EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

Original article:

THE : AN EARLY REQUISITE FOR EFFICIENT IN

Niraj Kumar Singh1,$, Ravi Raghav Sonani2,$, Rajesh Prasad Rastogi2,*, Datta Madamwar2,*

1 Shri A. N. Patel PG Institute (M. B. Patel Science College Campus), Anand, Sardargunj, Anand – 388001, Gujarat, India 2 BRD School of Biosciences, Sardar Patel Maidan, Vadtal Road, Post Box No. 39, Sardar Patel University, Vallabh Vidyanagar 388 120, Anand, Gujarat, India

* Corresponding authors: Tel.: +91 02692 229380; fax: +91 02692 231042/236475; E-mail addresses: [email protected] (R.P. Rastogi), [email protected] (D. Madamwar) $ These authors have contributed equally. http://dx.doi.org/10.17179/excli2014-723

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/).

ABSTRACT Cyanobacteria trap light energy by arrays of pigment molecules termed “phycobilisomes (PBSs)”, organized proximal to "reaction centers" at which perform the energy transduction steps with highest quantum efficiency. PBSs, composed of sequential assembly of various chromophorylated (PBPs), as well as nonchromophoric, basic and hydrophobic polypeptides called linkers. Atomic resolution structure of PBP is a heterodimer of two structurally related polypeptides but distinct specialised polypeptides- α and β, made up of seven alpha-helices each which played a crucial step in evolution of PBPs. PBPs carry out various light de- pendent responses such as complementary chromatic adaptation. The aim of this review is to summarize and discuss the recent progress in this field and to highlight the new and the questions that remain unresolved.

Keywords: protein, phycobiliproteins, , linker polypeptide, complementary chromatic adaptation

Abbreviations: PBS – ; PBP – ; LHC – Light harvesting complex; PE – Phyco- erythrin; PC – ; AP – ; F-αCPE – Fragmented α PE; PEB – ; PCB – ; PUB – ; PVB – Phycoviolobilin; RC – Reaction center; CCA - Comple- mentary chromatic adaptation

INTRODUCTION credited for the evolution of existing aerobic life on the Earth’s surface due to their inher- Cyanobacteria are the oldest group of ent capacity of photosynthesis mediated ox- oxygenic prokaryotes in the history of photo- ygen evolution (Olson, 2006). Adaptive di- synthetic life having cosmopolitan distribu- versification of cyanophyceae to optimize tion in both aquatic and terrestrial ecosys- their oxygenic photosynthesis in a range of tems (Whitton, 2012). They are most im- ecological niches over the last 3.5 billion portant component of photoautotrophic mi- years (Fischer, 2008; Feyziyev, 2010) has croflora in terms of total biomass and led to the evolution of a large and diverse productivity, maintaining the trophic energy dynamics of an ecosystem (Iturriaga and array of photosynthetic pigments, each with Mitchell, 1986; Stock et al., 2014). They are

268 EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

specialized functions to compete them suc- Phycobilisomes cessfully on the planet. The PBSs are supramolecular complex The oxygenic photosynthetic organisms composed of a core substructure and peri- such as cyanobacteria contain two distinct pheral rods, and account for up to 60 % of reaction centers (RCs) i.e., P700 and P680 of the total protein mass in cyanobacteria. The photosystem I (PSI) and photosystem II molecular weight of PBS varies from 7 to 15 (PSII), respectively. Each RC is associated MDa which is far larger than the reaction with an antenna of light-harvesting protein- centers i.e., PSI and PSII (Mullineaux, 2008) pigment complexes (LHC) (Bearden and and utilizes 30-50 % of the total light- Malkin, 1975). (Chl a) is the harvesting capacity of cyanobacterial and red universal reaction center pigment biomole- algal cells and transfers the energy to reac- cule in oxygen-evolving organisms. In high- tion centers (Wang et al., 1977). Apart from er plants and green algae, the major LHC light absorption and transduction, an addi- consists of membrane associated Chl a/b- tional function of the PBS so far identified is binding (Cab) proteins which are integral as an emergency source of nutrients in case membrane proteins, arranged in trimeric ar- of nitrogen, sulfur or carbon starvation rays (Kuhlbrandt and Wang, 1991). In ma- (Parmar et al., 2011). Ordered disassembly rine algae such as diatoms, dinoflagellates, of the PBS complex during starvation re- brown algae, and chrysophytes, the major quires the presence of the products of a light-harvesting complex contains xantho- number of non-bleaching (nbl) genes phylls, such as fucoxanthin or peridinin, Chl (Grossman et al., 2001). However, different a and Chl c. The LHC of cyanobacteria, eu- disassembly pathways may occur in different karyotic , cryptophytes and glauco- organisms. Soni et al. (2010) identified a 14 phytes consist of Chl a and phycobilisomes kDa truncated fragment of α-subunit of phy- (PBSs). The PBSs are light harvesting an- coerythrin (F-αPE) from the degraded and tennae of cyanobacterial photosystem which stored samples of 19 kDa protein of C-phy- consist of key photosynthetic pigment mole- coerythrin (C-PE). Even so, the specific cules, the phycobiliproteins (PBPs). These mechanism of nbl-A or nbl-B gene products intensely colored are homoge- is still unclear (Baier et al., 2001). nous family of light harvesting proteins which absorb visible light in the range of 450 Phycobilisome morphology to 670 nm. They transfer excitation energy The structure and size of PBSs vary with high quantum efficiency to photosystem widely. Three classes of structures have been II and I in the photosynthetic lamellae described to date: (a) hemi-discoidal phyco- (MacColl, 1998). Quality of incident radia- bilisomes; so far seen in red algae, cyanobac- tion and efficient energy transfer involved in teria, and certain cyanelles; (b) "rod bundle" light harvesting evolve PBSs to gain the abil- shaped phycobilisomes - represented until ity to survive in broad range of environments now only by those of the thylakoid-less cya- including different depths of sea and fresh nobacterium Gloeobacter violaceus (Gysi water. Many cyanobacteria have shown a and Zuber, 1974) and (c) hemi-ellipsoidal; so tendency to respond to a change in light far seen only in red algae. Hemi-ellipsoidal quality by changing the composition of their phycobilisomes have molecular weights of antenna in such a manner that it increases the 20 MDa, whereas others are in range of 5-8 proportion of the pigment with absorption MDa (MacColl, 1998). The family of exten- spectrum more nearly complementary to the sively found hemidiscoidal PBSs has been available light (Kehoe, 2010). further divided into three sub-groups accord- ing to structural differences of the core do- main (Sidler, 1994). This domain is com- posed of 2, 3 or 5 cylinders (Figure 1) of

269 EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

stacked allophycocyanin trimers (α3β3), with tra cylinders having only two discs (Figure a diameter of about 110-117 Å of each and 1). A series of 6 to 8 rods radiate from the its length is given by the stacking of two to core which is composed of cylinders of four discs of thickness ~30 Å each (Arteni et stacked hexameric (α3β3)2 discs. Each rod al., 2009). Each of the cylinders belonging to consists of two to four discs having diameter the core substructure is composed of four 110-120 Å and thickness 60 Å each (Adir, trimeric allophycocyanin (AP) discs. How- 2005) (Figure 2). ever, pentacylindrical core contains two ex-

