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Planta

, Volume 249, Issue 1, pp 31–47 | Cite as

A hypothesis about the origin of carotenoid lipid droplets in the green algae Dunaliella and Haematococcus

  • Uri PickEmail author
  • Aliza Zarka
  • Sammy Boussiba
  • Lital Davidi
Review
Part of the following topical collections:
  1. Terpenes and Isoprenoids

Abstract

Main conclusion

Hypercarotenogenesis in green algae evolved by mutation of PSY that increased its transcription at high light, disintegration of the eyespot in Dunaliella and acquisition of the capacity to export carotenoids from chloroplasts in Haematococcus.

Carotenoids (Car) are lipid-soluble pigments synthesized in plants, algae, bacteria and fungi. Car have strong antioxidative properties and as such are utilized to reduce the danger of different diseases in humans. Two green microalgae are utilized as rich natural sources for Car: Dunaliella salina/bardawil accumulates 10% (w/w) β-carotene (βC), which is also pro-vitamin A, and Haematococcus pluvialis accumulates 4% (w/w) astaxanthin (Ast), the strongest antioxidant among Car. D. bardawil accumulates βC in plastoglobules within the chloroplast, whereas H. pluvialis deposits Ast in cytoplasmic lipid droplets (CLD). In this review we compare the hypercarotenogenic responses (HCR) in Dunaliella and in Haematococcus and try to outline hypothetical evolutionary pathways for its origin. We propose that a mutation in phytoene synthetase that increased its transcription level in response to high light stress had a pivotal role in the evolution of the HCR. Proteomic analyses indicated that in D. bardawil/salina the HCR evolved from dissociation and amplification of eyespot lipid globules. The more robust HCR in algae that accumulate carotenoids in CLD, such as H. pluvialis, required also acquisition of the capacity to export βC out of the chloroplast and its enzymatic conversion into Ast.

Keywords

Carotenogenic response Carotenoid lipid droplets Dunaliella salina/bardawil Haematococcus pluvialis Eyespot Phytoene synthase 

Abbreviations

Ast

Astaxanthin

Ast-TA

Ast transacylase

βC

β-carotene

βC-PG

βC plastoglobules

BKT

βC ketolase

Car

Carotenoid/s

Chl

Chlorophyll

CLD

Cytoplasmic lipid droplets

CRTR-B

ΒC hydroxylase

FA

Fatty acids

HCR

Hypercarotenogenic response

HL

High light

ND

Nutrient deprivation

PS-II

Photosystem II

PSY

Phytoene synthase

ROS

Reactive oxygen species

TAG

Triacylglycerol

Introduction

Carotenoids (Car) have two major functions in photosynthetic organisms: they are essential components in light harvesting and in photoprotection in the photosynthetic system (primary Car) and they may also accumulate in lipid droplets inside or outside plastids, to serve different functions such as photoprotection, phototaxis and as natural dyes (secondary Car). Car are also essential ingredients for humans serving as pro-vitamins and as protectants against oxidative damage. The main natural sources of Car are fruits and vegetables; however, several green algae also accumulate secondary carotenoids to much higher levels, and there are two algae species which accumulate high levels of two Car: the halotolerant D. bardawil (and D. salina), which are the richest natural source for β-carotene (βC) (Ben Amotz et al. 1982; Jin and Polle 2009) and H. pluvialis, which is the richest natural source for astaxanthin (Ast) (Boussiba 2000; Solovchenko 2015; Shah et al. 2016). Recent proteomic, metabolomic and lipidomic analyses of these algae and of their Car-containing lipid globules shed new light on the metabolic changes involved in the HCR and on their evolutionary origins. The purpose of this review was to briefly summarize the current knowledge and gaps in our knowledge about Car accumulation in these green algae. We will discuss the similarities and the differences in Car accumulation between Dunaliella and Haematococcus and the possible evolutionary origins of secondary Car accumulation in these and in other green algae.

Benefits of secondary Car for humans

At least seven algae-derived Car have already been approved for human studies and use including: βC, lutein, zeaxanthin, Ast, lycopene, canthaxanthin and fucoxanthin (reviewed in Gong and Bassi 2016; Galasso et al. 2017). βC is essential for human health, since being a pro-vitamin A it is the precursor of our visual pigment (11-cis retinal), prevents human blindness, and it also gives rise to all-trans retinoic acid and of 9-cis retinoic acid which control cell differentiation and metabolism (Dufosse et al. 2005; Plutzky 2011). Lutein and zeaxanthin are used to improve the protection against excessive irradiation damage to our eyes, which can lead to cataract and to age-related macular degeneration (Bone and Landrum 2003; Manayi et al. 2016). Ast is the most potent antioxidant Car and reactive oxygen species (ROS) scavenger. It has many commercial applications including food additives, animal feed, cosmetics and pharmaceuticals (Capelli et al. 2013; Husssain et al. 2006; Yuan et al. 2011). It also has anti-inflammatory and antioxidant benefits for human health for lowering the risks of cancer, cardiovascular disease, inflammatory disease, diabetes and obesity (Liu et al. 2014; Solovchenko 2015). Lycopene has been shown to protect against radiation, cardiovascular diseases and cancer (Story et al. 2010). Canthaxanthin creates tan color (Garone et al. 2015), and fucoxanthin is being used against diabetes and obesity (Maeda et al. 2009; Muradian et al. 2015). All these Car effectively neutralize ROS and as such potentially protect our body against different degenerative diseases including cancer. The natural origins of Car are flowers, fruits and vegetables; however, some microalgae produce specific Car in much larger quantities. In fact, to date, Car are the most valuable commercial products derived from microalgae (Borowitzka 2013; Henríquez et al. 2016).

Primary and secondary Car in green algae

Car in microalgae and in higher plants function primarily as accessory pigments in light-harvesting antenna, by absorbing blue light that is poorly absorbed by chlorophyll (Chl), thus improving light absorption (Hashimoto et al. 2016). Specialized xanthophylls: violaxanthin, antheraxanthin and zeaxanthin, function in photoprotection of the photosynthetic system, by deactivation of exited singlet Chl, while lutein quenches triplet Chl, resulting in dissipation of excessive absorbed light from the light-harvesting antenna proteins as heat (Demming-Adams and Adams 1996; Jahns and Holzwarth 2012; Duffy and Ruban 2015). βC functions in photoprotection of the photosynthetic reaction center II (PS-II), probably by quenching singlet oxygen that is produced in PS-II (Telfer 2002; Trebst 2003). These types of Car are associated with specific photosynthetic protein complexes and are termed primary Car, being an integral part of the photosynthetic system.

