Phycobilisome Mobility and Its Role in the Regulation of Light Harvesting in Red Algae

Red algae represent an evolutionarily important group that gave rise to the whole red clade of photosynthetic organisms. They contain a unique combination of light-harvesting systems represented by a membrane-bound antenna and by phycobilisomes situated on thylakoid membrane surfaces. So far, very little has been revealed about the mobility of their phycobilisomes and the regulation of their light-harvesting system in general. Therefore, we carried out a detailed analysis of phycobilisome dynamics in several red alga strains and compared these results with the presence (or absence) of photoprotective mechanisms. Our data conclusively prove phycobilisome mobility in two model mesophilic red alga strains, Porphyridium cruentum and Rhodella violacea. In contrast, there was almost no phycobilisome mobility in the thermophilic red alga Cyanidium caldarium that was not caused by a decrease in lipid desaturation in this extremophile. Experimental data attributed this immobility to the strong phycobilisome-photosystem interaction that highly restricted phycobilisome movement. Variations in phycobilisome mobility reflect the different ways in which light-harvesting antennae can be regulated in mesophilic and thermophilic red algae. Fluorescence changes attributed in cyanobacteria to state transitions were observed only in mesophilic P. cruentum with mobile phycobilisomes, and they were absent in the extremophilic C. caldarium with immobile phycobilisomes. We suggest that state transitions have an important regulatory function in mesophilic red algae; however, in thermophilic red algae, this process is replaced by nonphotochemical quenching.Photosynthetic light reactions are mediated by pigment-binding protein complexes located either inside the thylakoid membrane (e.g. chlorophyll-binding proteins of both photosystems) or associated on the membrane surface (e.g. phycobilisomes [PBsomes] in cyanobacteria and red algae). Recent progress in structural biology has allowed the construction of high-resolution structural models of most photosynthetic protein complexes (for review, see Fromme, 2008) together with their large-scale organization into supercomplexes (for review, see Dekker and Boekema, 2005). However, the dynamics of these supercomplexes and the mobility of particular light-harvesting proteins in vivo are still poorly understood (for review, see Mullineaux, 2008a; Kaňa, 2013; Kirchhoff, 2014) The importance of protein mobility in various photosynthetic processes, like nonphotochemical quenching and state transitions, has been explored mostly based on indirect in vitro experiments, including single-particle analysis (Kouřil et al., 2005), or by biochemical methods (Betterle et al., 2009; Caffarri et al., 2009). Recent studies on the mobility of light-harvesting proteins using live-cell imaging (for review, see Mullineaux, 2008a; Kaňa, 2013) have elucidated the importance of protein mobility for photosynthetic function (Joshua and Mullineaux, 2004; Joshua et al., 2005; Goral et al., 2010, 2012; Johnson et al., 2011). In addition, the redistribution of respiratory complexes in cyanobacterial thylakoid membranes plays an essential role in controlling electron flow (Liu et al., 2012).It is generally accepted that the mobility of most of the transmembrane photosynthetic proteins is very restricted in the thylakoid. The typical effective diffusion coefficient of photosynthetic proteins is somewhere between 0.01 and 0.001 μm⁻² s⁻¹ (Kaňa, 2013). A similar restriction in membrane protein mobility has also been described for bacterial membranes (Dix and Verkman, 2008; Mika and Poolman, 2011). In fact, this is very different in comparison with what we know for other eukaryotic membranes (e.g. plasma membrane and endoplasmic reticulum), where membrane-protein diffusion can be faster by 1 or 2 orders of magnitude (Lippincott-Schwartz et al., 2001). Therefore, macromolecular crowding of proteins has been used to rationalize the restricted protein mobility in thylakoid membranes of chloroplasts (Kirchhoff, 2008a, 2008b). Indeed, atomic force microscopy studies have shown that there is a dense packing and interaction of complexes in the photosynthetic membranes (Liu et al., 2011). Therefore, the diffusion of photosynthetic proteins in the thylakoid membrane is rather slow, and it increases only in less crowded parts of thylakoids (Kirchhoff et al., 2013). The current model of photosynthetic protein mobility thus proposes the immobility of protein supercomplexes, such as PSII (Mullineaux et al., 1997; Kirchhoff, 2008b), with only a small mobile fraction of chlorophyll-binding proteins represented by external antennae of photosystems, including light harvesting complex of PSII in higher plants (Consoli et al., 2005; Kirchhoff et al., 2008) or iron stress-induced chlorophyll-binding protein A in cyanobacteria (Sarcina and Mullineaux, 2004).The restricted mobility of internal membrane supercomplexes (photosystems) contrasts with the relatively mobile PBsomes (Mullineaux et al., 1997; Sarcina et al., 2001). PBsomes are sizeable biliprotein supercomplexes (5–10 MD) attached to the thylakoid membrane surface with dimensions of approximately 64 × 42 × 28 nm (length × width × height; Arteni et al., 2008; Liu et al., 2008a). PBsomes are composed of chromophore-bearing phycobiliproteins and colorless linker polypeptides (Adir, 2005; Liu et al., 2005). They serve as the main light-harvesting antennae in various