palmitate to the metabolically active acyl-ACP (acyl carrier protein) derivatives (Moseley & Thompson Jr., 1980). This means that palmitate is not the best choice as a radiotracer in order to measure lipid metabolism since it will not become metabolically active when incorporated into the membrane lipids.

On the other hand, shorter chain fatty acids such as lauric acid have been found to enter the metabolically accessible lipids of other green algae such as Chlorella. To investigate this, |14C|lauric acid was added to Volvox spheroids and the lipids following this addition also had high radioactivity in DGTH (Moseley & Thompson Jr., 1980). Lauric acid is a straight 12-carbon, saturated medium-chain fatty acid and it has a role as an algal metabolite (National Library of Medicine, 2023). Unlike with |14C|palmitate, there was higher radioactivity content in other membrane lipids, and more elongation and desaturation of the incorporated |14C|lauric acid. This shows that lauric acid entered a more metabolically active lipid compartment than longer chain fatty acids such as palmitate (Moseley & Thompson Jr., 1980). This means that lauric acid is a more interesting fatty acid chain to measure lipid metabolism in Volvox since it binds to the metabolically active part of the lipids. From these results, the lipid that is most involved in the metabolism of Volvox is DGTH.

To the depths of the Volvox ECM

The extracellular matrix (ECM) of a multicellular organism is an organelle that tends to fill in the space between its cells. In the case of olvox, it occupies extent of available space, taking up more than 99% of a colony's total volume (Ender et al. 2002). A Volvox colony's ECM is what defines its shape and size. It is not the number of cells that grow throughout its lifetime, but the size of the ECM. A structure so large and important certainly must play a crucial role in the survival of the colony, and certainly, a Volvox's ECM allows for control and coordination of colony activity through pheromone/protein expression and helps define and maintain the colony's structure, all while protecting the organism. All these tasks are performed thanks to numerous types of hydroxyproline-rich glycoproteins (HRGP) that fill the ECM. Kirk et al. (1986) have proposed a system of nomenclature for the structure of a Volvox colony and separated the ECM into four zones: the flagellar zone (FZ), the boundary zone (BZ), the cellular zone (CZ), and the deep zone (DZ) (see Fig. 10). Now, let us venture deep into this ECM ourselves and discover what a Volvox colony truly looks like.

Fig. 10 Stylized representation of the structure of the Volvox ECM. In (a), the S's stand for somatic cells and the G stands for a gonidium. The small box in (a) is what we can observe up close in (b) (. [Adapted from Sumper and Hallmann (1998))].

A surface-level view of the surface: the flagellar & boundary zones

Starting off at the surface of the colony, we are surrounded by thousands of flagella, with each pair pointing out of the spherical colony from a somatic cell located deeper into the ECM. This is the flagellar zone (FZ), as described by Kirk et al. (1986). At the base of the flagella (FZ3 in the figure) we can find a thicker layer, composed of SSG 185 (which we shall discuss soon), which helps withstand the mechanical forces induced on the layer during flagellar beating. While there is not much to say about these flagella in terms of their chemistry, they have some fascinating physical properties, such as the fact that they can all take part in a synchronous beating pattern that helps the colony swim while minimizing its energy usage.

The boundary zone (BZ), is the thin, continuous layer that coats the colony, separating it from the outside world. The layer of the boundary zone that has often been focused on and the one that is common among all organisms of the order Volvocales is the tripartite layer, or BZ2 (Kirk et al., 1986), which is a crystalline layer composed of two types of hydroxyproline-rich glycoproteins (HRGPs) (Goodenough & Heuser, 1988), which are glycoproteins that have amino acid chains in which an excessive amount of them are prolines with a hydroxyl (OH) group. Goodenough & Heuser (1988) analyzed the structure formed by the HRGPs up close and observed that there seems to be a pattern made up of parallel rows of oval granules with fibrils either interconnecting the granules, or coursing between them, creating a three-dimensional meshwork (see Fig. 11 below).