Figure 1: Schematic representation of the three types of hemi-discoidal PBS. a, PBS with bicylindrical core. Bicylindrical core consists of two identical asymmetric cylinders arranged in anti-parallel manner, which is made up of four allophycocyanin trimer disk, arranged in sequence of T8-T-M-B8; b, PBS with tricylindrical core. Tricylindrical core consists of two types (asymmetric T8-T-M-B8 and symmetric T8-T-T-T8) of allophycocyanin cylinders. Two similar types of cylinders are arranged in same manner as in bicylindrical core, whereas third symmetric cylinder (T8-T-T-T8) is arranged on them as shown in figure; c. PBS with pentacylindrical core. Pentacylindrical core contains two extra asymmetric half- cylinders (only two trimer disks- T-T8) beside the symmetric cylinder as shown in figure, oriented in anti-parallel manner to each other. T8-disk is made up of α- and β-subunits of allophycocyanin and 8.9 core linker peptide (LC ); T-disk contains an α- and β-subunit of allophycocyanin without any linker peptide; M-disk is composed of α-, β- and variant β-(16 kDa)subunits of allophycocyanin and core- 72-127 membrane linker peptide (LCM ). B8-disk comprises of α-, variant α- and β-subunits of allophycocy- 8.9 anin and core linker peptide (LC ).

270 EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

Figure 2: Structural model of a hemi-discoidal PBS, as seen from the thylakoid membrane upwards. The architecture exhibits a tri-cylindrical core, from which six rods composed of PC and PE hexamers radiate outwards.

The rods from various organisms are types of cores present in Mastigocladus lam- quite variable in the composition with num- inosus and Anabaena sp. PCC7120, Syn- ber of discs and the ratio of PE to PC, de- echoccous sp PCC7942 (Kaňa et al., 2009) pending on the growth environment (Lüder are less common (Figure 1c). Due to lack of et al., 2001, 2002). Single particle microsco- integral membrane domain in PBS, it diffus- py studies showed that the interaction be- es through the cytoplasm and interacts with tween AP trimer is looser than that of PC thylakoid membrane. Diffusion reflects fluid trimmers forming hexamers (Arteni et al., nature of PBS, which is influenced by their 2009). The PBSs containing tri-cylindrical size but not by temperature (Kaňa, 2013). cores are widely spread in cyanobacteria such as Synechocystis sp. PCC6803 (Re- Phycobiliproteins decker et al., 1993). Tri-cylindrical cores PBSs are composed of - consist of two anti-parallel basal cylinders associated water soluble acidic polypeptides and one upper cylinder with internal two- called PBPs and nonchromophoric basic fold symmetry (Arteni et al., 2009) (Figure polypeptides called linker proteins that are 1b). Whereas bi-cylindrical cores contain hydrophobic. PBPs function as the principal only cylinders arranged anti-parallel to each photoreceptor in cyanobacteria and some other (Lundell and Glazer, 1983) (Figure 1a). other algae (Chaiklahan et al., 2011). PBPs The penta-cylindrical cores consist of two are linked to characteristic anti-parallel basal cylinders, one upper cyl- chromophores called bilins, which are cova- inder with internal two-fold symmetry and lently bound to the apoprotein via one or two two anti-parallel additional cylinders. These thioether bonds (Scheer and Zhao, 2008).

271 EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

PBPs may comprise more than 60 % of the (PE – bright pink - λmax 540-570 nm), (2) total soluble cellular protein fraction, which intermediate energy-phycocyanin (PC – dark is equivalent to almost 20 % of the total dry cobalt blue – λmax 610-620 nm), and (3) low weight of cyanobacteria (Soni et al., 2008). energy-allophycocyanin (AP – brighter aqua The assembly of PBS is initiated with chro- blue - λmax 650-655 nm) (Moraes and Kalil, mophorylation of PBPs, followed by methyl- 2009; Sonani et al., 2014a, b) (Figure 3). The ation and oligomerization, and linker protein visible absorption spectra of the biliproteins insertion (Miller et al., 2008). PBS assembly are determined by the open-chain tetra- and architecture are driven to facilitate ener- pyrrole chromophore present and by the en- gy transfer from higher to lower energy state. vironment in which they are located. Interac- On a functional level, PBPs are a group tion of chromophore, apoprotein, nonchro- of intensely colored proteins that occur in mophoric linker polypeptide and level of or- cyanobacteria and other algae. Based on their ganization makes PBS especially suitable for colour, absorption maxima and energy the energy absorption, transfer and funneling PBPs can be sub-divided into three main to the chlorophyll-containing reaction cen- groups i.e., (1) High energy- ters within the membrane.

A 0.6 APC 0.5 PE PC 0.4

0.3

Absorbance 0.2

0.1

0 250 350 450 550 650 750 Wavelength (nm)

B 0.25 0

APC

3 PC 0 0.2 x 10 PE 0 Figure 3: UV-visible (A) and fluorescence spectra 0.15 0 (B) of purified phyco- erythrin, phycocyanin, allo- phycocyanin isolated from 0 0.1 Lyngbya sp. A09DM. Phy- coerythrin, phycocyanin 0 and allophycocyanin were Fluorescence Intensity Intensity Fluorescence 0.05 exited at 559, 589, 645 nm 0 respectively, to measure their fluorescence emission spectra. 0 0 400 450 500 550 600 650 700 750 800 Wavelength (nm)

