Quinn 1 Chloroplast evolution, divergence, and functional maintenance in algae and plants: adaptations and implications for carbon

sequestration, improved crop productivity and global warming

Abstract: Chloroplasts are the organelles within photosynthetic organisms that are responsible for photosynthesis. They are descended from cyanobacteria, and were incorporated into eukaryotes via endosymbiosis. Green algae are the most ancestral of the photosynthetic eukaryotes and gave rise to both red algae and land plants, two very different groups. The primary endosymbiotic event likely took place between an ancestor of current green algae and a cynaobacterium. Since then the chloroplasts in the red algal, green algal, and land plant lineages have diverged significantly from each other, especially in regards to their proteins and pigments. Rubisco stands out as the most important chloroplast protein. It catalyzes the fixation of carbon, and is a particularly challenged enzyme: it is likely to perform the fixation of oxygen, which is a detrimental reaction, when oxygen competes with carbon dioxide for the enzymes active site. The mechanisms algae and plants have developed to deal with this enzymatic problem are indicative of their particular environmental challenges. In regards to their pigments, each of these lineages has evolved to best utilize the light wavelengths available to them. Land plants can capture enough light with mostly chlorophylls, so they do not need to maintain a wide range of accessory pigments. Marine algae live a light-limited environment, so they must utilize a range of accessory pigments to capture sufficient light. Trans-membrane transport systems and strategies for preventing photoinhibition and photorespiration also differ among these lineages. Algae and plants live in distinct environmental niches, and understanding the adaptations of their photosynthetic systems to these environments can help us understand how these various primary producers may respond to challenges such as global warming. Keywords: Chloroplasts, photoinhibition, photorespiration, Rubisco, red algae, green algae, land plants, Tic/Toc translocation, pigments, light harvesting complexes

Quinn 2 Introduction Chloroplasts are the central organelles for the production of energy across Plantae. Today's chloroplasts began as independent organisms that were captured by an early ancestor of plants and algae at least 1.6 billion years ago. 1,2,3,4 Numerous efforts have been made to document the evolution of chloroplasts across the plant and bacterial kingdoms, and it is believed that chloroplasts are likely descended from primitive forms of cyanobacteria which were incorporated into eukaryotes through a single endosymbiotic event.5,6 Chloroplasts operate using a variety of processes with the primary functions of the organelle being the production of energy and the fixation of carbon. In order to successfully manufacture organic compounds in the many environments inhabited by plants and algae, chloroplasts necessarily have evolved to suit the circumstances of these diverse groups. 7,8,9,10 There are many reasons to be interested in chloroplast processes, among which are their role as the primary energy producer for life on earth, curiosity about the origins and relations of cellular organisms, and even hopes of using genetic engineering to improve chloroplast metabolic processes to raise crop yields and address global warming through optimization of CO2 extraction from the atmosphere.11 Plants and algae have spent approximately one and a half billion years adjusting to atmospheric changes, specializing, and evolving to function across the many environmental niches of the planet. Among the key photosynthetic processes that have been tuned and adjusted in the evolutionary process is the Calvin cycle. Within that cycle, a particular family of proteins known as Rubisco is both essential and seemingly flawed in their function. Rubisco is essential in carbon fixation, but it operates only under very constrained conditions, conditions limited by the availability of CO2 or bicarbonate and under the constant threat of mis-function, as Rubisco is nearly as attracted to O2 as it is to CO2.12 Green algae are the ancestors of all plants and other algal groups, including the red algae (Figure 1).1 Green algae are more closely related to land plants than to red algae, though both types of algae are marine. This indicates that red and green algae have developed distinct mechanisms for surviving in similar habitats. Land plants survive in a particularly different environment from either algae group. These three groups differ in some of their basic chloroplast proteins: the light-harvesting complexes and those proteins used to prevent photorespiration and photoinhibition. 13 Chloroplast Evolution:
Figure 1: This shows the evolutionary history of red algae, green algae (the Chlorophytes and Charophytes), and land plants (the Bryophytes, Ferns, Gymnosperms, and Angiosperms).1 There was only one endosymbiotic event for all Plantae, and it occurred with the incorporation of a cyanobacteria (CB) by an early eukaryote. The secondary endosymbiotic event led to the divergence of brown algae, among other photosynthetic species.