Fig. 11 Close-ups of a Volvox carteri spheroid. The “W” tags represent different layers (or walls) of the ECM. W2-W6 represent the BZ (Sumper & Hallmann, 1998). W6 is the crystalline layer. (a) Continuing down the bottom right, we would eventually reach a somatic cell, while up to top left, we would leave the colony. x37 000 magnification. (b) W6 is separated into an upper (up) layer and under (un) layer. The thick arrows represent the axis that the parallel rows of granules follow. x145 000 magnification (. [Adapted from Goodenough and Heuser (1988))].

As seen in Fig. 11, there are two crystalline layers (up & un) forming the BZ. Goodenough and Heuser (1988) proposed that their superposition helps thicken the wall. Further, the granules create bulkier areas which could help maintain the integrity of the layer, while the meshwork originating from the fibrils could serve as a barrier preventing water or any undesired solutes or pathogens from gaining access to the inside of the colony. Finally, while analyzing and attempting to replicate the crystalline layer in vitro, Goodenough and Heuser (1988) sometimes observed distortions within the pattern, which they hypothesized could occur within the BZ itself in the case of impacts with particles in water, where reversible deformations of the BZ could help in absorbing shocks. Overall, it appears as though the crystalline structure of the BZ is thick in order to help isolate and protect the colony, through its network of fibrils and presence of bigger granules, while potentially having the ability to absorb impacts through local deformations, preventing damage to the colony.

Through the honeycomb-like cellular zone

Venturing beneath the crystalline layer, we reach the cellular zone (CZ), where the cells are each spread out in their own cellular compartments (Kirk et al., 1986), displaying a honeycomb-like organization. The compartments are filled up by components with no real distinct morphology (CZ2), though they are delimited by a filamentous network surrounding the cells and attached to their plasmalemma (CZ1) and another fibrous network creating the compartments themselves by outlining and separating them from each other (CZ3) (see Fig. 12) (Kirk et al., 1986). The CZ3 structure has been examined in more depth and a glycoprotein serving as a monomeric precursor (or building block) for it has been discovered (Wenzl et al., 1984) and analyzed (Ertl et al., 1989).

Fig. 12 The cellular compartment of a somatic cell in Volvox globator. x10,000 magnification (. [Adapted from Kirk et al. (1986))].

This glycoprotein has been termed SSG 185 by Wenzl et al. (1984), which stands for a sulfated surface glycoprotein with molecular mass 185 kDa. It is once again a hydroxyproline-rich glycoprotein, with Ertl et al. (1989) determining that its main polypeptide chain of 80 amino acid residues consists almost entirely of hydroxyprolines. They proposed that the secondary structure of this polypeptide forms a polyproline helix conformation, as do most proteins with several consecutive proline residues. More specifically, they considered the II helix conformation, which is a left-handed helix, with 3 residues per turn and a pitch (the height between two turns) of 0.94nm. Such a conformation would take on a rod-shaped form, which they have indeed observed through electron microscopy. From this rod also extends a polysaccharide, 21.4 nm long, with a backbone consisting of over 40 mannose units, each one having a di-arabinoside group extending from it (see Fig. 13). Surprisingly, this side chain is excessively sulfated, with 5 sulfate groups per unit.

Fig. 13 Representation of a unit of the polysaccharide side chain with the molecular structures for mannose and arabinose.

The high sulfation of the side chain leads to a high concentration of negative charges. However, Goodenough and Heuser (1988), have found that the Volvox crystalline layer consists of positively charged glycoproteins, therefore Ertl et al. (1989) proposed that the high number of sulfate groups is there to help in the aggregation of the crystalline BZ and hold everything together. Additionally, it is only natural that a glycoprotein requiring this many sulfates has a backup plan in case the colony runs low on sulfur. Indeed, when that is the case, an enzyme called arylsulfatase is synthesized in the ECM in order to mineralize additional sulfur (Hallmann & Sumper, 1994).