272 EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

PBPs are typically composed of various ic complexes along with chromophores at- aggregates of αβ where α and β are homolo- tached to them. Each of α- and β-subunits of gous polypeptide chains (or ‘subunits’) the PBP complex are generally attached to which are encoded by genes usually present two bilin chromophores. In most PE contain- in an operon, with molecular masses in the ing cyanobacterial strains isolated to date, range of 12–20 and 15–20 kDa, respectively the distal part of the PBP rod is composed of (Singh et al., 2009). The α- and β-subunits of two types of PE (PE I and PE II). PE I binds PBPs are uniform in length with 160 to 180 either only to phycoerythrobilin (PEB) or to amino acid residues, respectively. X-ray both PEB and phycourobilin (PUB) (Amax = crystallographic studies have shown that the 495 nm) whereas, PE II always binds to both fold of α and β subunit of PBPs are a well- PEB and PUB. The PE can be classified into defined helical globin-like domain with sev- four classes: C-PE (cyanobacterial PE), B- en helices (A, B, E, Fʹ, F, G and H) and heli- PE (Bangiophyceae PE, containing PEB on- cal hairpin domains (X and Y) at the N- ly), B-PE (Bangiophyceae PE, containing teminus of each chain which are responsible PEB and PUB) and R-PE (Rhodophyta PE), for providing the stability of (α/β) monomers based on their origins and absorption proper- (Kikuchi et al., 2000). These subunits along ties (Gantt and Lipschultz, 1974). The C-PE, with bilins, oligomerize themselves to form which also assembles to discs, has α-poly- hexameric and trimeric discs which further peptide with two chromophore and β with participate in the construction of rod and three (Table 1). Pan et al. (1986) have re- core. The different absorption and emission searched the spectral properties of PEs in spectra of the consecutive disc ensures a rap- marine red algae and reported that R-PEs id flow of energy from the donor bilins to the have two spectral types i.e., ‘‘two-peak’ type acceptor bilins. Position of PBPs and the in- and “three-peak” type (Pan et al., 1986). fluence of the linker polypeptide contribute Soni et al. (2010), Parmar et al. (2011) and to rapid directional flow of the excitation en- Sonani et al. (2015) have reported the 14 ergy from one acceptor bilin to the other kDa (fragmented-PE, F-PE) and 15.45 kDa moving towards the core. The organization (truncated-PE, T-PE) single subunit PE ge- of the core is not very well known, but fea- nerated due to in vivo and in vitro truncation. tures are similar to those seen in the rods Structural (Soni et al., 2010) as well as dena- governing the polar transfer of energy. Ami- turation kinetics (Sonani et al., 2015; Anwer no acid sequence data (Zuber, 1983) and et al., 2015) study revealed the maintenance DNA sequence data (Bryant et al., 1985) of F-PE and T-PE functionality even after shows considerable conservation of sequence the truncation. among the various PBPs (AP, PC and PE) as well as between α- and β-subunits of a given Phycocyanin PBP. These findings suggest that PBP genes Phycocyanin is found in complex of tri- arose via duplications of a single ancestral mers (α β ), hexamers (α β ) and other oli- sequence. It has also been suggested that an 3 3 3 3 2 gomers plus chromophore attached to them, insertion of short DNA-fragments plays a generally one to α- and two to β-subunits. role in the evolution of the β-subunit ances- Trimers (α β ) are ring-like assemblies of tral gene from that for α-subunit (Zhao and 3 3 three monomers (αβ) with a three-fold sym- Qin, 2006). Another way of PBP diversity is metry. The hexamers (α β ) are disc-shaped due to variation in the structure of the chro- 3 3 2 and formed by face-to-face assembly of tri- mophore (bilin) prosthetic groups. mers. Rods are formed by face-to-face as-

sembly of these discs. In most of the PE con- Phycoerythrin taining marine cyanobacterial species char- Phycoerythrin (PE) occurs as a trimeric acterized so far, PC makes up the basal disc (α3β3), hexameric (α3β3)2 and other oligomer- at the core proximal end of the rod. There are

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B many types of phycocyanin depending on the Lcm and α biliproteins receive energy from type of attached chromophore, found in cya- AP and energy from them is transferred to nobacteria like C-PC (contain only PCB chlorophyll in the thylakoid membrane. (λmax: 620 nm)), R-PC II (contain both PEB These two biliproteins have emission maxi- (λmax: 540 nm)) and PCB in the molar ratio ma of about 680 nm, which allows them to of 2:1) and R-PC III (an optically unusual transfer energy efficiently to chlorophyll PC that contains both PEB and PCB in the (MacColl, 2004). Cyanobacteria also contain molar ratio of 1:2) (Six et al., 2007a) (Ta- orange protein (OCP) to protect ble1). photosynthetic system against photo-oxidat- ion (Kirilovsky and Kerfeld, 2012). This Allophycocyanin process requires the binding of the red active Allophycocyanin (AP), organized as tri- form of the OCP, which can effectively mer (α3β3) at neutral pH, consists of three β- quench the excited state of one of the AP polypeptides; each of this polypeptide is as- bilins (Tian et al., 2012). However, Jallet et sociated with one of bilin, which forms the al. (2012) demonstrated that terminal emit- major building blocks of the core. The fluo- ters (ApcD, ApcF and ApcE) are not re- rescence properties of adjacent PC and ab- quired for the OCP-related fluorescence sorption properties of AP ensure that latter quenching and they strongly suggested that function as an efficient acceptor. Moreover, the site of quenching is one of the AP trimers there are three other core proteins with emitting at 660 nm. 16 B chromophores, namely Lcm, β and α . The

Table 1: Subunit structure, type, number and location of chromospheres on the Phycobiliprotein Biliproteins Type of Chro- Subunits No. of Chromo- Chromophore mosphores phores on a sub- binding site on unit cysteine residue from N - terminal α β γ

C-Phycoerythrin Phycoerythrobilin (α3β3)2 2 3 or 4 na α – 82/84, (C-PE) (PEB) α – 139/143, β – 50/61, β – 84, β – 155,

b-Phycoerythrin Phycoerythrobilin (αβ)n 2 4 na α – 84, α – 140, (b-PE) (PEB) β – 50/61, β – 84, β – 155

B-Phycoerythrin Phycoerythrobilin (α3β3)2γ 2PEB 4PEB 2PEB& α – 84, α – 140, (B-PE) (PEB) & Phy- 2PUB β – 50/61, courobilin (PUB) β – 84, β – 155

C-Phycocyanin Phycocyanobilin (α3β3)2 1 2 na α– 84, β – 84, (C-PC) (PCB) β – 155

R-Phycocyanin Phycocyanobilin (α3β3) 1PCB 1PCB& na α – 84, (R-PC) (PCB) & Phyco- 1 PEB β – 84, erythrobilin (PEB) β – 155

Phycoerythrocyanin Phycobiliviolin (α3β3) 1 2 PCB na α – 84, (PEC) (PXB) & Phycocy- PXB β – 84 (PXB) anobilin (PCB) β – 155 (PCB)

