Chloroplast magnesium transporters play essential but differential roles in maintaining magnesium homeostasis.

Magnesium (Mg2+) is essential for photosynthesis in the chloroplasts of land plants and algae. Being the central ion of chlorophyll, cofactor and activator of many photosynthetic enzymes including RuBisCO, magnesium-deficient plants may suffer from leaf chlorosis symptoms and retarded growth. Therefore, the chloroplast Mg2+ concentration is tightly controlled by magnesium transport proteins. Recently, three different transporters from two distinct families have been identified in the chloroplast inner envelope of the model plant Arabidopsis thaliana: MGT10, MGR8, and MGR9. Here, we assess the individual roles of these three proteins in maintaining chloroplast Mg2+ homeostasis and regulating photosynthesis, and if their role is conserved in the model green alga Chlamydomonas reinhardtii. Phylogenetic analysis and heterologous expression revealed that the CorC-like MGR8 and MGR9 transport Mg2+ by a different mechanism than the CorA-like MGT10. MGR8 and MGT10 genes are highest expressed in leaves, indicating a function in chloroplast Mg2+ transport. MGR9 is important for chloroplast function and plant adaptation in conditions of deficiency or excess of Mg2+. Transmission electron microscopy indicated that MGT10 plays a differential role in thylakoid stacking than MGR8 and MGR9. Furthermore, we report that MGR8, MGR9, and MGT10 are involved in building up the pH gradient across the thylakoid membrane and activating photoprotection in conditions of excess light, however the mechanism has not been resolved yet. While there are no chloroplast MGR-like transporters in Chlamydomonas, we show that MRS4 is a homolog of MGT10, that is required for photosynthesis and cell growth. Taken together, our findings reveal that the studied Mg2+ transporters play essential but differential roles in maintaining chloroplast Mg2+ homeostasis.

Alleviation of high light stress in shade-treated tea leaves by acclimation to light before shade removal.

Shade cultivation of tea plants (Camellia sinensis L.) is employed for the production of high-quality green tea which increases the content of chlorophylls and free amino acids, including theanine. However, shaded tea plants suffer from photooxidative stress caused by sudden exposure to high light (HL) when the shade is removed. In this study, we tried to acclimatize shaded tea plants to light prior to shade removal to alleviate HL-induced stress. Acclimated tea plants showed milder photoinhibition in response to HL exposure than the shaded plants without acclimation. Moreover, there were no large differences in the total chlorophylls and free amino acids (including theanine) content between acclimated and non-acclimated plants. These results indicate that acclimation of shaded tea plants can alleviate subsequent HL stress without causing large changes in the content of chemical components associated with tea quality.

Stress Responses of Shade-Treated Tea Leaves to High Light Exposure after Removal of Shading.

High-quality green tea is produced from buds and young leaves grown by the covering-culture method, which employs shading treatment for tea plants (Camellia sinensis L.). Shading treatment improves the quality of tea, but shaded tea plants undergo sudden exposures to high light (HL) at the end of the treatment by shade removal. In this study, the stress response of shaded tea plants to HL illumination was examined in field condition. Chl a/b ratio was lower in shaded plants than nonshaded control, but it increased due to exposure to HL after 14 days. Rapid decline in Fv/Fm values and increases in carbonylated protein level were induced by HL illumination in the shaded leaves on the first day, and they recovered thereafter between a period of one and two weeks. These results revealed that shaded tea plants temporarily suffered from oxidative damages caused by HL exposure, but they could also recover from these damages in 2 weeks. The activities of antioxidant enzymes, total ascorbate level, and ascorbate/dehydroascorbate ratio were decreased and increased in response to low light and HL conditions, respectively, suggesting that the upregulation of antioxidant defense systems plays a role in the protection of the shaded tea plants from HL stress.

The direction of protein evolution is destined by the stability.

Protein evolution is potentially governed by protein stability. Here, we investigated the relationship between protein evolution and stability through the random mutational drift of a thermophilic bacterial protein, an esterase of Alicyclobacillus acidocaldarius (Aac-Est), at high and low temperatures. In the first random mutation of Aac-Est, few proteins exhibit increased activity at 65 °C, indicating that the wild-type (WT) Aac-Est is located on the peak of a mountain in a fitness landscape for activity at high temperature. To obtain higher active variants than those of WT, it must go down the mountain once and climb another, higher mountain. In the second and third generations from lower active templates, the evolvability (the proportion of variants with higher activity in all the variants obtained in a given generation than a parent protein) depended on the stability of the template proteins. Compared to WT, the stability-maintaining template could recover the activity more. Thus, a low-activity variant with high stability is able to drift vastly in sequence space and reach the foot of a higher mountain. Meanwhile, random mutations in stability-loss templates produced several variants with higher activity at 40 °C than those produced by WT, via cold adaptation. Our results indicate that maintaining protein stability enables the protein to search sequence space and evolve in the original environment, and proteins with lost stability use a cold adaptation path.

