Photosystem II Photochemistry and Phycobiliprotein of the Red Algae Kappaphycus alvarezii and Their Implications for Light Adaptation

Photosystem II photochemistry and phycobiliprotein (PBP) genes of red algae Kappaphycus alvarezii, raw material of κ-carrageenan used in food and pharmaceutical industries, were analyzed in this study. Minimum saturating irradiance (I k) of this algal species was less than 115 μmol m−2 s−1. Its actual PSII efficiency (yield II) increased when light intensity enhanced and decreased when light intensity reached 200 μmol m−2 s−1. Under dim light, yield II declined at first and then increased on the fourth day. Under high light, yield II retained a stable value. These results indicate that K. alvarezii is a low-light-adapted species but possesses regulative mechanisms in response to both excessive and deficient light. Based on the PBP gene sequences, K. alvarezii, together with other red algae, assembled faster and showed a closer relationship with LL-Prochlorococcus compared to HL-Prochlorococcus. Many amino acid loci in PBP sequences of K. alvarezii were conserved with those of LL-Prochlorococcus. However, loci conserved with HL-Prochlorococcus but divergent with LL-Prochlorococcus were also found. The diversities of PE and PC are proposed to have played some roles during the algal evolution and divergence of light adaption.


Introduction
Kappaphycus alvarezii (Doty) Doty (Rhodophyta, Solieriaceae) is a commercially important marine alga for its high production of polysaccharide -carrageenan, which is commonly used in food, medicine, and cosmetic industries [1,2]. Extensive application and increasing demand make K. alvarezii widely cultivated around Southeast Asia, East Africa, and South America and promote approximately 8% growth rates annually in the carrageenan industry [3,4]. Photosynthetic responses of K. alvarezii have frequently been detected to clarify the optimal conditions that would maximize its photosynthesis and growth. For example, the effects of temperature, salinity, and UVB radiation on different morphotypes of K. alvarezii were evaluated under controlled conditions [5][6][7]. However, little is known about their physiological changes associated with light intensities, and the optimal photosynthetic available radiation levels of many locally cultivated strains remain to be determined. Photosystem (PS) II photochemical parameters measured by pulse amplitude modulation (PAM) fluorometry have long been used for evaluating the photosynthetic physiology of land plants [8,9]. Moreover, its application development can easily be found in algal research [10][11][12]. It should be a feasible tool for in vivo testing of the photosynthetic behaviors of K. alvarezii to different radiations as well as for ascertaining the best light conditions. Photosynthetic behaviors and light response of plants are largely determined and regulated by their photosynthetic units [13,14]. Phycobilisomes (PBSs) are major photosynthetic units that endue K. alvarezii and other red algae, cyanobacteria, several cryptomonads, and brown algae with  the capability of light-harvesting and energy migration [15]. For their biomedical value, combinational biosynthesis and purification of recombinant PBSs have been carried out with the help of modern biotechnology [16,17]. PBSs are composed of linker polypeptides and phycobiliproteins (PBPs), mainly allophycocyanin (APC), phycoerythrin (PE), and phycocyanin (PC). PBSs are one of the ways by which algae survive in varying and sometimes extreme habitats [18]; some examples would be Prochlorococcus and Synechococcus, two genera of abundant unicellular organisms. Prochlorococcus consist of two ecotypes specifically adapted to either low-light (LL) or high-light (HL) conditions. Their pigmentation and gene complement are quite different [19]. By altering their PBPs structure and PBSs composition, such as the PE divergence in LL-Prochlorococcus and HL-Prochlorococcus [20,21], algae evolve to accommodate different light conditions. Prior studies demonstrated that PE genes in LL-Prochlorococcus suffer from positive selection, and the selected sites are related to light-harvesting or energy-transferring, which is consistent with the findings in other PBPs [22]. Similarly, based on their PBS structure and composition, Synechococcus were divided into three types: type 1, rods are composed of C-PC only; type 2, rods are composed of either C-PC or R-PCIII and a PEI-like PBP; type 3, rods are composed of R-PC and two PE types (PEI and PEII) [23]. Accordingly, nucleic or amino acid sequences of PBPs and PBSs linker family may have hidden some helpful information related to algal photosynthesis and light adaptation [24,25]. Unfortunately, so far, only PBP genes of very few red algae such as Aglaothamnion neglectum and Gracilaria tenuistipitata were sequenced [26,27]. The information of K. alvarezii is still unknown. The present study measured PSII photochemistry to evaluate photosynthetic efficiency and light response of K. alvarezii. In addition, PBP genes were firstly sequenced and aligned with those of other algae. The results of this study would improve our understanding of the impact of changing light conditions on K. alvarezii and the potential mechanism of light adaption of this red alga.  Relative electron transport rate (rETR) was calculated as rETR = yield × PAR × × 0.5, in which PAR symbolizes responses to photosynthetic active radiation ( , absorbancy index = 0.85 in this study). From the RLC, relative maximum electron transport rate (rETRmax) and initial slope ( ) were obtained. Minimum saturating irradiance ( ) = rETRmax/ . Results were expressed as mean ± SD, in which data from three independent experiments were analyzed with the statistical software STATISTICA7.0 ( -test, < 0.05).