Allophycocyanin Phycocyanobilin (α3β3) 1 2 na α – 84, (APC) (PCB) β – 84

Allophycocyanin B Phycocyanobilin (α3β3) 1 1 na α – 84, (APC-B) (PCB) β – 84

274 EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

Evolution of phycobiliproteins stress condition. In rod region, the phyco- Different components of PBS evolved cyanin gene seems to have evolved before independently from each other according to the phycoerythrin gene (Six et al., 2007a). necessities of cyanobacteria under different This evolutionary adaptation has led to the stress conditions (like high light condition, development of a modular and flexible an- low light condition, UV light, high tempera- tenna system that can acclimate to a wide ture, etc.) (Six et al., 2007b). During the range of environmental conditions to opti- course of evolution, cyanobacteria appear to mize photosynthesis (Neilson and Durnford, have acquired more and more sophisticated 2010). Koziol et al. (2007) proposed LHC light-harvesting complexes, from simple C- evolutionary studies of photosynthetic organ- PC rods to elaborate rod structure compris- isms in great details. The gene cluster for ing three distinct PBP types viz PC, PE I and rods, in blue green strain lacking PE, is PE II (Six et al., 2007a). On the basis of mainly composed of two identical cpcB-A amino acid sequences as well as the identity operons encoding the C-PC α- and β-sub- and similarities in protein folds, it was sug- units and gene encoding three rod linkers, gested that the PBP scaffold evolved from an one rod core linker and two types of phyco- ancient globin family of proteins (Pastore bilin lyase (Zhao et al., 2000). Whereas a and Lesk, 1990; Apt et al., 1995). part of these PC gene cluster is found to be All PBPs can be traced to a most recent replaced in PE containing strain by a set of common ancestor protein, which diverged, 19 genes, likely involved in synthesis and duplicated, fused and mutated during the de- regulation of PE I like complex. This PE re- velopment. Apt et al. (1995) have provided a gion can also be found in all PE II containing detailed analysis on sequence comparison strain, but it is interrupted by additional sub and phylogenetics, which suggests that at region containing 5 to 9 genes, likely in- least three steps of gene duplication may volved in synthesis and regulation of PE II have occurred during the evolution of PBPs complex (Six et al., 2007b). On the other (Apt et al., 1995). First, a duplication of the hand, the fine tuning of chromophore also ancestral gene generated a pair of tandem plays a significant role in the evolution as PBP genes. Second, this heterodimer gene well as function of PBPs. Methylation of gave rise to two separate lines of descent chromophorylated β subunit monomer of PC through duplication, generating the core at Asn72 position restricts the conformation- (AP) and the rod PBPs. Third, the rod pre- al flexibility of the Cys82 chromophore and cursor duplicated again to form the PC and gives the rigid and extended conformation. PE subfamilies. Core of PBS i.e., AP This change in conformation increases the evolved long before the evolution of PE and photosynthetic efficiency by decreasing the PC, quite probably together with the ances- excited state proton transfer reactions and tral genome of cyanobacteria (Zhao and Qin, intersystem photoisomerization (Zhao et al., 2006). It implies that light-energy transfer 2000). Since Asn72 is very close to the sur- from the PBS core to PSII is an evolutionary, face of the protein, methylation of Asn72 ancient and conservative mechanism that has also restricts the approach of surrounding not allowed much phenotypic variability dur- solvents and oxygen to the chromophore i.e., ing the course of evolution (Six et al., photo oxidation in presence of very high 2007a). In contrast, rod region appears to light intensity (Miller et al., 2008). have evolved later through complex episode of gene duplication, horizontal gene transfer Atomic resolution structure and evolution or gene acquisition from cyanophage. It sug- of phycobilisome gests that the process of light-energy transfer The basic “repeating unit” of the PBS is from PBS rods to PBS cores evolved accord- PBP consisting of two related but distinct ing to necessities of bacteria under different polypeptide chains of α and β. The α- and β-

275 EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

subunits are made up of six alpha-helices at α-Cys84, β-Cys84 and β-Cys155 that are each. These six alpha-helices (A, B, E, F, F’ responsible for the spectral properties of PC and H) form a globular core of phycobilipro- (Table 1). The trimeric organization of PC teins, plus there are two additional helices brings the chromophore linked to β-Cys84 of (X, Y) that extend from the core and they are monomer in close proximity (~21Å) to the α- necessary for assimilation. The heterodimers Cys84 chromophore of the neighboring of α and β are assemble into (α3β3) trimer monomer (Duerring et al., 1991). The β- and two such trimmers constitute an (α3β3)2 Cys84 chromophore is oriented into the cen- hexamer. Structural studies suggest that tral cavity of the disc while the β-Cys155 Asp13 of α-subunit plays an important role linked chromophores are located at the pe- in the formation of stable α-β dimer. Fur- riphery of the trimer. In this organizational thermore, Asp13 also plays a role in prevent- scheme, the inter-chromophore distances are ing β-β homodimerization by destabilizing of the order of 20-50 Å, which are too large the interaction of the N-termini of identical for effective excitonic coupling and energy β-subunits (Soni et al., 2010). The positive transfer may be accomplished by Forster charge at the N-terminus of the α-subunits type of interaction (Soni et al., 2010). Inter- may similarly be unfavorable for α–α associ- trimer energy-transfer is attributed to β- ation. However, two α-subunits in truncated Cys155 chromophores while inter-hexamer conditions are reported to interact via two energy-transfer occurs via chromophores at- ionic interactions and two hydrogen bonds tached to β-Cys84 positions. The structures together with a few van der Waals contacts of PEC share several features with PC struc- (Figure 4) (Soni et al, 2010). The structure of tures (Ficner et al., 1992) except one of the PC (PDB ID: 1CPC, Fremyella diplosiphon) attached bilins. The alpha-subunit of PEC is composed of α helices connected via β- contains a phycobiliviolin (PVB) chromo- turns and has a globin like fold. The mono- phore with a pi-conjugation that gives it meric unit has three chromophores attached unique spectral properties.

Figure 4: Interaction between two crystallographically individual molecules of F-αPE; A and B is shown by ribbon diagram constructed using PYMOL. The interaction between molecule A and B is indicated by the dotted lines. A and B molecules were found to be interactive via two ionic interactions, two ionic bonds and few van der Waals contacts.

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The overall structure of PE bears resem- PBP. This process, known as complementary blance to PC and has conserved globin fold chromatic adaptation (CCA) (Kehoe and (Duerring et al., 1990) except the attached Grossman, 1994) (Figure 5), increases pho- bilin group: chromophores attached at α- tosynthetic efficiency under a range of light Cys82, α-Cys139 and β-Cys50/61, β-Cys84, conditions by allowing the organism to pre- β-Cys155. Wilk et al. (1999) crystallized and cisely tailor its light-harvesting pigment defined the structure of cryptophytes PE hav- composition to closely match the ambient ing β-chain structure is similar to α- and β- light spectrum. Some thermotolerant cyano- chains of other known PBPs. But the overall bacteria also exhibit change of pigmentation structure of PE 545 is novel with α chain in response to temperature (Singh et al., forming a simple extended fold with an anti- 2012). The molecular principles that underlie parallel β-ribbon followed by an α-helix such molecular adaptations are only partly (Wilk et al., 1999). AP shares modest se- understood. But preliminary investigations quence similarity with the other subunits of suggest extensive inactivation of chromo- the PBS i.e., PC and PE, but shows a greater phore protein turn over, by which chromo- degree of structural conservation as the over- phore conformation and dynamics are modu- all fold of the molecule is similar to that of lated (Miller et al., 2008). The CCA is PC and PE (Apt et al., 1995). Both α- and β- grouped on the basis of PC and PE content subunit of AP bound to one bilin each (Table of chromatically adapting cyanobacterial 1). The α-PCB of AP has a different envi- species in red and green light respectively ronment as compared to PC and it has been (Kehoe, 2010) into following categories: suggested that this is responsible for the Group I species have unaltered PC and spectral shift of AP. Bryant et al. (1985) de- PE levels during growth in red/green light scribed the physiological significance and conditions and comprise 27 % of total cya- existence of AP hexamer or trimer (Liu et nobacterial species e.g., Red alga. al., 1999) in details. The α-β monomer is as- Group II species have PE levels that are sembled to form (α3β3) trimer and further high in green light and low in red light, (α3β3)2 hexamer. Hexameric assembly of AP whereas PC levels are not affected by light is found to be less well-packed than that of colour and comprise 16 % of total cyanobac- PC and PE (Bryant et al., 1979). Loosely- terial species e.g., Nostoc punctiforne. packed AP core has high flexibility, which The most complex Group III species allows the possible mechanism of interaction comprise 57 % of total cyanobacterial spe- between the AP cores and rods. The interac- cies; they also have PE levels that are high in tions that stabilize the AP hexamer are also green light and low in red light, like group II different in two crystal structures. In PDB e.g., F. diplosiphon. However, additional 1ALL, the β-subunits mediate the formation regulation is exercised in PC content in re- of an AP hexamer while in PDB 1KN1, the sponse to light color-PC levels are high in inter-monomer interface is formed by the α- red light and low in green light, which con- subunits. trasts PE regulation. Group IV was discovered more recently Complementary Chromatic Adaptation and is distinct from group I-III, as PC and PE (CCA) do not change; instead, a green-light absorb- Many, but not all, cyanobacterial species ing bilin is added to PE in green light and a are capable of acclimating to changes in am- blue-light-absorbing bilin is added to PE in bient light quality by incorporating several blue light (Kehoe, 2010) e.g., different PBSs and linker proteins into their strain.