Several species of microalgae, mostly belonging to green microalgae (Chlorophyceae), may also accumulate high levels of secondary Car under stress conditions, such as high light (HL) stress, nutrient deprivation (ND) or at the stationary growth phase (Table 1). This response is termed the HCR and it is usually accompanied by accumulation of secondary carotenoids, by a decrease in size of the chloroplast and in Chl content, resulting in a high Car/Chl ratio. Secondary Car are accumulated in lipid droplets, composed mostly of neutral lipids, are not directly associated with the photosynthetic system, and are usually located outside the chloroplast. The major function of secondary Car in green algae is to screen the photosynthetic system against excessive irradiation, thus preventing photodamage, as was shown for D. bardawil and for H. pluvialis (Ben-Amotz et al. 1989; Wang et al. 2014a).
Table 1

Accumulation of secondary Car in green algae

Species

Major Car

Car/DW

Car Car/Chl

Stress

Organelle

References

(%)

(pg/cell)

(w/w)

Dunaliella bardawil

βC

Up to 10%

  

HL, N deprivation, S deprivation, P deprivation

PLG

Ben Amotz et al. (1982, 1989)

Dunaiella salina Teodoresco

βC

Up to 10%

36.5

 

HL, N deprivation, S deprivation, P deprivation

PLG

Orset and Young (2000); García-González et al. (2005)

Haematococcus pluvialis

Ast- ester

2–4%

  

HL, N deprivation, S deprivation, P deprivation

CLD

Boussiba (2000), Orosa et al. (2001), Aflalo et al. (2007)

Chromochloris

(Chlorella) zofingiensis

Ast-ester

Lutein, ketolutein

0.15–0.7%

0.4%

  

Stationary N-deprivation

CLD

Orosa et al. (2001); Del Campo et al. (2004); Mulders et al. (2015)

Neochloris wimmeri

Ast

1.8%

  

HL, N-deprivation, high NaCl

CLD

Orosa et al. (2001)

Protosiphon botryoides

Ast

1.5%

  

HL, N-deprivation, high NaCl

CLD

Orosa et al. (2001)

Chlamydomonas nivalis

Ast + Ast- ester

  

8–25

HL + low temperature

CLD

Remias et al. (2005)

Scotiellopsis oocystiformis

Ast

0.7%

  

HL, N-deprivation, high NaCl

CLD

Orosa et al. (2001)

Scenedesmus vacuolatus

Ast

0.25%

  

HL, N-deprivation, high NaCl

CLD

Orosa et al. (2001)

Scenedesmus sp.

Not identified

1.75%

  

Fe-SO4

 

El-Sayed (2010)

Scenedesmus komarekii

Canthaxanthin

Ast

 

0.65

0.1

 

HL + N limitation

CLD

Hanagata and Dubinsky (1999)

Chlorella emersonii

Canthaxanthin

  

3–20

HL + N deprivation

CLD

Arad et al. (1993)

Parietochloris incisa

βC

 

1.1

 

HL + N limitation

CLD

Merzlyak et al. (2007)

Botryococcus brauni

Adonixanthin

Lutein

0.23%

0.1–0.5%

  

Stationary N-deprivation, P-deprivation

CLD

Grung et al. (1994); Ambati et al. (2018)

PLG plastoglobules, CLD cytoplasmic lipid droplets, DW dry weight

Among green algae, Dunaliella and Haematococcus stand out as the two species which accumulate particularly high levels of secondary Car: D. bardawil and D. salina Teodoresco, the dominant algae species in hypersaline and in most brackish water niches worldwide (Polle et al. 2009), accumulate up to 10% (w/w) βC in small plastoglobules within the chloroplast under HL/ND stress (Ben Amotz et al. 1982). H. pluvialis is transformed under HL/ND stress from green to red cells, which contains large cytoplasmic lipid droplets (CLD) composed of neutral lipids and Ast-esters (Ast), totaling up to 30% and 4% of the cell dry weight, respectively (Aflalo et al. 2007). Other notable examples of green algae that accumulate secondary Car up to 1% of the biomass are the Ast-accumulating “snow algae” Chlamydomonas nivalis (Table 1, Remias et al. 2005); Chromochloris (previously Chlorella) zogfingiensis, which accumulates Ast, canthaxanthin and ketolutein, considered as a future alternative source for Ast (Del Campo et al. 2004; Liu et al. 2014; Mulders et al. 2015), some species of Scenedesmus, which accumulate canthaxanthin and Ast (Table 1, Hanagata and Dubinsky 1999; El-Sayed 2010); Parietochloris incisa, a fresh-water green alga, which accumulates large amounts of the polyunsaturated fatty acid (FA) arachidonic acid as well as βC (Table 1; Merzlyak et al. 2007; Solovchenko and Neverov 2017); and Botryococcus braunii, which accumulates neutral lipids and Car including lutein (Grung et al. 1994; Ambati et al. 2018). Since Dunaliella and Haematococcus are the only hypercarotenogenic algae in which the HCR has been extensively studied, we take them as representatives of Car accumulating algae and limit the discussion mostly to these species.

Most microalgae, which do not produce secondary Car, or which do produce low levels of secondary Car under non-stressed conditions, never exceed a Car/Chl ratio of 0.3. The reason for that is that primary Car are synthesized in stoichiometric amounts similar to Chl, and their levels are strictly controlled by the levels of antenna proteins (Caffarri et al. 2014). Massive biosynthesis of a secondary Car may inhibit photosynthesis by interference in the synthesis of primary Car by feedback inhibition or by screening the light. Therefore, there is a need to efficiently eliminate the secondary Car from the photosynthetic membranes as soon as they are synthesized. This is achieved by storing the Car in special lipid bodies, which act as sinks for Car accumulation. Dunaliella and Haematococcus evolved different mechanisms to achieve this goal as will be discussed below.

βC accumulation in D. bardawil/salina

Dunaliella bardawil and D. salina Teodoresco accumulate βC when exposed to HL or to deprivation of nutrients such as nitrogen, phosphate or sulfate (Ben Amotz et al. 1982; Jin and Polle 2009). Smaller levels of βC accumulation can be obtained also under other growth-limiting conditions such as iron depletion, high NaCl concentration (exceeding 1.5 M, the optimal salt concentration for this species) or extreme temperatures (Ben Amotz and Avron 1983; Jin and Polle 2009). It has been demonstrated that the level of βC accumulation depends primarily on the light intensity within the visible light spectrum that is absorbed by the photosynthetic system (Ben-Amotz and Avron 1989), whereas far-red light and UV-A light promote βC accumulation and inhibit growth (Sanchez-Saavedra et al. 1996; Jahnke 1999). βC protects D. bardawil cells against photoinhibition in HL intensities by screening the photosynthetic system, since inhibition of βC biosynthesis makes the algae vulnerable to HL (Ben-Amotz et al. 1989; Ghetti et al. 1999).