Quinn 3 All of the photosynthesis within plants and algae is performed by chloroplasts, which are semi-independent organelles derived from photosynthetic bacteria.14 These bacteria were incorporated into an ancestor of plants and algae via phagocytosis. This engulfment and incorporation of the photosynthetic bacteria by an ancestor of photosynthetic eukaryotes is known as endosymbiosis and is believed to have occurred around 1.6 billion years ago.1 There was only one primary endosymbiotic event that resulted in the chloroplasts for Plantae.5 Over time, the chloroplasts within the various algal and plant lineages have diverged from each other, but they continue to serve the same fundamental role within their hosts. Land plants and other algal lineages are descended from green algae.1 Plantae, which is the kingdom that encompasses green and red algae and plants, is monophyletic (Figure 1).5,15 All of the chloroplasts throughout Plantae have a common cyanobacteria ancestor resulting from a single primary endosymbiotic event. Despite the fact that the chloroplasts all derive from a single ancestor, there is a great deal of variation among the chloroplasts of green algae, red algae, and land plants. An example of this is the number of grana per stack in a thylakoid: red algae have single grana, while both green algae and plants have multiple grana per stack.16 This variation is also evident in the differences in translocation mechanisms between chloroplasts and their hosts and in the diversity of the Rubisco proteins found within Plantae. Chloroplasts now contain only a small number, from 50 to 200, of their original component of genes.17 The other one to two thousand proteins used in chloroplasts are now encoded for by genes in the nuclei of the host eukaryotes. The fact that the hosts manufacture chloroplast proteins indicates that there must be mechanisms to transfer these proteins into the chloroplasts. One example of such a mechanism is the Toc and Tic translocators (the translocon at the outer chloroplast envelope and the translocon
Figure 2: Diagram of the evolution and current state of the Toc/Tic translocator systems found in Plantae lineages.6 C. reinhardtii is the green alga.

Quinn 4 at the inner chloroplast envelope, respectively). Many of the proteins involved in these and other complexes are homologous among green algae, red algae, and plants, but there are significant differences in the number and type of proteins involved. The Tic/Toc translocator system, for example, is mostly conserved across groups, but there have been important changes in the alternate evolutionary paths.18 Toc proteins have been particularly conserved between the green algae and plants. There are fifteen major proteins involved in the vascular plant Toc system. Of these, two are present solely in the plant lineages, and one of the proteins is slightly modified from the green algal version.19,20,21 Thus, the mechanism for translocation across membranes functions mostly the same for these two groups. Red algae are more modified compared to land plants than the green algae, but the system is still quite similar (Fig. 1). Another example of variation in photosynthetic eukaryotes proteins is Rubisco. The types of Rubisco among these groups appear to be descended from three major plastid lineages. Green algae and plants contain Rubisco-encoding genes from a single lineage that is related to cyanobacteria, and red algal Rubisco-encoding genes come from a distinct other lineage that is more closely related to proteobacteria.22 These distinctions are particularly based on differences among the rbcL genes (which encode the large subunit of Rubisco and are located within the chloroplast) and are not observed in other proteins. The fact that the Rubisco-encoding genes are divided into multiple evolutionary lineages (i.e. that they are polyphyletic) is in contrast with other chloroplast lineage determinations, which Figure 3: The larger yellow circles represent endosymbiotic events, demonstrate that these either primary or secondary.25 The smaller yellow circles represent plastids have a gains of LHC genes. The red squares represent the loss of LHC genes. monophyletic In particular, note that all groups except the red algae and the background.5 Thus, Glaucophytes (freshwater algae) lost PBP, phycobilin proteins. Green algae are the Chlorophyceae and Ulvophyceae. Land plants are the there is some support Embryophytes. for the idea that there were either multiple horizontal gene transfer events between photosynthetic groups or gene duplications leading to paralogous Rubisco-encoding genes in these groups.23