Protein Evolution is Potentially Governed by Protein Stability: Directed Evolution of an Esterase from the Hyperthermophilic Archaeon Sulfolobus tokodaii.

The study of evolution is important to understand biological phenomena. During evolutionary processes, genetic changes confer amino acid substitutions in proteins, resulting in new or improved functions. Unfortunately, most mutations destabilize proteins. Thus, protein stability is a significant factor in evolution; however, its role remains unclear. Here, we simply and directly explored the association between protein activity and stability in random mutant libraries to elucidate the role of protein stability in evolutionary processes. In the first random mutation of an esterase from Sulfolobus tokodaii, approximately 20% of the variants displayed higher activity than wild-type protein (i.e., 20% evolvability). During evolutionary processes, the evolvability depended on the stability of template proteins, indicating that protein evolution is potentially governed by protein stability. Furthermore, decreased activity could be recovered during evolution by maintaining the stability of variants. The results suggest that protein sequence space for its evolution is able to expand during nearly neutral evolution where mutations are slightly deleterious for activity but rarely fatal for stability. Molecular evolution is a crucial phenomenon that has continued since the birth of life on earth, and mechanism underlying it is simple; therefore, this could be demonstrated by our simple experiments. These findings also can be applied to protein engineering.

Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain.

Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the determination of the structure of the long α-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long α-helical domain has three α-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight into the Lip system is discussed.

Folding and Unfolding Kinetics of Unpurified Proteins by Pulse Proteolysis.

It has been reported that pulse proteolysis may be used to investigate protein unfolding kinetics in cell lysate. However, the method has not become popular and we could not judge whether or not it is effective for protein folding study. In this work, we examined the folding and unfolding kinetics of a protein and its variants without purification by pulse proteolysis. The unfolding and refolding rates of the unpurified proteins were similar to those of the purified proteins determined by pulse proteolysis and circular dichroism. Furthermore, because we used a super-stable subtilisin as a protease, we could evaluate the kinetics at 50°C. The present work demonstrates the validity of pulse proteolysis for folding and unfolding studies of unpurified proteins.

Light-dependent expression of flg22-induced defense genes in Arabidopsis.

Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30%) genes strongly induced by flg22 (>4.0) require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid (SA), indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB). Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controlling the light-dependent expression of flg22-inducible defense genes.

Abundant sequence divergence in the native Japanese cattle Mishima-Ushi (Bos taurus) detected using whole-genome sequencing.

The native Japanese cattle Mishima-Ushi, a designated national natural treasure, are bred on a remote island, which has resulted in the conservation of their genealogy. We examined the genetic characteristics of 8 Mishima-Ushi individuals by using single nucleotide polymorphisms (SNPs), insertions, and deletions obtained by whole-genome sequencing. Mapping analysis with various criteria showed that predicted heterozygous SNPs were more prevalent than predicted homozygous SNPs in the exonic region, especially non-synonymous SNPs. From the identified 6.54 million polymorphisms, we found 400 non-synonymous SNPs in 313 genes specific to each of the 8 Mishima-Ushi individuals. Additionally, 3,170,833 polymorphisms were found between the 8 Mishima-Ushi individuals. Phylogenetic analysis confirmed that the Mishima-Ushi population diverged from another strain of Japanese cattle. This study provides a framework for further genetic studies of Mishima-Ushi and research on the function of SNP-containing genes as well as understanding the genetic relationship between the domestic and native Japanese cattle breeds.

Next-generation sequencing reveals genomic features in the Japanese quail.

The Japanese quail has several advantages as a laboratory animal for biological and biomedical investigations. In this study, the draft genome of the Japanese quail was sequenced and assembled using next-generation sequencing technology. To improve the quality of the assembly, the sequence reads from the Japanese quail were aligned against the reference genome of the chicken. The final draft assembly consisted of 1.75 Gbp with an N50 contig length of 11,409 bp. On the basis of the draft genome sequence obtained, we developed 100 microsatellite markers and used these markers to evaluate the genetic variability and diversity of 11 lines of Japanese quail. Furthermore, we identified Japanese quail orthologs of spermatogenesis markers and analyzed their expression using in situ hybridization. The Japanese quail genome sequence obtained in the present study could enhance the value of this species as a model animal.

Chloroplast-mediated activation of plant immune signalling in Arabidopsis.