The pigment that accumulates in small lipid droplets within the chloroplast, that we termed βC plastoglobules (βC-PG), are composed of 40–50% (w/w) TAG, 50–60% (w/w) βC, a small amount of proteins and are surrounded by a monolayer of polar lipids (Ben Amotz et al. 1982; Davidi et al. 2015). The pigment is composed of two stereoisomers, all-trans βC and 9-cis βC, in approximately equal amounts (Ben-Amotz et al. 1988). The two stereoisomers differ in their spectral properties and in their physical properties: 9-cis βC is more lipid-soluble than all-trans βC and probably acts as a solvent for the all-trans isomer in βC-PG, since the latter tends to crystalize at high concentrations (Ben-Amotz et al. 1988). Hypercarotenogenic mutants of D. bardawil, which accumulate high levels of βC also under optimal growth conditions, have been isolated in our laboratory (Shaish et al. 1991, supplemental material S1). These mutants produce higher relative levels of the 9-cis βC isomer under low light conditions and appear to be hypersensitive to HL stress (supplemental material S1).

The accumulation of βC is dependent on the accumulation of TAG, since inhibition of TAG biosynthesis inhibits βC accumulation; however, inhibition of βC biosynthesis does not impact TAG formation (Rabbani et al. 1998). The reason for the dependence of βC accumulation on TAG biosynthesis is that the lipid droplets act as a sink for βC, driving the transfer of the pigment from chloroplast membranes by their higher lipid solubility. Thus, this process prevents the accumulation of βC or its intermediates in chloroplast membranes which could inhibit the synthesis of primary Car by feedback inhibition (Cazzonelli and Pogson 2010). In a recent study we have investigated the metabolic changes involved in TAG biosynthesis during N deprivation in Dunaliella and the interrelation between the different carbon pools. We found that the metabolic response to N deprivation can be divided into two stages: In the early stages (24 h), the cells synthesize starch by photosynthetic CO2 assimilation, whereas in the later stages, photosynthesis is repressed, and starch is utilized as the major carbon and energy source for synthesis of TAG (Pick and Avidan 2017).

The formation, composition and origin of βC-PG were not clear until very recently. We found that the formation of βC-PG is a step-wise process: It starts with the appearance of tiny lipid droplets within the chloroplast, which seem to originate from pre-formed CLD, because the molecular species composition of βC-PG is almost identical to that of CLD and their accumulation is correlated with a parallel decrease in TAG level of CLD (Davidi et al. 2014; depicted in Fig. 1). The lipid globules then assemble with over 100 different proteins. In parallel, upregulation of phytoene synthase (PSY) in chloroplast membranes leads to accumulation of phytoene, which is transferred into the plastoglobules and is utilized for synthesis of all-trans βC and 9-cis βC (Fig. 2).
Fig. 1

Biogenesis of Car lipid droplets in D. bardawil and in H. pluvialis. In D. bardawil (right), exposure of vegetative (Vg) cells to HL/ND stress induces first the accumulation of starch (St) within the chloroplast. Starch is utilized to synthesize TAG in CLD in the cytoplasm. Next plastoglobules are formed by transfer of TAG from CLD into the chloroplast (S1). This is followed by synthesis and accumulation of βC within the plastoglobules (S2). In H. pluvialis (left), stress induces first loss of flagella and encystment of the cell forming the palmeloid cell (Pl). This stage is accompanied by accumulation of starch in the chloroplast. Part of the starch is utilized next to form TAG creating CLD, and it is accompanied by transfer of βC from the chloroplast into CLD, synthesis of Ast which is converted to Ast-ester. At extended stress, the palmeloid cell accumulates massive amounts of Ast-ester and is converted into a heterocyst (Hc) or aplanospore cell

Fig. 2

Biosynthetic pathways of βC in D. bardawil and of Ast in H. pluvialis. The abbreviated biosynthetic pathways depict the formation of phytoene through the 2-methyl-d-erythriol-4-phosphate (MEP) pathway (Rohmer 1999), and from phytoene into βC, which in H. pluvialis takes place within the chloroplast (Chl). In D. bardawil, phytoene is transferred from inner chloroplast membranes into pre-mature plastoglobules and is converted into all-trans βC and 9-cis βC creating the βC-PG. Notice that different enzymes convert phytoene into βC in chloroplast membranes and in βC-PG. In H. pluvialis, βC is transferred from the chloroplast into CLD, and is converted into Ast and next into Ast-ester, creating Ast-CLD. Enzymes are spelled in italics. Stress-upregulaed enzymes are marked in bold red. GA-3P glyceraldehyde-3-phosphate, Pyr pyruvate, IPP isopentenylpyrophosphate, GGPP geranylgeranyl Pyrophosphate, DXS 1-deoxy-d-xylulose phosphate synthase, DXR 1-deoxy-d-xylulose phosphate reductase, IspD 4-diphosphocytidyl-2C-methyl-d-erythritol synthase, IspE 4-diphosphocytidyl-2C-methyl-d-erythritol kinase, IspF 2C-methyl-D-Erythritol-2,4 cyclodiphosphate synthase, IspG 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase, IspH 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, GGPPSY geranylgeranyl pyrophosphate synthase, PSY phytoene synthase, PDS phytoene desaturase, ZDS zeta-carotene desaturase, LYCB lycopene cyclase, 9-cis βC-ISO all-trans/9-cis βc isomerase, BKT βC ketolase, CRTR-B βC hydroxylase, Ast-AT astaxanthin acyltransferase

The biosynthetic pathway of βC biosynthesis in D. bardawil was clarified from the accumulation of carotenoid intermediated under inductive conditions in the presence of different inhibitors (Ben-Amotz et al. 1988; Shaish et al. 1990). These results indicated that phytoene is converted into βC via the following intermediates: phytoene, phytofluene, ζ-carotene, neurosporene, lycopene, γ-carotene, βC. Interestingly, all these carotenoid intermediates were found in two isomeric forms, all-trans and 9-cis, suggesting that the isomerization probably takes place already at the stage of phytoene (Fig. 2).