Quinn 5 The type of Rubisco contained by each of these groups has been classified in detail. All of the photosynthetic eukaryotes have chloroplasts that contain form 1 of Rubisco, which is distinct from the Rubiscos found in dinoflagellates and Archaea (Lisa M Nigro, Masters thesis, The Graduate School, University of Maine, 2006). There are four subgroups of Rubisco within form 1: 1A, 1B, 1C, and 1D. Plants and green algae contain form 1B. Red algae mostly contain form 1D but have also been found to contain form 1C. There is little variation in the sequences of form 1B, and form 1C contained much more genetic variation. These distinct Rubiscos have differing affinities for CO2 and varied rates of fixation.24 Though Rubisco has been modified across Plantae, the Calvin Cycle, the major pathway for carbon assimilation, is mostly conserved in all photosynthetic organisms.11 Light Harvesting Complexes (LHCs) and Pigments: LHCs demonstrate both evolutionary connections between the algae and plants and adaptations to environmental conditions. These complexes are composed of pigmentbinding proteins.25 Though there are comparable LHC structures in green algae and plants, the antennae systems in these two groups likely evolved independently since there are few shared orthologs. Green algae have a lager photosystem I (PSI) complex, with nine polypeptides, whereas plants PSI uses six polypeptides (Table 1). There are two types of pigments in green algae and plants, chlorophyll and carotenoids, and an additional family of pigments in red algae called phycobilins. Plants and algae use carotenoids in photoprotective roles and dissipate excess excitation energy through them in a process known as non-photochemical quenching (NPQ).26 There are over 600 known carotenoids. Those used in the xanthrophyll cycle differentiate plants from algae.27 Red light, which is absorbed by both chlorophylls a and b, is limited in its penetration of water. Green algae compensate by absorbing more green light through their carotenoids.28 Phycobilins, which absorb light in the green and yellow wavelengths, are the primary light harvesting pigments of red algae, which typically live in a more lightlimited environment than green algae or plants (Figure 4).

Figure 4: These show (a) the depth to which particular wavelengths of light penetrate the ocean and (b) the absorbance spectra of various pigments found in algae and plants. Also in (b) the black line represents the (a) is from http://oceanexplorer.noaa.gov/explorations/04deepscope/background/deeplight/media/diagram3.htm, and b. is from http://course1.winona.edu/sberg/ILLUST/fig15-5.jpg.

Quinn 6 The core antennae system of the light harvesting compounds developed early in green algae evolution and included chloroplast proteins that today are functionally involved in PSI and PSII across all green algae and plant species. The core antennae system includes chloroplast proteins 29 and 26 (CP29 and CP26), which are coded for by light harvesting complex genes Lhcb4 and Lhcb5, respectively (Table 1). CP26 and CP29 are associated with PSII and are present in green algae and higher plants. There exist, however, chloroplast proteins specific to green algae with early evolutionary roots, as demonstrated in their widespread presence in algal species, which are not present in higher plants. Light harvesting complex genes Lhca2 and Lhca9 are green algae specific, and the fact that there is no evidence of them in red algae demonstrates that red and green algae diverged early. Chlamydomonas reinhardtii, the model green alga, contains 11 light harvesting complex genes that program for LHCI, while Arabidopsis thaliana, the model plant, only contains 3 genes that perform corresponding work.29 Photorespiration: Photorespiration in plants and algae is impacted by the types of Rubisco and the carbon concentrating mechanisms (CCMs) that the respective groups have evolved. There are four major classes of Rubisco. The Rubisco found in plants and nearly all algae, form1, discriminates poorly between oxygen and carbon dioxide.22 Algae and plants use different CCMs. In land plants, there are two main methods for dealing with Rubiscos low specificity: C3, and C4 photosynthesis. Plants with C3 use a form of Rubisco that is more specific for carbon dioxide.24 C4 plants have developed cellular processes that result in higher concentrations of carbon dioxide around Rubisco, so the enzymes specificity does not matter as much. Algae have increased requirements for carbon concentration over plants since water contains a lower concentration of CO2 than air30,31 Many algae have developed carbon concentrating mechanisms to help deal with the issue of photoinhibition.24,32,33,34 Those algae that have not developed such mechanisms have forms of Rubisco with a higher affinity for CO2. The type of Rubisco found in red algae also has a lowered affinity for O2. . Both plants and algae use CCMs to increase the concentration of CO2 around Rubisco so that the enzyme will preferentially bind CO2 instead of O2. Marine algae do not appear to be carbon-limited with their CCMs maintaining steady cellular access to carbon in variable environmental conditions. 35 Within algae, there are multiple levels of carbon concentration. There is evidence for active transportation of inorganic carbon, either in CO2 or HCO3- (bicarbonate) form, across the chloroplast membranes and across the pyrenoid membranes in algae. The diffusion of carbon dioxide in water is 104 times slower than in air, consequently algae must actively transport CO2 into their cells, in contrast with the passive diffusion of CO2 into C3 plants. 36 One of the main problems for marine algae is that the carbon available in water exists mostly as HCO3-.37 Thus, it must be converted to CO2 before being used by Rubisco. Marine algae use carbonic anhydrase to perform this reaction. Rubisco may also exist freely in the stroma or be localized within a pyrenoid. The efficiency of Rubisco is not only dependent on CO2 concentrations but also on the surrounding temperature. Heat stress can result in the decrease in efficiency of Rubisco.38 This occurs through the deactivation of Rubisco activase and the subsequent decrease in activity of Rubisco. Increases in temperature can also lower the exchange rate of CO2 across membranes, which further negatively impacts Rubisco.39, 40