species, including cyanobacteria, red algae, glaucocystophytes, and cryptophytes. Although a single PBsome is composed of hundreds of biliproteins, absorbed light energy is efficiently transferred toward a specific biliprotein that functions as a terminal energy emitter (Glazer, 1989). From there, energy can be transferred to either PSI or PSII and used in photosynthesis (Mullineaux et al., 1990; Mullineaux, 1992, 1994). In typical prokaryotic cyanobacteria and eukaryotic red algae, PBsomes are composed of two main parts: (1) allophycocyanin (APC) core proteins adjacent to the thylakoid membrane; and (2) peripheral rod proteins made from phycocyanin only or from a combination of phycocyanin together with phycoerythrin. Such complex and modular composition allows for different spectroscopic properties of PBsomes and thus their complementary absorption in the spectral region that is not covered by chlorophyll-binding proteins.PBsome mobility has been studied only in a few types of cyanobacteria (for review, see Kaňa, 2013). PBsomes have been recognized as a mobile element with an effective diffusion coefficient of about 0.03 μm² s⁻¹ for Synechococcus sp. PCC 7942 (Mullineaux et al., 1997; Sarcina et al., 2001). The effective diffusion coefficient value depends on lipid composition, temperature, and the size of the PBsome (Sarcina et al., 2001). The diffusion coefficient reflects PBsome mobility, but it is not affected singularly by physical diffusion processes, and the role of PBsome-photosystem interaction is an open question (Kaňa, 2013). PBsome mobility seems to be related to the requirement of light-induced PBsome redistribution during state transitions (Joshua and Mullineaux, 2004). The mechanism of state transitions in cyanobacteria is still rather questionable (for review, see Kirilovsky et al., 2014). As PSII seems to be immobile, it has been suggested that PBsomes interact with photosystems only transiently and that physical redistribution (diffusion) of PBsomes is crucial for the state transition (Mullineaux et al., 1997). The importance of such long-distance diffusions, however, should be tested experimentally in more detail (Kaňa, 2013), as an alternative theory of the state transition proposed only slight PBsome movement (shifting) between photosystems (McConnell et al., 2002). However, in both cases, PBsome mobility (i.e. the PBsome’s ability to move) is required (Kaňa, 2013).Red algae are the eukaryotic representatives of phototrophs containing PBsomes (Su et al., 2010). They represent the ancestor of photosynthetic microorganisms from the red clade of photosynthesis (Yoon et al., 2006; Wang et al., 2013), which includes various model organisms such as diatoms, chromerids, or dinoflagellates. Red algae contain a unique combination of antennae systems on their membrane surfaces, which are formed mostly by hemispherical PBsomes (Mimuro and Kikuchi, 2003; Arteni et al., 2008). Red algae also contain transmembrane light-harvesting antennae (Vanselow et al., 2009; Neilson and Durnford, 2010; Green, 2011) associated mostly with PSI (Wolfe et al., 1994). Therefore, red algae represent a functionally important eukaryotic model organism; however, few facts are known about the regulation of its light-harvesting efficiency, although it seems to be connected with photoprotection in the reaction center (Delphin et al., 1996, 1998; Krupnik et al., 2013). The presence of photoprotective NPQ in PBsomes of prokaryotic cyanobacteria has been conclusively proven (Kirilovsky et al., 2014); however, this mechanism seems to be missing in eukaryotic phycobiliproteins of cryptophytes (Kaňa et al., 2012b) and red algae. Moreover, the presence (or absence) of PBsome mobility has not been confirmed conclusively (Liu et al., 2009).Therefore, we carried out a detailed study of PBsome mobility in red algal chloroplasts to determine the role of mobility in the regulation of light-harvesting efficiency. We found that red alga PBsomes are a mobile protein complex with effective diffusion coefficient between 2.7 × 10⁻³ and 13 × 10⁻³ μm⁻² s⁻¹ in all studied mesophilic strains. It contrasted with PBsomes in extremophilic red algal strains (Cyanidium caldarium), where PBsome mobility under physiological conditions was highly restricted (effective diffusion coefficient of approximately 0.6 × 10⁻³ μm⁻² s⁻¹). The restriction of PBsome mobility in extremophilic C. caldarium was due to a tight interaction of PBsomes with both photosystems and not to changes in lipid desaturation, an effect typical for extremophiles. The PBsome-photosystem interaction was weakened for C. caldarium grown at suboptimal temperatures, resulting in a pronounced increase in PBsome mobility thanks to PBsome decoupling from the photosystem. This result shows that PBsome mobility in this strain is limited by the strength of the PBsome-photosystem interaction rather than by the restriction of diffusion by factors such as macromolecular crowding. Moreover, our study allows us to describe two different models of light-harvesting antenna regulation in red algae. In mesophilic strains (Porphyridium cruentum and Rhodella violacea), absorbed light is redistributed between photosystems in a process of state transition that requires PBsome mobility. On the contrary, in extremophilic C. caldarium, PBsome are strongly coupled to photosystems and excess light is dissipated by a process of nonphotochemical quenching, as has been described recently (Krupnik et al., 2013).     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