In our proteomic analysis of βC-PG, we identified 124 different proteins, including the major carotene-globule-associated proteins (CGP) which stabilize βC-PG (Katz et al. 1995), and βC biosynthesis enzymes (Davidi and Pick 2012; Davidi et al. 2015). These enzymes include phytoene desaturase (Dusal.0029s00014-PDS1), ζ-carotene desaturases (Dusal.0023s00041-ZDS1, Dusal.0023s00041-ZDS2, Dusal.0592s00005-ZDS3), lycopene cyclases (Dusal.0649s00010 LCY1, Dusal.0018s00028 and Dusal.0018s00026-LCY2), which differ from the β-carotene biosynthesis enzymes, identified in a chloroplast membrane fraction (Dusal.0163s00010-PDS2, Dusal.0372s00006-ZDS4) (Fig. 2, Davidi et al. 2015, 2017). PSY was not identified in the βC-PG proteome. These results indicated that D. bardawil has two different Car biosynthetic pathways: the constitutive pathway, for synthesis of primary Car, probably localized in photosynthetic membranes, and the inductive pathway, for synthesis of βC from phytoene, in βC-PG. Based on these results we proposed that under stress conditions, PSY in the chloroplast membranes is upregulated, the phytoene is then transferred to the plastoglobules and there is converted to βC (Fig. 2). Since D. bardawil/salina have two distinct class II PSY proteins (PSY-1, PSY-2) (Tran et al. 2009), it is still uncertain what is their function and whether one of these enzymes acts as the house-keeping PSY in the constitutive pathway and the other is induced only under stress and initiates the HCR. In our βC-PG proteome, we identified also two putative 9-cis/all-trans βC isomerases and demonstrated that these proteins catalyze the isomerization of all-trans-βC to 9-cis-βC (Davidi and Pick 2017). These results indicated that 9-cis-βC may be produced within βC-PG either by isomerization from all-trans-βC or, as proposed before, from 9-cis phytoene (Ben-Amotz et al. 1988; Shaish et al. 1990) (Fig. 2).

Ast accumulation in H. pluvialis

The induction of Ast accumulation in H. pluvialis by different stress conditions, including HL stress and ND, has been extensively studied and reviewed (Boussiba 2000; Wang 2003; Aflalo et al. 2007; Lemoine and Schoefs 2010).

Exposure to HL stress and/or to ND induces a robust response, revealed by changes in the morphology, motility and physiology of H. pluvialis: the green vegetative cells lose their flagella and motility and are converted to rounded motionless palmeloid cells, enclosed by a primary wall (PW) beneath the thick layers of the extracellular matrix. However, some H. pluvialis strains show flagellated cells only a short time after cell division, mostly in the form of palmeloid cells, also in their vegetative stage. These palmeloid cells gradually accumulate Ast and loose Chl, and change from green to brown and finally, under prolonged stress, to a thick-walled red cyst or aplanospore, filled with Ast-containing cytoplasmic lipid droplets (Ast-CLD), starch granules and a partly degraded chloroplast. The red aplanospore is surrounded by two impermeable layers: a trilaminar sheath which contains the sporopollenin like polymer algaenan and a secondary cell wall, which provide remarkable protection against physical and chemical damage (review by Shah et al. 2016). In parallel, the cells undergo also prominent changes in the organization and function of the photosynthetic system and in their metabolism. At the early stages of stress, photosynthesis persists, and the cells accumulate starch granules within the chloroplast, amounting to 63% (W/W) (Recht et al. 2012) and also start to accumulate lipid droplets in the cytoplasm. At subsequent stages of stress, photosynthetic activity is down-regulated, Chl level decreases, there is an increase in respiration and glycolysis activities, starch level partly decreases, whereas neutral lipids (TAG) and Ast accumulate in Ast-CLD in the cytoplasm (Recht et al. 2012; Solovchenko 2015; Chekanov et al. 2016; Wang et al. 2014a). Metabolomic and metabolic profiling analyses indicate that at this stage accumulated starch is partly degraded in synchrony with synthesis of FA, indicating formation of TAG from starch (Recht et al. 2014). Ast synthesis is kinetically correlated and coupled to TAG biosynthesis: inhibition of TAG accumulation by a FA biosynthesis inhibitor inhibits Ast accumulation, but not vice versa (Zhekisheva et al. 2005). This finding is not surprising because CLD serve as the sink for Ast accumulation in the form of Ast-ester.

One of the least understood stages of Ast biosynthesis concerns the coordination between the chloroplast and the CLD. It is well established that the first stages in Ast biosynthesis, until βC, take place in the chloroplast (Grünewald et al. 2001). The final two stages, namely introduction of two keto groups by βC-ketolase (BKT) and of two hydroxyl groups by βC-hydroxylase (CRTR-B), as well as the esterification of Ast with a FA to Ast-acyl-ester, occur in the CLD (Fig. 2; Fan et al. 1995; Grünewald et al. 2001). What is not clear is how is βC transferred from the chloroplast to CLD and how is the process controlled (Grünewald and Hagen 2001). BKT and CRTR-B are the rate-limiting steps in Ast accumulation and the activity of the esterase is also essential for Ast accumulation and deposition in Ast-CLD (reviewed in Solovchenko 2015; Shah et al. 2016). In a recent functional analysis of photosynthetic pigment-binding complexes of H. pluvialis, it was found that both PS-I and PS-II in induced cells contain small amounts of bound Ast and Ast-ester, which seem to partially replace βC and to destabilize PS-I and PS-II (Mascia et al. 2017). It is not clear if this plastidic Ast is synthesized within the plastids or is retro-transported from CLD into the plastids.

The transformation of H. pluvialis first to a palmeloid cell and finally to an aplanospore cyst, affords enhanced protection against diverse stress conditions, including excessive irradiation, oxidative stress and high NaCl. At the palmeloid stage, enhanced stress resistance is obtained by screening the photosynthetic pigments and by channeling of excess redox energy from the photosynthetic system, to avoid over-reduction, which can lead to photooxidative damage. This is achieved by decreasing of Chl level, by decreasing the level of Cyt b6f which down-regulates linear electron flow, by diverting excess electron flow to chlororespiration via the plastid terminal oxidase (PTOX) (Han et al. 2012; Wang et al. 2014a) and by channeling excessive redox energy and assimilated carbon for synthesis of starch and of neutral lipids (Recht et al. 2012). The massive accumulation of Ast, which peaks in the aplanospore stage, provides further protection against stress by two different mechanisms: First, it provides an internal sunscreen which protects the photosynthetic pigments from photooxidative damage by absorbing excessive light (Wang 2003; Wang et al. 2014a). The screening effectiveness is further enhanced by the light-induced migration of Ast-containing CLD to the periphery of the cell shielding the chloroplast (Peled et al. 2012). Ast may also have a second important function of protecting nuclear DNA against photodamage, as suggested before (Hagen et al. 1993; Wang 2003). In its natural habitat, Haematococcus is frequently exposed to drought and ND stresses and is transformed into aplanospores. However, once conditions become favorable for growth, the aplanospores germinate to produce 4–64 daughter cells. Notably, other microalgae which accumulate Car in CLD also undergo similar multimeric fission during recovery from stress. This multiple fission process is preceded by a unique single-DNA replication step for all daughter cells prior to mitosis, followed by one consecutive cytokinesis (Reinecke et al. 2018). This intensive DNA synthesis is vulnerable to photodamage by UV and to oxidative damage by ROS. Since Ast is not a UV-absorbing pigment, it cannot act as a direct screen against UV light, but it can screen the photosynthetic system against HL which generates ROS and it can directly quench photosynthetically generated ROS, thus protecting DNA. Moreover, in HL-exposed aplonospores, the concentration of Ast-CLD around the nucleus under low light in H. pluvialis (Wang 2003; Peled et al. 2012) brings the pigment into close vicinity with DNA, thus affording better protection against ROS. In correlation with this hypothesis is the finding that the Ast-rich aplanospores are tolerant to UV-B irradiation, and to UV-B-induced cell damage, which correlates with degradation of Ast (Kobayashi and Okada 2000).