Finally, it has been observed that SSG 185 forms a very robust and stable polymer, as it is insoluble in the ECM, and does not dissociate when faced with chaotropic agents (solutes that disrupt hydrogen bonding and reduce stability of proteins) or detergents (Ertl et al., 1989). While it makes life tough for researchers, it certainly offers some robust scaffolding to contain the cells.

Deep into the deep zone

We have now escaped our compartment of the cellular zone and after having gone through multiple layers of ECM, we have reached our final destination: the deep zone (DZ). In most Volvox species, the DZ consists of 90% of the colony's entire volume (Kirk et al., 1986). This is where we find the pherophorin-based network of the colony (Ender et al., 2002), as well as many more HRGPs, which have been a common feature of the Volvox ECM (Sumper & Hallmann, 1998).

Pherophorins are ECM glycoproteins with a C-terminal domain homologous (similar in structure and protein sequence) to the sex-inducing pheromone (mentioned earlier) (Amon et al., 1998). They are believed to often serve as signal amplifiers once the sex-inducing pheromone has decided that it is time for the colony to prepare for sexual reproduction (Amon et al., 1998). It is important to note that not all pherophorins are synthesized via release of the sex-inducer. For example, pherophorin I and III can be found in the CZ of asexual colonies, serving as structural units (Sumper & Hallmann, 1998), though under the release of the sex-inducing pheromone, pherophorin III is cleaved and pherophorin II is synthesized (Godl et al., 1995). These glycoproteins are found in all four zones of the ECM (von der Heyde & Hallmann, 2023), though they are more common in the DZ (Ender et al., 2002). Nearlyall pherophorins have HRGP-like composition, often forming rod-like structures that help create sets of frameworks within the ECM, just like SSG 185 (Ender et al., 2002). An experiment by Ender et al. (2002) has shown the discovery of two rod-shaped pherophorins within the DZ that had 500 amino acid long and 2750 amino acid long hydroxyproline-rich polypeptide chains. Further, they concluded that there are at least nine members in the Volvox pherophorin family. Recently, this number has been dwarfed when von der Heyde and Hallmann (2023) performed an analysis of the phylogeny of a whopping 118 different pherophorins in Volvox carteri. The recent study even refers to SSG 185 as being part of this family. SSG 185 was therefore the first pherophorin to be discovered in Volvox all along, though at the time, it was not known to be one.

Interestingly, pherophorins are not the only biomolecules synthesized as a response to the sex-inducing pheromone, as Amon et al. (1998) have also observed the presence of a member of the chitinase/lysozyme family and a cysteine protease with three chitin binding domains. They observed that these compounds were similar to the ones induced in higher plant cells as a defense mechanism against pathogens. In other words, the sex-inducing pheromone also induces wound healing genes. Similarly, wounding would also induce the genes expected to arise from the release of the sex-induced pheromone, including pherophorins, implying a common signal transduction pathway for both. They believed that this relates to the idea that Volvox only undergo sexual reproduction when they are in danger, and a wound would be a good indication of that. As an example, they explained that Volvox carteri live in ponds that tend to dry out in the summer, and switching to sexual reproduction as a response to heat would allow them to produce dormant zygotes that could survive the drought.

Many HRGPs within the DZ have been observed to have a similar combination of phosphodiester bridges between two arabinose compounds within their saccharide side chains (see Fig. 14a) (Sumper & Hallmann, 1998). This has brought about speculation that there could be crosslinking involved between saccharide side chains, where an intramolecular phosphodiester bridge could be turned into an intermolecular bridge and vice-versa through a transesterification, without the need to spend any energy (see Fig. 14b) (Sumper & Hallmann, 1998). This would allow for the easy insertion of a monomeric unit within the polymeric structure, enabling the structure to expand, which could greatly help in the uniform growing of the Volvox colony.

Fig. 14 (a) Structure of the phosphodiester bridge between arabinose molecules. (b) Transition from an intramolecular phosphodiester bridge to an intermolecular one (. [Adapted from Sumper and Hallmann (1998))].