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A White light Red light

CCA

B 0.35 White light 0.3 Red light 565 nm 616 nm 0.3 0.25 0.25 0.2 0.2 0.15 0.15 Absorbance Absorbance 0.1 0.1 0.05 0.05 0 0 450 500 550 600 650 700 750 450 500 550 600 650 700 750 Wavelength (nm) Wavelength (nm)

Figure 5: (A) The cyanobacterium Lyngbya sp. A09DM, grown in liquid media and fully acclimated to white (left) and red light (right). The color of crude extract (shown along with cell mass) indicated the alteration in phycobilisome pigment composition in a complement manner to absorb the available light. (B) The absorbance spectra of crude extract of white and red light acclimated Lyngbya sp. A09DM. Red light exposure switches the dominance of phycoerythrin by phycocyanin in phycobilisomes.

Given the fairly ubiquitous distribution harvesting apoproteins, several unidentified of the property of light harvesting, it is re- membrane-associated soluble proteins and markable that although red macro algae use chromophore (Stowe-Evans et al., 2004). PBS for light-harvesting, none are known to Radio-isotopic pulse-chase experiments undergo CCA (Kehoe and Gutu, 2006). CCA demonstrate that chromatic adaptation does is the massive reconstruction of the photo- not involve degradation of pre-existing PBSs synthetic light-harvesting antennae, or PBSs, (Glazer, 1977). When phycoerythrin synthe- which are responsible for providing light en- sis is halted by transfer of a culture to red ergy primarily to photosystem II reaction light, the content of this pigment is decreased centers (Kehoe and Gutu, 2006). per cell by cell multiplication and cell death. Chromatic adaptation is a result of Change in pigmentation is achieved through changes at the level of specific gene expres- the induction of hormogonia formation, sion. It includes biosynthesis of light- which are resistant to adverse environmental

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condition. Regulation of CCA is based on Rca pathway in red light results in green two distinct mechanisms: (a) Redox control phenotype. However non-phosphorylated and (b) Photoreceptor control. Regulation state in green light results in blue-green phe- through redox state of photosynthetic elec- notype (Kehoe and Gutu, 2006). tron transport chain is controlled by oxida- Till date, very few components of Cgi tion/reduction of plastoquinone (PQ) (Glaz- and CcaSR systems have been identified or er, 1977). In both red/green lights, PQ-pool isolated, although this might be expected redox poise favors oxidation which in turn given that it controls steady-state CCA re- reduces photosynthesis efficiency by 40 %. sponses. Gutu and Kehoe (2012) also pro- The transient reduction in photosynthetic ef- posed that CCA in Group II is regulated only ficiency immediately after a shift in light by utilizing Cgi and CcaSR systems whereas color (red/green) lasts for ~ 48 hours and is in Group III, CCA is controlled by Rca and known as “acclimation state”. Once the PBS Cgi systems (Gutu and Kehoe, 2012). So fur- composition is optimized for absorbance of ther study of Cgi pathway may help to estab- the ambient light colour, the photosynthetic lish whether Group II strains were derived activity gets recovered and PQ retains its re- from Group III by loss of the Rca system, or duced state, called “steady state”. Therefore, if group III strains arose from group II the long-term CCA responses that persist strains through the acquisition of Rca path- during the steady state would be predomi- way, or if both events occurred (Gutu and nantly photoreceptor-controlled. Analysis of Kehoe, 2012). Rca system and its interaction photoreceptor-controlled mechanism leads to with Cgi system is discussed in great detail the identification of light dependent regula- by Kehoe and Gutu (2006). tor for complementary chromatic adaptation (Rca), CcaSR and control of green light in- Bilin duction (Cgi) systems which regulate PBP Bilins are open chain cova- synthesis during CCA (Gutu and Kehoe, lently attached to the cysteine residue of 2012; Hirose et al., 2013; Rockwell et al., apoprotein via at least one thioether linkage, 2012). These systems asymmetrically con- sometimes two (Glazer, 1989), and serve as trolled the expression of PE and PC gene op- the site for fluorescence origin. In cyanobac- eron. The Rca system comprised of different teria, up to four bilin chromophores are post- regulatory proteins designated as Rca family translationally attached to specific cysteines proteins, encoded by rca gene cluster. It is a of as many as a dozen or even more individ- complex photoreceptor based two-compo- ual proteins (Scheer and Zhao, 2008). Apart nent regulatory system, of which the recep- from phycocyanobilin and phycoerythrobil- tor, Rca E, contains histidine kinase domain in, there are two other types of bilin found on and chromophore binding domain at C- certain biliproteins in the cyanobacteria, terminal and N-terminal, respectively. Rca E phycourobilins and phycoviolobilin (Sam- senses the variation in light wavelength and sonoff and MacColl, 2001), which allow bet- initiates the Rca cascade by conveying the ter light-harvesting in particular regions of message to the response regulators. It either the spectra. The diverse colours generated in phosphorylates or dephosphorylates the this simple manner cover almost the total downstream element of Rca system. Where- range of visible wavelengths from 495 nm as response regulators are transcriptional for phycourobilin (PUB) to 650 nm for phy- controlling elements which further govern cocyanobilin (PCB). PE and PC operon expression. Rca F and Rca C proteins are such response regulators Properties and biosynthesis of bilins of Rca system, which are substrates for Rca In heterotrophs, the formation of open E. A series of experiments suggested that the chain tetrapyrrole products is considered a predominantly phosphorylated state of the catabolic process and it is often related to