Similarities and differences in Car accumulation between D. bardawil/salina and H. pluvialis

The similarities between the HCR in D. bardawil/salina and in H. pluvialis are quite striking (summarized in Fig. 3): Both microalgae accumulate Car under HL and/or ND (-N, -P, -S); the HCR in both algae is associated with growth arrest and inhibition of photosynthesis which are fully reversed under permissive growth conditions. In both algae, triggering the HCR involves ROS, since it can be mimicked by external ROS generators like hydrogen peroxide or Rose Bengal; in both species the accumulated Car serve to protect the photosynthetic system against photoinhibition by acting as a sunscreen against HL; and in both the pigment is accumulated within lipid droplets composed primarily of TAG.
Fig. 3

Similarities and differences between the HCR of D. bardawil and H. pluvialis. Arrows indicate the differences between D. bardawil (Db) and H. pluvialis (Hp)

Both βC and Ast accumulation are coupled to lipid biosynthesis, as inhibition of FA biosynthesis during the HCR inhibits Car accumulation. Yet, inhibition of Car biosynthesis does not inhibit TAG biosynthesis (Rabbani et al. 1998; Zhekisheva et al. 2005). There are also close similarities in the kinetics and metabolic interrelations between starch, TAG and Car biosynthesis in the two species: Starch precedes TAG accumulation and serves as the major carbon source for synthesis of FA and TAG (Recht et al. 2012, 2014; Pick and Avidan 2017), whereas TAG and Car accumulation are kinetically synchronized. A prominent metabolic change preceding TAG accumulation in both algae is the large increase in the level of acetyl-CoA, the key precursor in FA biosynthesis (Avidan et al. 2015; Su et al. 2014).

However, there are also major differences in the HCR and in Car accumulation between these species: First, the HCR in H. pluvialis is far more robust and involves major morphological changes, complete growth arrest and stop of motility and massive DNA synthesis in preparation for multinuclear cell division which are missing in Dunaliella. These differences reflect different strategies to resist stress: Whereas D. bardawil maintains its flagella and motility also under prolonged stress conditions, H. pluvialis loses its flagella and motility and encysts itself first in an Ast-producing palmella cell which finally develops into an Ast-haematocyst or aplanospore (reviewed in Shah et al. 2016). D. bardawil/salina may also form aplanospores, but under different stress conditions (low salt concentrations, no effect of light, accumulation of the ketocarotenoid canthaxanthin rather than βC), suggesting a different mechanism (Borowitzka and Huisman 1993). Once culture conditions return to normal, D. bardawil/salina cells lose their βC and divide vegetatively into two daughter cells, whereas H. pluvialis distributes the pigment between the newly formed 4–64 flagellated zoospores. Second, the Car accumulate in different cellular domains and structures: in D. bardawil/salina βC accumulates in tiny plastoglobules within the chloroplasts (βC-PG), whereas in H. pluvialis Ast accumulates in large cytoplasmic lipid droplets (Ast-CLD). Also, H. pluvialis, but not D. bardawil/salina, has the capability to reversibly translocate the Ast-CLD from the cell periphery and the center. βC-PG and CLD differ in their functions, as reflected by their different proteome compositions (Davidi et al. 2015). The biosynthetic pathway also differs: while the synthesis of phytoene takes place in the chloroplast membrane in both D. bardawil and H. pluvialis, its conversion to βC in D. bardawil under the inductive pathway, takes place in the βC-PG, whereas in H. pluvialis phytoene is converted to βC in the chloroplast and then transported into CLD where it is further converted into Ast (Fig. 2).

Phytoene synthase (PSY) and the HCR

Secondary Car accumulation is common in microalgae, higher plants, fungi and in some archaea and eubacteria (Sandmann 2002; Solovchenko and Neverov 2017).

The major rate-limiting step of Car biosynthesis in photosynthetic organisms is the formation of phytoene by PSY, which is generally recognized as the key regulatory enzyme in the process (reviewed in Cazzonelli and Pogson 2010; Nisar et al. 2015). PSY is one of the most conserved enzymes in Car biosynthesis throughout evolution, and a phylogenetic analysis can trace its evolution back from archaea and bacteria to higher plants (Sandmann 2002; Lao et al. 2011). However, the structural organization of PSY changed during the course of evolution (Han et al. 2015) and so did its photoregulation. Carotenogenic genes such as psy and pds in bacteria, fungi and green algae such as Chlamydomonas reinhardtii are photoregulated by blue light (Fig. 4; Schmidhauser et al. 1994; Botella et al. 1995; Bohne and Linden 2002). In contrast, in higher plants, PSY is upregulated by red light, through phytochrome photoreceptors, via phytochrome-interacting factors (Toledo-Ortiz et al. 2010). Although phytochrome signaling was observed also in some algae species, it is absent from most Chlorophyte algae genomes (Duanmu et al. 2014). The HCR to HL stress in algae such as D. bardawil and H. pluvialis, seem to be controlled by distinct stress-responsive elements, which probably evolved independently, as indicated by their different gene promoters and different gene structures and organization, as will be described below (Botella et al. 1995; Liang and Jiang 2017).
Fig. 4

Proposed evolutionary origin of carotenogenesis in D. bardawil and in H. pluvialis. The proposed first function of secondary Car (βC) in green algae is in eyespot lipid droplets, which is part of the cell visual system. Phytoene synthase (PSY), which controls the synthesis of Car, was originally controlled by blue light (blue arrowhead). A critical stage in the evolution of the HCR to HL stress (yellow arrowhead) was a mutation in psy, which elevated its transcription level in response to HL. In D. bardawil (left), accumulation of βC–PG evolved by disintegration and amplification of eyespot plastoglobules. In H. pluvialis (right), formation of Ast-CLD evolved by acquisition of a Car transporter in chloroplast envelope membranes and by insertion of BKT, CRTR-B and Ast-TA (Ast-TA) into CLD