Crosslinking is not limited to structures in the DZ, as practically all HRGPs, which constitute a large percentage of the Volvox ECM, are involved in crosslinking. It has been previously mentioned that SSG 185 can resist different agents that attempt to break it apart. This is in fact due to covalent crosslinking between the saccharide chains, making it very difficult for the molecule to be dissociated (Ertl et al., 1989). Similarly, other pherophorins, such as the ones discovered by Ender et al. (2002) would crosslink into fibrous networks, a process that has been observed to be autocatalytic (where the pherophorins themselves act as catalysts for the formation of a self-assembled, crosslinked structure). Sumper et al. (2000) managed to dissociate these glycoproteins using Ellman's reagent (a sulfhydryl reagent) and found that they even self-assemble and crosslink in vitro. Ender et al. (2002) used Ellman's reagent to analyze the reassembly of slit colonies. Those incubated in the presence of the reagent would stay slit open, while those incubated in the absence of it would heal almost perfectly (see Fig. 15). This confirms that the HRGPs within the ECM are truly robust and tight structures that are not easily disturbed and that would do anything to stay in their shape and maintain the integrity of the Volvox colony.

Fig. 15 Wound healing in Volvox. (a) Intact colonies, (b) presence, and (c) absence of Ellman's reagent after the colonies are slit (. [Adapted from Ender et al., (2002))].

Photosynthesis in Volvox

The ATP molecule

One area where Volvox's strong use of chemistry is apparent is when observing its photosynthesis process. Volvox is an aquatic plant cell organism and therefore it performs photosynthesis as well as cellular respiration to produce energy. It is important to first understand the adenosine triphosphate (ATP) molecule and its use for storing energy in plant cells. ATP consists of a nitrogenous base (adenine) paired with a ribose sugar and is given its function by the three phosphate molecules bonded to the ribose sugar (Fig. 16). The bond between the second and third phosphates contains significant energy and is easily released through hydrolysis and can then be used to serve any required cell function. Volvox uses ATP throughout the photosynthesis process to store and transfer energy when required. Also, it generates ATP by cellular respiration for use throughout the cell. Glucose is the main molecule providing the energy for ATP, making it equally important. As photosynthesis is the process by which glucose is generated, it shares this high importance as well (Dunn J, 2023).

Fig. 16 A diagram showing the structure of an ATP molecule produced at a pH of around 3 (Haimovich et al.).

Note: Phosphorylation is the process of adding phosphate to an organic compound. The phosphate bond contains energy and will now allow the molecule to take part in reactions it would not have otherwise been able to go through due to not meeting the energy barrier required beforehand. It will change the reaction from endergonic to exergonic.

Light-dependent reactions

It is now important to see how the photosynthesis reaction is conducted in plants. Photosynthesis can be divided into two parts: the light-dependent and light independent reactions (also called dark reactions). The chemical equation for the overall process of photosynthesis is created by the coupling of the light reaction:

2H_2O + \text{light} \rightarrow O_2 + 4H^+ + 4e^- \quad (\Delta G^\circ = +317 \, \text{kJ/mol}),

and dark reaction:

CO_2 + 4H^+ + 4e^- \rightarrow CH_2O + H_2O \quad (\Delta G^\circ = +162 \, \text{kJ/mol}),

to produce:

H_2O + \text{light} + CO_2 \rightarrow CH_2O + O_2 \quad (\Delta G^\circ = +479 \, \text{kJ/mol}).

Note: G represents standard free energy and is a value used to assess if a reaction will proceed spontaneously or not. A negative G represents a spontaneous reaction. Its value represents the maximum amount of work performed by the given reaction.