Quinn 7

Photoinhibition: Photoinhibition is a significant problem for plants and to a lesser extent also affects marine algae. When plants and algae are exposed to extreme conditions, from heat stress to drought to excess light, they can suffer from photoinhibition.41 This means that the electron transport chain (ETC) of photosynthesis, involved in the production of a proton motive force (pmf), is functioning non-optimally. The ETC is being damaged faster than it can be repaired. The damage is believed to result from the evolution of radical oxygen species, which slow the rate of protein production and thus slow the rate of repair to systems damaged by stress from excess energy uptake. Two of the main mechanisms for photosynthetic organisms to defend themselves from excess light energy are regulating the rate of photosynthesis and dissipating heat through NPQ. State transitions are regulatory mechanisms that are used to balance the activities of PSII and PSI in order to optimize photosynthesis under variable conditions.42 State transitions are used to prevent reactive oxygen species from forming. State transitions refer to the phosphorylation and movement of the light harvesting complexes (LHC) from PSII to PSI and back. This transfer of the LHCs is a mechanism for equilibrating the output of the two photosystems. This process is moderated by the relative rates of reaction of the photosystems and so is dependent on the amount of light available. When LHCs are phosphorylated, some of them move from PSII to PSI. In plants, only about fifteen percent of the LHCs migrate to and from the PSI. In algae, up to eighty percent of the LHCs can be transferred. This system works both to maximize photosynthetic output and protect the organism from the effects of photoinhibition. The process is catalyzed by a protein kinase; in plants it is called STN7 and in algae Stt7.43,44,45,46 There is a high degree of similarity between these proteins, but their efficiencies and results are markedly different.47 Another method of alleviating photoinhibition via regulation of the photosynthetic rate is cyclic electron flow.48 This cycling of electrons around PSI takes place when the damage to PSII is occurring faster than the rate of repair. PSII, and especially its D1 protein, is the preferential site of damage during photoinhibition. Damage to the D1 protein shuts down the electron transport chain and thus prevents the formation of reactive oxygen species. This pattern is conserved across algae and plants. When cyclic electron flow is inhibited, the rate of damage to PSII goes up under high light conditions. The formation of a pmf continues under cyclic electron flow, but there is reduced conversion of NADP+ into NADPH or of ADP into ATP. This is the problem with photoinhibition prevention systems: they protect the photosystem proteins from damage but at the cost of a lower photosynthetic yield. A third method of preventing photoinhibition is heat dissipation (i.e. NPQ). CP24 is the most recently evolved minor antenna complex in PSII, and is found solely in land plants.49 It is believed to play a role in NPQ, particularly in the conversion of violaxathin to zeaxanthin. This reaction is central to the dissipation of heat, retarding photoinhibition.50 Green and red algae have alternate methods of NPQ since they do not have CP24 as plants do.49 Algae are not exposed to as much heat stress as plants (this is due to the different heat capacities of the ocean versus the atmosphere), so they do not need the most efficient protection against photoinhibition. They instead contain CP26, which is an ancestral protein to CP24. CP26 is also found in plants, and performs the same basic function as