Several lines of evidence suggest that PSY is indeed the major regulator controlling Car biosynthesis: First, PSY catalyzes the first committed step in the biosynthesis of Car from geranyl–geranyl-diphosphate (GGDP) to phytoene. PSY is subject to regulation by diverse environmental and developmental factors, including light, in both green algae and in higher plants and the regulation is mostly at the transcriptional level (reviewed in Cazzonelli and Pogson 2010). Second, overexpression of inserted psy genes, in both green algae and in higher plants, was shown to enhance and control Car accumulation (Couso et al. 2011; Chen et al. 2017; Paine et al. 2005). Higher plants have several psy genes that are expressed in different tissues and are differentially controlled, regulating primary and secondary Car biosynthesis (Li et al. 2009; López-Emparán et al. 2014; Fu et al. 2014; Wang et al. 2014b; Han et al. 2015). For example, of the three PSY paralogs in maize, PSY3 regulates abiotic stress-induced carotenogenesis in roots (Li et al. 2008), whereas PSY1 controls the developmentally regulated carotenogenesis in maize endosperm (Li et al. 2008). Most green algae have just one psy gene’; however, a few microalgae, including some green algae, were reported to have two or even multiple forms of psy genes (Tran et al. 2009). Notably, D. salina/bardawil encode at least two distinct class II psy genes, which may represent differentially regulated enzymes (Tran et al. 2009). Protein sequence homology analyses of D. bardawil PSY with other green algal PSY shows that the sequence of the C-terminus is relatively conserved, whereas the N-terminus is more variable indicating that the latter may be involved in the evolution of the HCR (Tran et al. 2009; Liang and Jiang 2017).

Recent studies are starting to elucidate the molecular details of the regulation of PSY: Analysis of the promoter region of D. bardawil psy identified salt-regulated elements (SRE), light-regulated elements and W-box cis-acting elements. The latter are known to bind to WRKY transcription factors, which have also been identified, and were shown to be upregulated under salt stress (Lao et al. 2011; Liang and Jiang 2017). Notably, this study identified light-regulated elements and W-box cis-acting elements in seven different genes involved in βC biosynthesis in this alga. Similarly, in H. pluvialis the psy gene promoter region also contains cis-regulatory elements potentially involved in stress regulation (C-repeat/DRE, abscisic acid-regulatory elements, ethylene-regulatory elements, W-box elements) and light regulatory elements (I-box, GC-box, TCCC-box) (Liang et al. 2006). These findings are consistent with the similar responsiveness of PSY to high salt and to HL stresses in D. bardawil and in H. pluvialis. In higher plants, the multiple forms of PSY are differentially regulated both transcriptionally (as described above) or post-transcriptionally via the ORANGE protein (OR), a regulator of chromoplast differentiation, which binds and activates PSY (Zhou et al. 2015).

Origin of the HCR in green algae

Eyespot plastoglobules

The original functions of Car in predecessors of green algae were probably to serve as accessory light-harvesting pigments in photosynthetic antenna proteins as discussed above (Fig. 4). However, the origin of secondary Car accumulation is not clear. Plastoglobules were most probably the first organelles that accumulated βC, since they are present in all microalgae and in higher plants and function as versatile biosynthesis and shuttling organelles of lipids and of pigments including Car in the chloroplast (Bréhélin et al. 2007; Rottet et al. 2015). Notably, plastoglobules also have a high storage capacity for βC. Green algae have a specialized form of plastoglobules that store βC in the eyespot. The eyespot is part of the visual system in green algae and combined with rhodopsin photoreceptors it activates the flagella and controls motility in green algae. The eyespot consists of two to three layers of lipid globules squeezed between chloroplast membranes in close proximity to the photoreceptors in the plasma membrane (Boyd et al. 2011; Kreimer 2009). The eyespot lipid globules are specialized forms of plastoglobules which resemble plastoglobules in both higher plants and in microalgae as indicated from their similar proteome compositions (Schmidt et al. 2006). The eyespot enabled both the phototactic response to weak light and the photophobic response of avoiding excessive irradiation, which are essential for survival in photoautotrophic organisms. Because the eyespot is common to all green algae, it probably originated early in the evolution of this family. Therefore, it is possible that the initial primary function of accumulated secondary Car in green algae was to create the eyespot to improve photosynthetic performance and to enhance the chances of survival. This stage in evolution should be common to all green algae (Fig. 4).

The hypercarotenogenic response (HCR)

The HCR to HL/ND and to nutrient deprivation in H. pluvialis is more robust than in D. bardawil/salina and involves drastic morphological changes and loss of motility as discussed above. Some other species of green algae as the “snow algae” C. nivalis, Scenedesmus strains and P. incisa (Table 1), can also accumulate carotenoids in CLD and resemble H. pluvialis in their robust HCR response. However, most green algae, including C. reinhardtii, do not accumulate secondary carotenoids, although some species, such as C. reinhardtii, seem to have the potential for the HCR, since it possess a functional bkt gene (Huang et al. 2012). Both H. pluvialis and D. bardawil/salina have in common a PSY that responds to HL/ND stress. We propose that a mutation in psy made it hyper-sensitive to HL, leading to enhanced psy transcription resulting in enhanced phytoene biosynthesis and in βC accumulation (Fig. 4). This could be brought about by a mutation in the psy gene promoter, such as introduction of a W-box cis-acting element enabling the interaction with a stress-activated WRKY transcription factor. This possibility is consistent with the central role of PSY in the control of Car biosynthesis and with the presence of light regulatory elements and of W-box cis-acting elements in the psy gene promoter and of stress-activated WRKY transcription factors in both D. bardawil and in H. pluvialis (Liang et al. 2006; Li and Zhang 2015; Liang and Jiang 2017).

Evolution of βC-PG in D. bardawil/salina

In a recent proteome analysis of βC-PG, in which we compared the proteome of βC-PG from D. bardawil to proteomes of other lipid droplets in plants and in algae, we found that βC-PG have the highest similarity to C. reinhardtii eyespot proteome, but minor similarities to proteomes of CLD in algae and in higher plants, indicating a different origin (Davidi et al. 2015; Lundquist et al. 2012; Nguyen et al. 2011; Schmidt et al. 2006). Out of 124 proteins in βC-PG, 40 are homologous to the proteome of eyespot lipid globules compared to 18 in plastoglobules of A. thaliana, ten of which are common to all three proteomes (Fig. 5a). A good example for the differences in proteomes of plastoglobules and CLD are lipid droplet structural proteins, which are highly divergent in structure, and as such are good indicators for evolutionary distances. For example, the major CLD protein in green algae, MLDP, has no homology to oleosins and to caleosins, the major CLD proteins in higher plants (Davidi and Pick 2012). In contrast, the major structural proteins in D. bardawil βC-PG, carotene globule protein (CGP) and PAP-fibrillins, have similar ortholog proteins in plastoglobules of other algae and higher plants (Fig. 5b, c). The similarities between D. bardawil βC-PG and the eyespot proteomes are quite striking: both proteomes contain similar βC biosynthesis enzymes, indicating that βC is produced within them, as well as eyespot-specific proteins such as EYE3. Interestingly, the βC in the eyespot of green algae is composed of 9-cis and of all-trans isomers, similar to βC-PG in D. bardawil (Kreimer 2009). Based on these results, we have proposed that βC-PG evolved from disintegration and amplification the eyespot lipid droplets structure (Fig. 4). The first step in this evolution, namely disintegration of the eyespot structure, may have evolved from a mutation in one of the scaffolding proteins in the eyespot that holds together the lipid droplets. For example, mutation in MIN1 or in EYE2, which in C. reinhardtii are essential for the association of eyespot globules (Lamb et al. 1999), could have led to disintegration of the eyespot in D. bardawil/salina. Following that, continuous exposure to HL and/or to ND could provide the evolutionary pressure for amplification of βC-PG, to effectively screen the photosynthetic system, affording protection against photoinhibition (Fig. 4). In screening of D. bardawil chloroplast proteome (Davidi et al. 2015), we did not identify any peptides related to MIN1 nor EYE2, even though potential genes with partial homology to MIN1 and EYE2 are encoded in the genome of closely related D. salina (phytozome Dusal.3563s00001.1 and Dusal.2052s00001.1, respectively). It is possible, therefore, that MIN1 and EYE2 in D. bardawil were lost during evolution due to loss of function.
Fig. 5