DNA Extraction and Sequencing of PBP Genes.
Algal samples were ground in liquid nitrogen and the genomic DNA was extracted with a Plant Genome DNA Kit (Tiangen, Beijing, China) according to the manufacturer's instructions.
Based on the conserved sequences of red algae A. neglectum (Z11906, Z11905), Ceramium boydenii (AF526383), Gracilaria lemaneiformis (AF275685), G. tenuistipitata var. liui (AY673996), Griffithsia monilis (Z98528), Porphyra haitanensis (DQ449071, AY372218), and Porphyra yezoensis (DQ666487), degenerate oligonucleotide primers of PBP genes were designed ( Table 1). The PCR program utilized an initial denaturation at 94 ∘ C for 5 min, followed by 35 cycles at 94 ∘ C for 1 min, 47 ∘ C for 1 min, and 72 ∘ C for 1 min 30 s, with a final elongation at 72 ∘ C for 10 min. Amplification reactions were performed in a Biometra Thermal Cycler (Biometra, Gottingen, Germany). PCR products were purified with a TIANgel Midi Purification Kit (Tiangen, Beijing, China) and then constructed into a pMD18-T vector system (Takara, Dalian, China). The ligation products were transferred into Escherichia coli TOP 10 strains. Five positive clones of each amplification product were checked by their electrophoretic mobility and PCR analysis after the recombinant plasmids were selected under a 100 g mL −1 concentration of ampicillin. Double strands of these target segments were sequenced by Shanghai Sunny Biotechnology Co., Ltd. (Sunny, Shanghai, China).

Multiple Sequence Alignment and Phylogenetic Analysis.
A twenty-four-cyanobacterial-genome database was accessed from JGI (http://www.jgi.doe.gov/) in FASTA format, and the species Gloeobacter violaceus (GV), Trichodesmium erythraeum (TE), Synechococcus (S.), and Prochlorococcus (P.) were examined. PBP sequences of Rhodophyta (including species of Bangiophyceae and Florideophycease), Cyanophora paradoxa (M11159, CP), and Guillardia theta (AM183803) were searched at NCBI [28,29]. PBP nucleotide sequences of K. alvarezii (KA) were cloned in this paper and translated to amino acid sequences by Omiga [30]. Protein sequences of the PBSs previously described were used as database queries. Each protein in this query dataset was used to search for potential novel sequences in above sequenced cyanobacterial species genomes using the BLASTP and TBLASTN programs [31]. Sequences giving better reciprocal BLAST hits were assumed to be capable of identifying homologous counterparts in these species if they could be aligned with at least the BLAST-Score >90 and the -value <1e −10 . The search was iterated until convergence and then examined individually.
Multiple protein sequences alignment was performed using ClustalX [32]. Neighbor joining (NJ) and maximum parsimony (MP) methods in MEGA5 were used to construct the phylogenetic tree [33], in which the confidence level of each branch was determined by analyzing 1000 bootstrap replicates. Bootstrap values >50% were generated.

Tertiary Structure Prediction.
Tertiary structure of PEand -subunits of K. alvarezii was analyzed using homology modeling. Next, -and -PE amino acid sequences of K. alvarezii were submitted to the protein-modeling server SWISSMODEL (http://swissmodel.expasy.org/) and predicted with PDB-1b8dK using PDB-1liaB as the model template. All the manipulations were performed using Pdb-Viewer.   (200 mol m −2 s −1 ), yield II retained a stable value, with a little higher value on the 3rd day ( Figure 1). The RLCs of K. alvarezii cultivated under different light intensities during four days are shown in Figure 2. Under the condition of low PAR, all rETRs rose rapidly; rETRs reached their maximum when actinic light was approximately 80 mol m −2 s −1 , declined significantly as soon as PAR exceeded 80 mol m −2 s −1 , and then climbed slightly. The rETRs were lower under dim light than under high light. It was highest under a cultivated light intensity of 100 mol m −2 s −1 . Regardless of the light intensity (dim or high), rETRs reached a relatively high value from the first day to the fourth day.