Selective measurement of α smooth muscle actin: why β-actin can not be used as a housekeeping gene when tissue fibrosis occurs

Background

Prevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of α smooth muscle actin (α-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate α-SMA and ß-actin from other actin isoforms.

Results

Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-SMA or ß-actin in the kidney of mice underwent UUO.

Conclusion

We developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat α-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of α-SMA is occur.

     

Chromosome phylogeny of the subfamily Pitheciinae (Platyrrhini,Primates) by classic cytogenetics and chromosome painting

Background  

The New World monkey (Platyrrhini) subfamily Pitheciinae is represented by the genera Pithecia, Chiropotes and Cacajao. In this work we studied the karyotypes of Pithecia irrorata (2n = 48) and Cacajao calvus rubicundus (2n = 45 in males and 2n = 46 in females) by G- and C-banding, NOR staining and chromosome painting using human and Saguinus oedipus whole chromosome probes. The karyotypes of both species were compared with each other and with Chiropotes utahicki (2n = 54) from the literature.     

Vegetal cover estimated by digital photographs related to biomass in a grassland site in northern Mexico

A regression model was used to determine the relationship between aerial herbaceous biomass and vegetation coverage estimated by digital images. Four samplings (n=36 each date) of vegetation cover and herbaceous biomass were performed during the growing season in 2011 in a grassland dominated by Bouteloua gracilis in La Cieneguilla, Municipality of Villa Hidalgo, Durango. Average production of dry biomass was 37.36 ± 9.66 g/m², and mean vegetation cover 30.02%. Dry biomass data were tested for normality using the test of Kolmogorov Smirnov, finding a lack of fit. The data were subjected to a logarithmic transformation and the model Ln(y) = 1.637926 + 0.08501X - 0.000586X² with an adjusted R² = 0.89 was found. In order to validate this model, another five samplings were carried out in 2013 at the same site during summer and autumn, using the same sampling size for each date as in 2011. Data collected in 2013 were analyzed with the model Ln (y) = β₀ + β₁X + β₂X². A comparison of regression coefficients was carried out between the 2011 and 2013 models with t (180+144-9-11-2=302, p<0.05) = 1.967. The results indicated that it is possible to use the 2011 regression model to estimate herbaceous aerial biomass from vegetation cover measurements with aerial photographs in La Cieneguilla site during summer and fall.     