Here, CH2O is the formula for any general carbohydrate, which is most commonly glucose. The process highlighted in Fig. 17 begins with photosystem II within the chloroplast. To note, chloroplasts make use of conjugated pi-system molecules like chlorophyll for light absorption as these molecules contain two easily accessible energy levels for electrons and can thus accept energy from light with ease. Photosystem II is a very special enzyme that makes excellent use of chemistry as it is one of the few in nature capable of splitting water into protons, electrons, and oxygen. It converts the water product into protons (H+) to be stored in the lumen and generates a proton gradient. Later on, the gradient will return to equilibrium and as this is a more favorable state, it will release energy which can be used for the synthesis of molecules. An electron from water will be transported via electron transport molecules and will end up in NADPH after a series of oxidation-reduction reactions and will be used to produce glucose in the dark reactions. Electrons are excited once again by light in photosystem I and are added to this electron transport chain as well. At the end, the machine-like enzyme ATP synthase shown in Fig. 18 is powered by a protein gradient which supplies energy to manufacture ATP molecules from ADP (two phosphate groups) and store energy for the next part of the photosynthesis process.

It is important to note that when photosystem II splits water, it produces 4 electrons, but the charge separation created only uses one electron. Therefore, four charge separations must be conducted. Transition metals have interesting properties allowing them to possess an array of positive charges. Therefore, one such element, manganese, which can range from +1 to +5 in charge is used to help maintain charge balance and catalyze the reaction (Johnson, 2016).

Fig. 17 Z-Scheme: The main components of the linear electron transfer pathway are shown on a scale of redox potential to illustrate how two separate inputs of light energy at PSI and PSII result in the endergonic transfer of electrons from water to NADP+ (Johnson, 2016).

Note:  An oxidation-reduction reaction is defined as the transfer of electrons between two compounds. The compound being reduced will gain electrons and the compound being oxidized will lose electrons. It is also important to note that the more a compound is reduced, the more energy it will contain.


Fig. 18 ATP synthase. The membrane is shown schematically in gray (Goodsell, 2005).

Light-independent/dark reactions

Following the production of ATP, the light-independent reactions occur. This procedure may once again be demonstrated in Fig. 19. The dark reactions called the Calvin-Benson cycle (or carbon fixation) use the energy from ATP along with the high energy electrons from NADPH produced in the light-dependent reactions to produce carbohydrates like glucose. In turn, this will regenerate ADP and NADP+ to be converted in the light-dependent reaction once again. The Calvin-Benson cycle is a delicate dance of reduction-oxidation reactions between different sizes of carbon-based molecules. To begin, CO2 is combined with a 5-carbon sugar (ribulose 1,5-bisphosphate) to produce a 6-carbon intermediate (fructose 1,6-bisphosphate) which will rapidly split into two 3-carbon molecules called 3-phosphoglycerate. These 3-carbon molecules will be converted into another two 3-carbon molecules called GAP (glyceraldehyde 3-phosphate). This process is only possible due to ATP driving phosphorylation of the initial molecule. The GAP molecule will then be converted into the needed carbohydrate. Finally, the initial 5-carbon molecule is regenerated (Johnson, 2016).

The powerful rubisco enzyme

The dark reactions of photosynthesis are all carried out with the help of the Enzyme Rubisco. This enzyme is another example of how Volvox uses chemistry to its advantage. The active site of Rubisco contains the element Mg2+, which catalyzes the conversion of CO2 to 6-carbon by changing the conformation of the molecules in a way to limit steric interaction and improve the likelihood of the reactions occurring.  Further, the colony also controls its pH in order to provide the optimal shape of the Rubisco enzyme for the reactions and improve the chance of it proceeding. Controlling the chemical properties of an environment has strong impacts on reactions that take place within it and is thus critical in controlling cell function (Johnson, 2016).


Fig. 19
Calvin-Benson cycle: Overview of the biochemical pathway for the fixation of CO2 into carbohydrates in plants (Chen et al., 2022).