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iron acquisition from . Bilin biosynthe- Bilin is widely distributed in all king- sis in phototrophs, on the other hand, may be doms of life except archaea and serves dis- classified as an anabolic pathway as bilins tinct functions. The initial step in the biosyn- function as chromophore and protein cofac- thesis of functional open chain tetrapyrroles tor with light-harvesting (PBSs) and/or light- is catalyzed by an enzyme hemeoxygenase sensing () properties (Dam- (HO) where heme serves as both the sub- meyer and Frankenberg-Dinkel, 2008). The strate and cofactor for its degradation. All numbers and types of chromophores associ- characterized HOs from photosynthetic or- ated with a particular type of PBP subunit ganisms cleave the heme, regiospecifically at are usually invariant, but are attached the α-meso carbon resulting in the IXα iso- through the cysteinyl residue of protein. Free mer of . and bilin (and bilin in denatured PBSs) fluo- are synthesized by reduction of biliverdin IX resces very weakly due to the preferred cy- α catalyzed by enzyme biliverdin reductase clohelical conformation in solution. This in- (BvdR) and phytochromobilin (PϕB) syn- dicates that the excitation energy is lost rap- thase using NADH and ferrodoxin as proton idly by radiation-less relaxation pathways donour respectively. Bilin biosynthesis from from free bilins and bilin associated with biliverdin IXα is catalyzed by feredoxin de- non-native protein environment. The chemis- pendent bilin reductase (FDBR) enzyme. try that is responsible for special spectral fea- Phycocyanobilin and phycoerythrobilin are tures of bilins is variations in the length of synthesized in two successive reductions by conjugated double bond system, which al- ferrodoxin dependent enzymes PcyA and lows light absorption at different wave- PebA/PebB respectively (Dammeyer and lengths. The bilin in PBPs are held rigidly in Frankenberg-Dinkel, 2008) (Figure 6). extended conformation (reduced freedom of molecules) which makes the excited state Chromophorylation of phycobiliproteins life-time longer for efficient energy transduc- Attachment of cofactor (bilin) to PBP tion between proteins. The spectroscopic and phytochrome like receptor is lyase medi- properties of each bilin within PBS are ated and autocatalytic respectively (Scheer strongly influenced by the conjugation and and Zhao, 2008). Although, spontaneous at- environment imposed on the bilin by the na- tachment of chromophore to PBS with low tive protein. Inter-αβ interaction makes an fidelity in vitro has been reported (Fairchild important spectroscopic contribution. For and Glazer, 1994). Various lyases are re- example, monomeric (αβ) allophycocyanin quired for chromophore attachment to specif- has λmax at 615 nm whereas λmax of (αβ)3 lies ic conserved cysteine residues of the protein at 650nm. This intermolecular energy trans- and yields only one attachment product (Soni fer within PBP trimer and hexamer is very et al., 2008). To date, four types of lyases fast and the steady state fluorescence emis- have been described: (1) E/F-type lyases, (2) sion originates in consequence. Crystallo- S(U)-type lyases and (3) T-type lyases (4) graphic and spectroscopic studies on numer- Y/Z-type lyase (Soni et al., 2008). The E/F- ous PBPs have led to the identification of lyases are heterodimeric and are required for donor and acceptor bilin in C-phycocyanin correct binding of chromophore to Cysα-84 (PDB ID: 1CPC, Fremyella diplosiphon) as of the PBS (Zhou et al., 1992; Fairchild and well as in other PBPs. For example, in C- Glazer, 1994). Nomenclature of different phycocyanin the bilin at Cysα-84 and Cysβ- E/F-lyases proposed by Wilbanks and Glazer 155 are donor (lie towards periphery) and the (1993) and others depends basically on the bilin at Cysβ-84 (extended in to center) is the kind of bilin whose attachment is catalyzed acceptor (Dammeyer and Frankenberg- by the lyase. It is of interest to note that E/F- Dinkel, 2008). lyases may also catalyze both release and isomerisation of attached chromophores

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(phycocyanobilin to phycoviobilin, and phy- hetrodimeric and catalyze the binding of coerythrobilin to phycourobilin). S-type ly- PEB to PE α- and β-subunits, but the precise ases are universal in distribution, and have site specificity of this enzyme is still un- an even larger spectrum of activity. They known (Scheer and Zhao, 2008). Moreover, may form hetero-dimers with V-type lyases. Shukla et al. (2012) have identified the iso- S-type lyases catalyze binding of chromo- merase Mpe Z with lyase activity in marine phores to α- and β-subunits of phycocyanin Synechococcus, attaches phycoerythrobilin and phycoerythrin, via Cys84. T-type lyases, to cysteine-83 of the α-subunit of phyco- on the other hand, specifically catalyze the erythrin II and isomerizes it to phycourobilin binding of PCB at cysteine 153 β-subunits of upon green to blue light shifting (Shukla et C-PC (Zhao et al., 2007). Y/Z-type lyases, al., 2012). also known as PE-I and PE-II lyases, are

Heme Oxygenase (HO)

Bilirverdin IXα

Heme b

Pcy A Peb S Peb A Fd (4e-) Fd (4e-) Fd (2e-)

H H

Phycocyanobilin (PCB) Phycoerythrobilin (PEB) 15, 16 - dihydrobiliverdin XIα (DHBV)

Peb B Fd (2e-)

Phycoerythrobilin (PEB)

Figure 6: The biosynthetic pathway of . Bilin synthesis starts from the cleavage of (bilin b) by hemeoxygenase (HO) to yield biliverdin IXα, the central molecule of bilin biosysnthesis pathways. Biliverdin IXα is further reduced by either NAD(P)H- or ferredoxin-dependent bilin reductas- es (FDBR). FDBR reductase like PebA synthase and PebB synthase transfer two electrons to their substrate to give phycoerythrobilin in two step catalysis. PcyA and PebS catalyze the transfer of four electrons from ferredoxin to two distinct double bonds of biliverdin to yield phycocyanobilin and phyco- erythrobilin, respectively in single step. Phycocyanobilin and phycoerythrobilin are further reduced to phycoviobilin and phycourobilin, respectively by PecE/F of Rpc G reductase.