Quinn 8 CP24: the production of zeaxanthin to defend the organism from excess light energy. CP26, however, is not as efficient at catalyzing this reaction.25,51. Photoinhibition is a common issue for both plants and algae. For algae, light intensity can vary dramatically during normal wave action and over tidal cycles and result in light stress.52 Similarly for plants, light and heat levels can increase over the course of the day and stress the photosynthetic systems.53 Because plants are primarily land-based and green and red algae are marine organisms they necessarily have to deal with different levels of stresses. The differences in environmental conditions permit algae to utilize less efficient systems than plants to mitigate the effects of photoinhibition. Conclusion: Red algae, green algae, and land plants, though they belong to a monophyletic group and have chloroplasts which perform similar functions, are distinct groups. There was only one endosymbiotic event which resulted in the formation of chloroplasts for all photosynthetic eukaryotes. However, the chloroplasts for each group have diverged from the others to better suit the environments of their hosts. All of these strategies for photosynthesis may be affected by global climate change in the future, Global warming is predicted to have dramatic effects on photosynthesis, particularly in regards to photoinhibition.54 The movement of CO2 into a plant, or through water, is dependent both on the concentration of CO2 in the surroundings and the temperature. At temperatures higher than the current climate, even with an increase in CO 2 concentration, Rubisco has been shown to be less efficient. Since both temperature and CO2 levels will change in the coming years, the impact on photosynthetic organisms may be significant. Due to the importance of photosynthesis to the biosphere, understanding the various mechanisms that photosynthetic organisms have evolved to perform this function is imperative.

Quinn 9

Table 1: A synthesis of the differences between green algae, red algae, and land plants in regards to photosynthetic and chloroplast machinery.
Pigments16 Green Algae Chl a Chl b carotenoids (examples): xanthophyll violaxanthin neoxanthin lutein loroxanthin Land Plants (vascular) Chl a Chl b carotenoids xanthophyll violaxanthin Red Algae Chl a carotenoids xanthophyll violaxanthin

phycobilins: phycocyanin phycoerythrin LHC1 (Lhcar1)* LHC1 (Lhcar2) LHC1 (Lhcar3) LHC1 (Lhcar4) LHC1 (Lhcar5) (Lhcf4)

LHC Proteins (genes)25,55,56,57

minor antennae proteins Kinase for the state transition of LHC58 Toc/Tic translocator proteins 16

LHCII type 1 (Lhcbm1-6) LHCII type 2 (Lhcbm8-9) LHCII type 3 (Lhcbm11) LHCI type 1 (Lhca1) LHCI type 3 (Lhca3) LHCI type 4 (Lhca2) LHCI type 5 (Lhca4-8) LHCI type 6 (Lhca9) LHCQ (Lhcq and LI818) CP29 (Lhcb4) CP26 (Lhcb5) Stt7

LHCII type 1 (Lhcb1) LHCII type 2 (Lhcb2) LHCII type 3 (Lhcb3) LHCI type 1 (Lhca1) LHCI type 2 (Lhca2) LHCI type 3 (Lhca3) LHCI type 4 (Lhca4) LHCI type 5 (Lhca5) LHCI type 6 (Lhca6) LHCQ (Lhcq) CP29 (Lhcb4) CP26 (Lhcb5) CP24 (Lhcb6) STN7

Stt7

80 80/75 (alt. combination) 80 159 159 159 75 75 34 34 34 64 12 22 22 22 22* 21 21 21 20 20 20 110 110 110 40 40 55 55 32 32 32 62 62* 62* ClpC ClpC ClpC * These genes and proteins correspond directly with those in green algae and plants. Red algae have not been extensively studied in this regard: analysis of their protein sequences and LHCs is still underway.59

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They do have CP26 and CP29, but not CP24, similar to green algae, but the genes encoding these minor antennae proteins do not appear to be known in red algae.

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