Studying the quantity component of seed dispersal effectiveness from exclosure treatments and camera trapping

The quantity component of effectiveness of seed dispersal by animals is determined by two events: fruit removal (intensity of the interaction) and animal visitation to the plant (frequency of interactions). Considering dispersal of Prosopis flexuosa seeds as case study, this work aimed at investigating the strengths and weaknesses of the two methods for assessing the quantity component of seed dispersal effectiveness: exclosures and camera traps. Prosopis fruits were offered for 48 hr. Exclosure treatments were performed using two types of wire‐screen cages, allowing access to ants (“closed exclosure”) and to small mammals up to 100 g (“open to small mammals”), and a treatment without exclosure (“open to all removers”). The camera trapping experiment was carried out using vertically oriented cameras placed at approximately 1.80 m height and focused on the fruits. The cameras were set in “motion detect mode,” taking series of three consecutive photographs. The exclosures largely allowed estimation of fruit removal by size‐based groups of animals, but did not provide information on species identity. In contrast, camera traps were able to identify all visitors to species level and could not only determine the number of visits by each species but also the proportion of visits, which resulted in removal of fruits. Camera trapping allowed discriminating among small mammals playing different roles, without underestimating fruit removal by scatter‐hoarding species. The quality of estimation of the quantity component of seed dispersal is remarkably better when the camera trapping method is applied. Additional information obtained, such as activity patterns of visitors, can contribute to a better understanding of the seed dispersal process.     

Effects of plant height and row spacing on kenaf forage potential with multiple harvests

Kenaf (Hibiscus cannabinus L.) forage potential can be enhanced through its regrowth capacity and higher production in narrow rows. A field experiment was conducted in Matamoros, Coahuila, Mexico, during 2 growing seasons (2004 and 2005) to study the effects of plant height and row spacing on kenaf forage potential with multiple harvests. This study evaluated the effects of (1) 2 plant heights at cutting (1.0-1.2 m and 1.8-2.0 m) and (2) 4 inter row spacings (0.19, 0.38, 0.57 and 0.76 m) using a 2 x 4 factorial arrangement of treatments in a completely randomized block design with 4 replications. Dry matter (DM) and crude protein (CP) yields, DM partitioning, neutral detergent fiber (NDF) and CP concentrations were determined. Heights at cutting × row spacing interactions were not significant for the monitored variables (p>0.05). Kenaf response to treatments was only relevant for main effects (p≤0.05). Row spacing and plant height affected DM and CP yields (p≤0.05), whereas only plant height affected chemical composition and DM partitioning (p≤0.05). Dry matter (17.0%-26.0%), and CP (12.4%-15.6%) yields were higher (p≤0.05) when plant heights had reached 1.8 to 2.0 m. Row spacing reduction from 0.76 m to 0.38 and 0.19 m increased DM yield (20.4-33.4%) and CP yield (24.2-38.5%) (p≤0.05). Kenaf forage potential increases when planted in narrow rows and harvested 2 or 3 times during the growing season.     

Mold challenge study in bottled natural mineral waters and spring waters

Microbiological challenge study was carried out to verify the microbial stability of bottled waters against four different mold species isolated from bottled water (Fusarium sp.; Cladosporium sp.; Penicillium chrysogenum and Aspergillus fumigatus) and to follow the growth of the molds in bottled water. Twelve types of bottled water with different mineralization and CO2 level in PET and glass packages were collected from 4 European countries. Three different inoculation levels of spore suspensions were used to contaminate bottled water samples. The surviving colony forming unit (CFU) numbers and visual growth were monitored during the investigation period (26 weeks). The results of surviving CFU showed that the fungal growth is mostly determined by the carbonation level and the type of the mold strain. Neither the inoculation level nor the mineral content had any significant effect on the survival of the different mold strains. Results showed decreased CFU numbers in carbonated waters, while slow decreasing, stagnation or even some growth in still waters. A. fumigatus was the most resistant test species. None of the other tested mold strains survived the first 12-week test period in carbonated water. Visual growth was not detected in carbonated water samples, in contrast to all of the non-carbonated samples.