The proteome of βC-PG from D. bardawil. a Similarities between the proteomes of D. bardawil βC-PG (brown), A. thaliana core PG (green) and C. reinhardtii eyespot lipid globules (cyan). Adapted from Table 4 in (Davidi et al. 2015). A. thaliana core proteome data are according to (Lundquist et al. 2012) and C. reinhardtii eyespot proteome data are from (Schmidt et al.(2006). Bold numbers indicate homologous proteins. b Phylogenetic tree of CGP compared to sequences form other green algae (Coccomyxa subellipsoidea, Chlamydomonas reinhardtii, Vovlox carteri, Ostreococcus lucimarinus, Chlorella variabilis) and plants (Arabidopsis thaliana). MLDP was added as reference. Sequences names followed by NCBI accession numbers: C. subellipsoidea (EIE18519.1), C. reinhardtii (XP_001691398.1), V. carteri (XP_002947474.1), O. lucimarinus (XP_001418356.1), C. variabilis (EFN56543.1), A. thaliana_PG (ABG48434.1), A. thaliana_Soul (NP_001190345.1). C. Phylogenetic tree of PAP-fibrillins in βC-PG together with proteins from Arabidopsis thaliana, Oryza sativa Chlamydomonas reinhardtii. Sequences names followed by NCBI accession no.: C. reinhardtii 1 (XP_001698259.1), C. reinhardtii 2 (XP_001693298.1), C. reinhardtii 3 (XP_001702245.1), C. reinhardtii 4 (XP_001698968.1), C. reinhardtii 5 (XP_001698965.1), C. reinhardtii 6 (XP_001692028.1), C. reinhardtii 7 (XP_001690132.1), FBN1a_A. thaliana (AT4G04020.1), FBN1b_A. thaliana (AT4G22240.1), FBN2_A. thaliana (AT2G35490.1), FBN4_A. thaliana (AT3G23400.1), FBN7a_A. thaliana (AT3G58010.1),FBN7b_A. thaliana (AT2G42130.4), FBN8_A. thaliana (AT2G46910.1), O. sativa 1 (NP_001054180.1). O. sativa 2 (EEE61457.1), O. sativa 3 (NP_001068210.1), O. sativa 4 (Q7XBW5.1), O. sativa 5 (AAO72593.1), O. sativa 6 (EEE51252.1), D. bardawil 1 (isotig09899), D. bardawil 2 (isotig15498), D. bardawil 3 (isotig16228), D. bardawil 4 (isotig04183.2). Adapted from Fig. 4 in Davidi et al. (2015, no permission required)

Interestingly, D. bardawil does not have an eyespot structure, in contrast to green Dunaliella species such as D. tertiolecta, which do have a clear eyespot structure similar to other green algae (Kreimer 2009). This finding is consistent with the idea that the eyespot in predecessors of D. bardawil disintegrated and proliferated to shield the photosynthetic system against photoinhibition.

bardawil/salina versus H. pluvialis

The remarkable similarities in the HCR responses between D. bardawil/salina and H. pluvialis resulting in massive accumulation of Car in lipid droplets indicates a common origin. The predicted first stage in this evolution may have been a mutation in psy gene which resulted in enhanced biosynthesis of phytoene leading to accumulation of βC as discussed above.

The major difference in Car accumulation between Dunaliella and Haematococcus is their intracellular localization: plastoglobules in Dunaliella and CLD in Haematococcus. Accumulation of massive amounts of carotenoids in the cytoplasm is advantageous relative to the chloroplast, because of the much larger capacity to accumulate oil, the sink for carotenoid accumulation, and also because it further separates the secondary carotenoids from the photosynthetic system, decreasing the danger of interference in the synthesis of primary carotenoids. Therefore, the question arises why does Dunaliella accumulate βC within the chloroplast and not in CLD? The simplest reason may be that Dunaliella lacks the capacity to transport βC out of the chloroplast. Maybe because of this, Dunaliella evolved an alternative solution of amplification of the eyespot lipid globules, which already possessed βC and the enzymatic machinery to produce it.

In contrast, H. pluvialis and other algae species that accumulate Car in CLD had to undergo a more complicated evolutionary track that included acquisition of the capacity to export carotenoids out of the chloroplast and their enzymatic conversion into Ast and other oxygenated carotenoids.

Origin of Ast-CLD in H. pluvialis

The accumulation of Ast in CLD in H. pluvialis seems to represent a more advanced evolutionary stage than βC accumulation in D. bardawil/salina since it includes three additional elements that are missing in Dunaliella: the capacity to transport βC from the chloroplast to extra-plastidic lipid bodies, the enzymatic conversion of βC to Ast and the development of a strong antioxidative capacity conferred by Ast. The ability to mobilize Ast in response to light intensity changes (Peled et al. 2012) confers H. pluvialis double protection against stress: screening of the photosynthetic system from HL, similar to βC in Dunaliella, and protection of the nucleus against oxidative damage, which are missing in Dunaliella (see below, in “Why βC? Why Ast?”). Secondary Car accumulation in CLD is not unique to H. pluvialis, and as was mentioned above, is common in several other green algae species, although at lower levels (Table 1). The light-dependent mobilization of Ast-CLD has been described so far only in H. pluvialis. Recent studies about Car accumulation in C. zofingiensis may be relevant to some of the open questions concerning the HCR in H. pluvialis (Takichi 2011; Roth et al. 2017). The genome of C. zofingiensis contains two copies of BKT and one CRTR-B, which by PredAlgo are predicted to be located outside the chloroplast, possibly in the cytoplasm (Roth et al. 2017). This can support the hypothesis that conversion of βC to Ast takes place outside the chloroplast also in this alga.