Phylogenetic Relationship Inferred by PE Sequences.
The relationship among PE sequences of K. alvarezii, 12 other red algae, and 22 completed sequenced cyanobacteria was investigated. With -PE of G. theta as the root, an alignment of 78 identified -and -subunits amino acid sequences of PE was followed by the generation of an NJ and MP phylogenetic tree (Figure 3). The -subunit PE (including -subunit PE of red algae, four clusters -subunit PEI of Synechococcus, PEII of Synechococcus, and -subunit PEIII of Prochlorococcus) and -subunit PE (including -subunit PE of red algae, three clusters PEIII of HL-Prochlorococcus, PEIII of LL-Prochlorococcus, and PEI and PEII of Synechococcus) were distinctly assembled into two monophyletic groups. In the -subunit group, red algae clustered first with the two cyanobacteria G. violaceus and T. erythraeum and then assembled with the PEI and PEII clades of Synechococcus. Compared with HL-Prochlorococcus, they have a closer relationship with LL-Prochlorococcus. Red algal PE sequences are more similar to LL-Prochlorococcus PEs than to HL-PEs. A possible explanation of this result is that HL-Prochlorococcus evolves faster than LL, and also exhibits much higher GC content. Hence, the similarities among red algal-, HL-, and LL-PEs do not necessarily reflect their photosynthetic properties and relationships. In the -subunit group, red algae successively assembled with PEI of Synechococcus, LL-Prochlorococcus, and T. erythraeum; PEII of Synechococcus formed a cluster by itself.

Tertiary Structure of PE.
Forty-eight amino acid loci conserved with LL-Prochlorococcus but diverged with HL-Prochlorococcus were mapped onto -PE tertiary structure of K. alvarezii in blue color (Figure 4(a)). These loci distribute to the following six domains: (1) N-terminal 2-10aa domain is obviously conserved, including the loci 3D function of interaction of N-terminus of -and -subunits; (2)
Judging from amino acid sequences in Figure 5, 80 conserved loci were shown, in which all reported that functional amino acids could be found. Loci 135 in -subunit APC and 150 in -subunit PC were highly divergent in all sequences aligned in this paper. In -subunit APC, -subunit APC,subunit of PC, and -subunit of PC, there were 5, 15, 20, and 28 divergent loci with known functions between K. alvarezii, G. tenuistipitata, P. purpurea, and other algae. The identity between -subunit PC in RS9917, WH5701 (without PE) and K. alvarezii, G. tenuistipitata, P. purpurea, and C. paradoxa was higher than other cyanobacteria, which were not found in -subunit PC and -and -subunits APC. Moreover, there are 13 and 10 loci identical in -and -subunits PC of RS9917, WH5701 but different from other cyanobacteria, which are more divergent than -and -subunits APC. Most of these divergent subunits are similar to K. alvarezii, G. tenuistipitata, and P. purpurea, which are conserved in red alga, while 10 loci are specially unique only in both RS9917 and WH5701.

Discussion
Based on the data of PSII photochemistry in this study, K. alvarezii is a low-light-adapted species, but it has some regulative mechanisms for both excessive and deficient light. The of K. alvarezii is less than 115 mol m −2 s −1 , which is consistent with the results of chlorophyll fluorescent measurement in Polyneura hilliae, Porphyra leucosticta, and Porphyra umbilicalis [34][35][36]. This revealed that these red algae favor relatively weak light conditions. Yield II values of K. alvarezii demonstrated the variation of photosynthetic efficiency under different light intensities. Electron transport rate decreased under deficient or excessive light; however, the rate increased after four days of adaptation. Similar results were shown by RLCs. The results implied that K. alvarezii possessed both high-and low-light adaptability.
Variation of composition and gene sequences may reflect the evolution of PBPs in response to light [37]. When comparing PE amino acid sequences of K. alvarezii with two ecotypes of Prochlorococcus (Figure 4), it is shown that many loci in K. alvarezii PE sequences were conserved with LL-Prochlorococcus. However, loci conserved with HL-Prochlorococcus but divergent with LL-Prochlorococcus were Highly conserved amino acid positions of which the function has been speculated Highly divergent amino acid positions Divergent amino acid in highly conserved positions Divergent amino acid positions in red algae Identical amino acid positions in Synechococcus RS9917 and WH5701 also found. The divergent loci were mainly located inhelix and had several reported functional domains (e.g., subunits' interaction, linkers' interaction, and chromophores' interaction). According to the previous studies [38], PE genes in LL-Prochlorococcus suffered from positive selection, and the selected loci were related to light-harvesting or energy-transferring. We speculate the mutative domains or loci were related to light responses of K. alvarezii. The extension domain near the chromophores' interaction area in PE-of red algae, lacking in LL-Prochlorococcus, is also hypothesized to have an effect on low-light response. Further experiments are still needed to prove these assumptions. Moreover, by comparing APC and PC ( Figure 5), we also found that APC sequences are more conserved than PC and -subunits are more divergent than -subunits in K. alvarezii and other algae. Although PBP sequences are divergent in all algae, we found that the loci with basic function are more conserved, such as subunits' interaction, linkers' interaction, and chromophores' interaction. The conservation may ensure PBPs' function in absorption and transfer of light and energy. The diversities of PE and PC may take part in algal surviving and reviving under different light conditions.
Our study evaluated photosynthetic characteristics of K. alvarezii and attempted to explain light adaptation by analyzing secondary and tertiary structure of photosynthetic protein. The results of this study would shed some light on understanding the potential mechanism of light response of K. alvarezii and other red algae.