Low CO2 environment solution

As has been shown, Volvox requires H2O, light, and CO2 in order to perform photosynthesis and survive. Light is not the only product that can limit photosynthesis. When in a low CO2 environment, Volvox uses chemical properties to manage this issue. It uses CCM (CO2 concentration mechanism), which is a process by which the cell will increase the concentration of CO2 around the Rubisco enzyme. This will prevent the enzyme from performing photorespiration (a process which expends energy) and will thus improve the carbon fixation cycle for the time being. The process by which this happens is complex but maycan be attributed to the fact that the CO2 concentration will affect gene transcription and favor certain enzymes that would otherwise not be present. Unfortunately, CO2 is required to create this induction of concentration around Rubisco and therefore the amount of CO2 can never drop too low (Wang et al., 2015). This process is driven by inorganic carbon and thus it can be studied in low CO2 to confirm that this process does indeed take place in Volvox. A test conducted confirmed that in low CO2 environments, Volvox colonies showed an affinity 38 times higher for inorganic carbon than in high CO2 environments. As such, this is proof that Volvox uses a CCM to deal with low CO2 environments. Once again, Volvox makes use of concentration differences to ensure survivability of the colony (Yamano et al., 2011).

Conclusion

The variety of environments in which Volvox thrive implies that the colony must be resilient against a diversity of conditions. One of the species' weaknesses is the fragile state of the juvenile spheroids during asexual reproduction. These are not fully developed and not protected to face droughts for instance, which often occur when ponds dry out in the summer. Therefore, Nature has designed a fascinating solution for Volvox, which consists of the activation of a sex-inducing pheromone that induces sexual reproduction in all cells of the colony. Hence, weak juveniles are replaced by zygospores encapsulated in a thick cell-wall that are able to remain in a dormant state until conditions are more favorable. By switching from one reproductive cycle to another, Volvox can continue to reproduce in harsh environments. Furthermore, in order to know when to switch to a reproductive cycle, Volvox have adopted the solution of having the wounding trigger and sex-inducing pheromone release the same set of genes, so that if ever the colony is wounded, they can instantly start the process of switching to a reproductive cycle.

Though, the colony cannot always switch cycles with the smallest of scratches, so it had to adopt a protective coating, especially since it lives in water, where many debris and bacteria float around. This is dealt with by the crystalline structure of the boundary zone of the ECM, which consists of thicker granules that allow for protection, as well as a meshwork that prevents undesired elements from entering the colony. The interior of the ECM is robust as well, consisting of many rod-like hydroxyproline-rich glycoproteins (mostly pherophorins), which help create a stable and compartmentalized structure for the cells within. These structures are even waterproof in case of leakage, as they are very difficult to break apart thanks to their strong levels of crosslinking. However, the colony would risk being too stiff if only consisting of rigid structures, which is solved by having the ability to alternate between intermolecular and intramolecular phosphodiester bridges, which are common throughout the ECM, facilitating the insertion of new monomers within the structure of the colony, allowing it to uniformly grow in size without any issues.

Volvox, like many other green algae, produces hydrogen gas and releases it into its environment. More specifically, Volvox evolves and consumes H2 with [FeFe]-hydrogenases, a group of enzymes that catalyze proton reduction and H2 oxidation. These proteins are inactivated in the presence of oxygen, so the green algae produce hydrogen only when oxygen gas is not present (under anaerobic conditions), which limits hydrogen production during photosynthesis. In a similar fashion to C. reinhardtii, Volvox solves this issue by channeling electrons to synthesize H2 by sulfur deprivation or using carbon stores obtained from photosynthesis. Also, it was shown in a study by Cornish et al. that in nature, Volvox can ferment carbon stores in order to drive hydrogen gas production under dark, anaerobic conditions.

Volvox desperately needs energy in the form of ATP to survive and thus uses photosynthesis to accomplish this goal. However, this poses multiple challenges. For one, it must deal with the mismatch of protons and electrons in the first phase of light-dependent reactions. To do so, it uses the transition metal manganese which has different numbers of possible positive charges that can help balance all charges. Also, the environment limits the amount of CO2 in the products, a conundrum for which Volvox once again has an elegant solution, as it induces a CO2 concentration around the Rubisco enzyme to lower the energy needed for it to conduct its reactions. Volvox appears to have a design solution for every problem that nature sends its way!

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