The critical stages in this evolution were the transfer of βC from the chloroplast to CLD, the integration of xanthophyll biosynthetic enzymes and amplification.

The ability to transport βC out of the chloroplast may be the major step in the evolution of the HCR response and the reason why D. bardawil/salina, lacking this capacity, accumulate carotenoids in plastoglobules, whereas H. pluvialis and other green algae accumulate them in CLD.

It is still not clear how is βC transported from the chloroplast into CLD. This requires transport through the chloroplast membranes and next incorporation into CLD. Transport of βC through membranes in other biological systems depends on a dedicated membrane transporter: In mammalian cells, βC uptake in intestinal cells is mediated by the scavenger receptor class B type I (SR-BI) protein transporter (During and Harrison 2007; Reboul 2013). Therefore, it may be expected that also the transport of βC in plants and in algae should be mediated by a specific protein transporter. In a recent transcriptome analysis made in C. zofingiensis, several genes encoding putative ABC-type transporters were found to be upregulated under carotenogenic stress conditions (Roth et al. 2017). It is possible that such proteins mediate transport of βC through the chloroplast envelope membranes. Therefore, we propose that the next event in the evolution of carotenogenesis in Car-CLD accumulating algae was the acquisition of a Car transporter (Fig. 4). The incorporation of βC into CLD after its export from the chloroplast may occur by diffusion through direct contacts between CLD and the chloroplast. Close associations between the outer chloroplast envelope membrane and CLD have been reported in many green algae including our own studies in H. pluvialis and D. bardawil (Peled et al. 2012; Davidi et al. 2014). Next, assembly of BKT and CRTR-B and finally Ast-acyltransferase (Ast-AT) with CLD could enable the conversion of βC into Ast-ester. Finally, prolonged exposure to HL and/or to ND could provide the evolutionary pressure for amplification of this process, to provide effective protection to the photosynthetic system against photoinhibition as discussed above for D. bardawil/salina (Fig. 4).

Why βC? Why Ast?

An interesting open question that comes to mind is why does D. bardawil accumulate βC, whereas H. pluvialis accumulates Ast, since both serve the same major function of screening the photosynthetic system against HL. Does Ast have any advantage over βC in screening the photosynthetic system that justifies the metabolic and energetic cost of conversion of βC into Ast? And if so, why does not D. bardawil/salina also accumulate Ast? In fact, D. bardawil/salina could benefit from accumulation of Ast in plastoglobules more than H. pluvialis, since the physical proximity of Ast plastoglobules to the photosynthetic system may afford significant protection against ROS, unlike the distant Ast-CLD in H. pluvialis.

The simplest reason why D. bardawil/salina accumulates βC and not Ast may be that it cannot transport βC out of the chloroplast and convert it to Ast in CLD as discussed above. But in fact, Dunaliella salina genome contains a bkt gene (Dusal.0138s00018), indicating that Dunaliella should have the capacity to convert βC into Ast. Why does it not happen in the plastoglobules? A possible reason for the preference of βC may be a better packaging efficiency in plastoglobules. D. bardawil accumulates a surprisingly high concentration of pigment in a small amount of oil compared to H. pluvialis. The calculated concentration of βC in plastoglobules is approximately 50% (w/w) (Ben Amotz et al. 1982; Davidi et al. 2015), whereas the concentration of Ast in CLD is at most 12%. βC is indeed more soluble than oxygenated carotenoids in apolar organic solvents such as hexane, but less soluble in polar organic solvents (Craft and Soares 1992). The very high solubility of βC in the plastoglobules may be enabled by the high lipid solubility of 9-cis βC isomer (Zechmeister 1962; supplement S3). Thus, the selection of βC as the light-screening pigment in D. bardawil/salina may have been dictated by the limiting ability for neutral lipid accumulation within the chloroplast.

The choice of Ast as the screening pigment in H. pluvialis and in most other microalgae that accumulate Car in CLD (Table 1) may be related to its high antioxidative activity, affording potential protection against damage of DNA, which may be essential for the unique multinuclear fission of the aplanospore into multiple zoospores, which involves active DNA synthesis, as discussed above. Thus, Ast provides both an effective screen against HL and can also afford effective protection against photooxidative damage due to its excellent ROS scavenging capacity. The light-induced migration of Ast-CLD in H. pluvialis (Peled et al. 2012) further enhances screening against HL and protection against ROS at low light. A second function of Ast may be to improve mitochondrial function by maintaining the optimal redox state in mitochondria (Wolf et al. 2009), further contributing to energy production during germination. A third possible reason for favoring Ast accumulation over that of βC may be to avoid destabilization of membranes by high levels of βC in the cytoplasm. Several studies have shown that βC can disorganize and destabilize the structure and dynamics of model membranes, whereas oxygenated Car, such as Ast, have an opposite effect, increasing the order and stability of membranes (Strzalka and Gruszecki 1994; Gruszecki and Strzalka 2005). Thus, it is possible that the proximity of βC in the cytoplasm to the plasma membrane and to the endoplasmic reticulum membranes creates significant threats to the cell integrity which justifies their conversion to the less toxic Ast.

Summary

In this brief review we propose a new hypothesis about the evolution of the HCR in green algae. Based on the central role of PSY in the regulation of carotenogenesis, we propose that the first stage in the evolution was a mutation that made PSY hyper-responsive to HL enabling massive Car biosynthesis. Next there was a split in evolution: Algae that acquired the capacity to export carotenoids out of the chloroplast accumulated βC within CLD and further metabolized it into Ast or to other xanthophylls, whereas D. bardawil/salina, missing the capacity to export βC, amplified eyespot plastoglobules giving rise to multiple βC-PG within the chloroplast. Both pathways resulted in screening the photosynthetic system against HL protecting the cells against photoinhibition.

Author contribution statement

UP organized and wrote most of the manuscript. LD prepared the figures and added part of the text. SB and AZ wrote the sections on H. pluvialis and contributed to the writing of other sections.

Notes

Acknowledgements

We wish to thank Dr. Aviv Shaish, The Bert W. Strassburger Lipid Center, Sheba Medical Center, Tel-Hashomer in Israel for critical comments and helpful discussions.

Supplementary material

425_2018_3050_MOESM1_ESM.docx (17 kb)
Supplementary material 1 (DOCX 17 kb)
425_2018_3050_MOESM2_ESM.pptx (116 kb)
Supplementary material 2 (PPTX 115 kb)

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© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biomolecular SciencesThe Weizmann Institute of ScienceRehovotIsrael
  2. 2.Microalgal Biotechnology Laboratory, French Associates Institute for Agriculture and Biotechnology of Drylands, The Jacob Blaustein Institutes for Desert ResearchBen-Gurion University of the NegevBeer-ShevaIsrael
  3. 3.Department of Chemistry and BiochemistryUniversity of CaliforniaLos AngelesUSA

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