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It is significant to note that in vitro stud- known as ApcE or anchor polypeptide, is ies performed by Kupka et al. (2009), have involved in the attachment of the PBSs to the suggested that lyses may function as chaper- membrane, and is envisaged as one type of ons, which binds the bilin chromophore to terminal acceptors of excitation energy with- the appropriate apo-protein in correct con- in the PBSs (de Lorimier et al., 1990). ApcE formation and stereochemistry to avoid syn- plays a vital role in interaction of PBS with thesis of unwanted by-products. the membrane as a proportion of ApcE re- mains membrane-associated even after all Linker polypeptides the other PBPs have been washed away Linkers are nonchromophoric basic pol- (Redlinger and Gantt, 1982). ApcE has mul- ypeptides which are hydrophobic in nature tiple large domains with similarity to rod and join the consecutive PBPs. Linkers are linker sequences that may support AP hex- involved in face-to-face aggregation of PE, amer formation (Houmard et al., 1990). PC and PEC trimers that are involved in ApcE (Lcm) also contains a chromophore, as higher order assembly of the PBS. The pro- well as some gamma subunits of PE that are cess is mediated by tail-to-tail joining of phylogenetically linker proteins. To under- hexameric assemblies into cylinders of rods stand the mechanism of assembly and func- and core. The linker itself is buried in the tion of linker polypeptides, several attempts central cavity of PBP discs by the combina- have been made to isolate different linker tion of hydrophobic and multiple charged polypeptide to its pure form. However, due interactions (Sidler, 1994), and thereby plays to its high hydrophobicity and their high sus- important roles in maintaining PBS structure ceptibility to proteolytic degradation, this and energy transfer (Tandeau de Marsac and purification is difficult and gives very low Cohen-Bazire, 1977; Sidler, 1994). Howev- yields. For this reason, linker proteins are er, the PBSs would not be able to arrange mostly studied in PBP-linker complexes themselves in the configuration necessary for (Fuglistaller et al., 1986). The principle for efficient energy transfer without linker pro- isolation of linker polypeptides from intact teins or peptides. Glazer (1985) provided a PBS is their basicity (pI > 7.0) as compared widely used classification system which de- to acidic (pI < 5.8) PBPs. Additionally, link- fines linker polypeptides with respect to their er isolation and purification by gel-permeat- location and molecular masses (Glazer, ion and reverse-phase chromatography has 1985) represented as subscript and super- been reported by Fuglistaller et al. (1986). script respectively. Using this classification Alignment results indicate that the prima- system, linker polypeptides can be divided ry sequences of linkers are less conserved into four groups according to their functions than those of their associated PBP subunits. and locations in the PBS: Group I, Lʀ poly- Among all linker polypeptide, LC shows a peptides (MW: 27 to 35 kDa, including small comparatively high level of sequence identi- rod linkers of 10 kDa), participates in the ty in different algae, indicating possible con- assembly of the peripheral rod substructure servation among ancestral linker polypep- and link PE/PC trimers to hexamers or tides. In case of LR polypeptide, many con- PE/PC hexamers to other PE/PC hexamers; served domains near the N-terminus are pre- Group II, Lʀc polypeptides (MW: 25 to 27 sumed to play crucial roles in packing into a kDa), is involved in attaching the peripheral central channel of the PBP hexamers, where- rods to the AP cores and may function in the as other regions may provide the assembly assembly of rod substructure; Group III, LC interface between rod discs (Sidler, 1994). polypeptides (MW: 7 to 8 kDa) is a portion LRC linkers are more distantly related to the of core components and plays key roles in rod linker polypeptides. However, six con- the assembly of the core substructure, and served domains (22-kDa fragment) have Group IV, LCM (MW: 70 to 120 kDa), also been identified within the N-terminal of

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these linker proteins, which were presumed transport chain. Kirilovsky and Kerfeld ΡC to occupy the central hole of (αβ)з and are (2013) reviewed and suggested the involve- largely protected in proteolytic treatment. ment of orange carotenoid protein (OCP), in Based on the sequence analysis and proteo- the energy transfer between PSII and PSI. lysis experiments of linker polypeptides, Presently it is unclear whether binding of Parbel and Scheer (2000) proposed an inter- PBS to PS I is mediated by specific interac- locking model for the PBS rod organization tions or by transient association (Aspinwall in which the linker polypeptides in the model et al., 2004). Indeed, direct measurements of are proposed to possess two distinctive do- fluorescence recovery using confocal mi- mains. The N-terminal domain is hypothe- croscopy has indicated that the diffusion of sized to be buried in the central hole of the PBS is quite rapidly as compared to photo- trimer and protected from proteolysis, systems (Mullineaux et al., 1997), indicating whereas the C-terminal domain is believed to that the association between antenna and protrude from the hexamer. Kondo et al. photosystems in cyanobacteria may be much (2007) found that Synechocystis sp. PCC looser than in other systems (Adir, 2005). 6803 possesses two types of PBSs that differ However the PBS diffusion is generally in their interconnecting ‘‘rod-core linker’’ slower in comparison to similarly sized pro- proteins (CpcG1 and CpcG2) (Kondo et al., teins from other eukaryotic membranes or 2007). CpcG1-PBS was found to be equiva- organelles (Kaňa, 2013). On the other hand, lent to conventional PBS, whereas CpcG2- an early study showed that PBSs are struc- PBS retains phycocyanin rods but is devoid turally associated with PSII only, and that of the central core. Sequence analysis re- energy is transferred onwards from PSII to vealed that CpcG2-like proteins containing a PSI by “spillover” or excitation exchange C-terminal hydrophobic segment are widely between the of the PSII and PSI distributed in many cyanobacteria. Recently core complexes rather than the change in as- Marx and Adir (2013) found that linker pro- sociation of the PBSs to PSII (mobile anten- tein actually stabilizes the interaction of rods na model). Vernotte et al. (1990) proposed and core cylinders in higher assembly (David that the excitation exchange between PSII et al., 2011; Marx and Adir, 2013). and PSI unit depends on the mutual orienta- Future studies on structure and function tion of photosystems rather than their dis- of linker polypeptides will require more ef- tance. However, existing evidence indicates fective approach that will allow their study that spillover is certainly not the only route in solution in the absence of PBP. for energy transfer to PSI (Mullineaux, 1994). Another possibility is that PBS core Organization of phycobilisome on stromal can interact directly with PSI much in the membrane and energy transfer same way that it is thought to interact with PSI and PSII are simultaneously present PSII (Su et al., 1992). Alternatively, there on the thylakoid membrane and provide the might be a direct interaction of PBS rods site for attachment of PBS. Low-temperature with PSI, allowing an alternative energy fluorescence spectra of excitation transfer pathway by-passing the PBS core indicate that a significant proportion of PBS- (Su et al., 1992). However in high light con- absorbed energy is delivered to PSII as well dition, orange carotenoid protein (OCP) has as PSI (Ashby and Mullineaux, 1999). Re- found to directly interact with PBS to trigger cent finding of Liu et al. (2013) suggests no photoprotective mechanism via increasing physical interactions between the PBS core thermal dissipation of excess absorbed ener- and PSII and I are required for energy trans- gy (Kirilovsky, 2010; Kaňa et al., 2012). fer. However the cytochrome b6f complex, Coupling and uncoupling of OCP-PBS com- which connects PSI and PSII by mobile- plex is regulated by 14-kDa thalakoid mem- electron carriers to complete the electron brane protein called as fluorescence recovery

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protein (FRP). FRP greatly accelerates the and RC is stabilized (Yang et al., 2007). This conversion of the active red OCP (attached suggests a dynamic model in which each in- to PBS) into the inactive orange OCP (free dividual PBS is coupled to a RC most of the form) in low irradiation, maintaining the or- time, but always with a strong probability of ange OCP pool (Boulay et al., 2010). decoupling. Decoupling of PBS from a RC is It was believed earlier that direct associa- followed by a brief period of rapid diffusion tion of the PBSs with lipid bilayer also takes before re-attachment to another reaction cen- place with the help of “ApcE” due to the ter, where the diffusion period depends on presence of membrane interacting domain. density of reaction centers (Sarcina et al., Nevertheless, analysis of amino acid se- 2003). Mullineaux (2008) and Kaňa (2013) quence of ApcE did not reveal any obvious have very nicely reviewed the numbers re- membrane-integral domains (Houmard et al., garding the diffusion velocity and fractions 1990). Furthermore, deletion of PB-loop decoupled PBS under normal conditions. from ApcE did not affect the interaction of A gene required for the short-term regu- PBSs with the lipid membrane (Ajlani and lation of photosynthetic light harvesting (the Vernotte, 1998). Study of PS mutant Syn- state transition) has been identified in the echocystis revealed that PBSs are assembled cyanobacterium Synechocystis sp. PCC6803, normally and are membrane-associated even encodes 16 kDa protein designated as Rpa C when no PSII or PSI reaction centers are pre- (regulator of phycobilisome association C). sent (Glazer, 1985). This observation sug- Moreover the in vitro [γ-32P]-ATP labeling gests a direct association of the PBSs with experiments suggest that Rpa C is not the 15 the lipid bilayer. Diffusion measurement ex- kDa membrane phosphoprotein previously periments below phase transition tempera- implicated in state transitions (Emlyn-Jones ture showed decline in membrane lipids dif- et al., 1999). Joshua et al. (2005) found the fusion (Sarcina et al., 2001), whereas PBS pool of free PBS that are neither functionally diffusion is not affected (Sarcina et al., nor structurally coupled to reaction centers 2003). This suggests an absence of integral after prolong iron-starvation (Joshua et al., membrane domain in PBS, but instead inter- 2005). On the other hand McConnell et al. acts with lipid head-groups at the membrane (2002) suggested intramembranous transfer surface. For efficient transfer and distribu- of excitation from Chl a holochromes of tion of energy to the reaction centers, PBS of PSII to Chl a holochromes of PSI - the so- several cyanobacterial strains also contains called spill-over phenomenon instead of di- one to two molecules of enzyme ferredoxin rect involvement of PBP diffusion NADP+ oxidoreductase (FNR). It has been (McConnell et al., 2002). Long distance dif- proposed that FNR is situated at the interface fusion of PBS require its decoupling from of the two lower PC rods closest to the tha- the membrane surface, which is induced ei- lakoid membrane and catalyzes the electron ther by excessive irradiance or by short-term transfer between ferredoxin and NADP (Lü- heat stress; therefore understanding of PBS der et al., 2001). decoupling mechanism requires additional Energy transfer and kinetics of PBS-RC research (Kaňa et al., 2009). The PBS de- complex at room temperature was measured couplings observed under very high and low by picoseconds time-resolved fluorescence. light are assumed to be a strategy of PBS to A relatively short and long overall lifetime prevent photodamage photosynthetic reac- suggests the presence of coupled and decou- tion centers (Liu et al., 2008). Some cyano- pled PBS-RC complex. Decoupled PBSs dif- bacteria which lake other photoprotective fuse rapidly on the membrane surface, but mechanism also use the state transition (S to are immobilized when cells are immersed in M fluorescence transition) as an alternative high-osmotic strength buffers, apparently mechanism for the dissipation of excess en- due to stabilized interaction between PBSs ergy (Kaňa et al., 2009). Decoupling of PBSs

284 EXCLI Journal 2015;14:268-289 – ISSN 1611-2156 Received: December 16, 2014, accepted: January 16, 2015, published: February 20, 2015

may serve as an emergency valve for protec- Postdoctoral fellowship. We also thank Er tion against photo-oxidative damage when Vijay Mungla for valuable help in drawing the protective capacity of all other light- the illustrations. The authors also thank Dr. adaptation responses has been exhausted Varun Shah (National Center for Antarctica (Tamary et al., 2012). Many attempts have and Ocean Research (NCAOR), Goa, India, been made to isolate the cyanobacterial for useful discussion and critical suggestions thylakoid membranes with functionally cou- on the manuscript. pled PBSs using buffer of very high osmotic (Katoh and Gantt, 1979) and ionic strength REFERENCES (Glazer, 1988) in the presence of detergent. Adir N. Elucidation of the molecular structures of However, Mullineaux (2008) has reviewed components of the phycobilisome: reconstructing a that the results of his own experiments are giant. Photosynth Res. 2005;85:15-32. extremely inconsistent and showing very Ajlani G, Vernotte C. Construction and characteriza- poor energy transfer in above mentioned tion of a phycobiliprotein-less mutant of Synechocyst- conditions. This effectively limits our in is sp. PCC 6803. Plant Mol Biol. 1998;37:577-80. vitro study of cyanobacterial thylakoid membrane with functionally coupled PBSs. Anwer K, Sonani RR, Madamwar D, Singh P, Khan F, Bisetty K, et al. Role of N-terminal residues on folding and stability of C-phycoerythrin: simulation CONCLUSION and urea-induced denaturation studies. J Biomol Str Sequential development of light-harvest- Dyn. 2015;33:121-33. ing antennae complexes in lower photosyn- Apt KE, Collier JL, Grossman AR. Evolution of the thetic organism made energy transduction phycobiliproteins. J Mol Biol. 1995;248:79-96. very efficient. Hydrophilic PBPs are a dis- tinctively coloured group of disk-shaped Arteni AA, Ajlani G, Boekema EJ. Structural organi- sation of phycobilisomes from Synechocystis sp strain macromolecular proteins orderly assembled PCC6803 and their interaction with the membrane. into PBS with the help of linker polypep- Biochim Biophys Acta-Bioener. 2009;1787:272-9. tides. High-resolution X-ray crystallography coupled with the knowledge of amino acid Ashby MK, Mullineaux CW. The role of Apc D and ApcF in energy transfer from phycobilisomes to PS I sequence of proteins such as F-αCPE, (often and PSII in a cyanobacterium. Photosynth Res. via gene analysis) has provided a detailed 1999;61:169-79. picture of the arrangement and interactions of PBP within PBS. Thus, biologist must Aspinwall CL, Sarcina M, Mullineaux CW. Phycobi- lisome mobility in the cyanobacterium Synechococcus begin to think more like engineers and in- sp. PCC7942 is influenced by the trimerisation of formation scientists in order to develop a photosystem I. Photosynth Res. 2004;79:179-87. meaningful understanding of energy transfer in cyanobacteria and understand the complex Baier K, Nicklisch S, Grundner C, Reinecke J, multimeric PBS. Lockau W. Expression of two nblA-homologous genes is required for phycobilisome degradation in nitrogen-starved Synechocystis sp. PCC6803. FEMS Acknowledgements Microbiol Lett. 2001;195:35-9. NKS gratefully acknowledges the De- partment of Science and Technology (DST), Bearden AJ, Malkin R. Primary photochemical reac- tions in photosynthesis. Q Rev Biophys. New Delhi for financial support in the form 1975;7:131-77. of DST (SERB) FAST TRACK YOUNG SCIENTIST fellowship. RRS acknowledges Boulay C, Wilson A, D'Haene S, Kirilovsky D. Iden- DST, New Delhi for financial support in tification of a protein required for recovery of full antenna capacity in OCP-related photoprotective form of INSPIRE fellowship [IF120712]. mechanism in cyanobacteria. Proc Natl Acad Sci RPR is thankful to the University Grant USA. 2010;107:11620-5. Commission (UGC), New Delhi, India, for financial support in the form of Dr. DSK

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