Chloroplasts have a critical role in plant immunity as a site for the production for salicylic acid and jasmonic acid, important mediators of plant immunity. However, the molecular link between chloroplasts and the cytoplasmic-nuclear immune system remains largely unknown. Here we show that pathogen-associated molecular pattern (PAMP) signals are quickly relayed to chloroplasts and evoke specific Ca(2+) signatures in the stroma. We further demonstrate that a chloroplast-localized protein, named calcium-sensing receptor (CAS), is involved in stromal Ca(2+) transients and responsible for both PAMP-induced basal resistance and R gene-mediated hypersensitive cell death. CAS acts upstream of salicylic acid accumulation. Transcriptome analysis demonstrates that CAS is involved in PAMP-induced expression of defence genes and suppression of chloroplast gene expression possibly through (1)O(2)-mediated retrograde signalling, allowing chloroplast-mediated transcriptional reprogramming during plant immune responses. The present study reveals a previously unknown chloroplast-mediated signalling pathway linking chloroplasts to cytoplasmic-nuclear immune responses.

Defense response of a pepper cultivar cv. Sy-2 is induced at temperatures below 24°C.

Temperature is one of the most important environmental factors that influence plant growth and development. Recent studies imply that plants show various responses to non-extreme ambient temperatures. Previously, we have found that a pepper cultivar cv. Sy-2 (Capsicum chinense) shows developmental defects at temperatures below 24°C. In this study, to gain new insights into the temperature sensitivity of cv. Sy-2, temperature-sensitive genes were screened using microarray techniques. At restrictive temperature of 20°C, almost one-fourth of the 411 up-regulated genes were defense related or predicted to be defense related. Further expression analyses of several defense-related genes showed that defense-related genes in cv. Sy-2 were constitutively expressed at temperatures below 24°C. Moreover, accumulation of high level of salicylic acid (SA) in cv. Sy-2 grown at 20°C suggests that the defense response is activated in the absence of pathogens. To confirm that the defense response is induced in cv. Sy-2 below 24°C, we evaluated the resistance to biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria and necrotrophic fungal pathogen Cercospora capsici. Cv. Sy-2 showed enhanced resistance to X. campestris pv. vesicatoria, but not to C. capsici.

High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 Å resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein.

Optimization of salt concentration in PEG-based crystallization solutions.

Although polyethylene glycol (PEG) is the most widely used precipitant in protein crystallization, the concentration of co-existing salt in the solution has not been well discussed. To determine the optimum salt concentration range, several kinds of protein were crystallized in a 30% PEG 4000 solution at various NaCl concentrations with various pH levels. It was found that, if crystallization occurred, the lowest effective salt concentration depended on the pH of the protein solution and the pI of the protein molecule; that is, higher salt concentrations were required for crystal growth if the difference between pH and pI was increasing. The linear relationship between the charge density of the protein and the ionic strength of the crystallization solution was further verified. These results suggested that the lowest effective concentration of salt in a crystallization solution can be predicted before performing a crystallization experiment. Our results can be a tip for tuning crystallization conditions by the vapor-diffusion method.

Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment.

Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.

High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors.

Human haematopoietic prostaglandin D synthase (H-PGDS; EC 5.3.99.2) produces prostaglandin D(2), an allergic and inflammatory mediator, in mast cells and Th2 cells. H-PGDS has been crystallized with novel inhibitors with half-maximal inhibitory concentrations (IC(50)) in the low nanomolar range by the counter-diffusion method onboard the Russian Service Module on the International Space Station. The X-ray diffraction of a microgravity-grown crystal of H-PGDS complexed with an inhibitor with an IC(50) value of 50 nM extended to 1.1 A resolution at 100 K using SPring-8 synchrotron radiation, which is one of the highest resolutions obtained to date for this protein.

Fluoroalkene modification of mercaptoacetamide-based histone deacetylase inhibitors.

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. The mercaptoacetoamide-based inhibitors are reported to be less toxic than hydroxamate and are worthy of further consideration. Therefore, we have designed a series of analogs as potential inhibitors of HDACs, in which the mercaptoacetamide group was replaced by (mercaptomethyl)fluoroalkene, and their HDAC inhibitory activity was evaluated. Subnanomolar inhibition was observed for all synthetic compounds.

Dual synchronization of chaos in Mackey-Glass electronic circuits with time-delayed feedback.

We experimentally and numerically demonstrate the dual synchronization of chaos in two pairs of one-way-coupled Mackey-Glass electronic circuits with time-delayed feedback. The outputs of the two drive circuits are mixed and used both for the feedback signal to the two drive circuits and for the transmission signal to the two response circuits. We investigate the regions for achieving dual synchronization of chaos when the delay time is mismatched between the drive and response circuits.

Crystallization of the archaeal transcription termination factor NusA: a significant decrease in twinning under microgravity conditions.

The transcription termination factor NusA from Aeropyrum pernix was crystallized using a counter-diffusion technique in both terrestrial and microgravity environments. Crystallization under microgravity conditions significantly reduced the twinning content (1.0%) compared with terrestrially grown crystals (18.3%) and improved the maximum resolution from 3.0 to 2.29 A, with similar unit-cell parameters. Based on a comparison of the crystal parameters, the effect of microgravity on protein crystallization is discussed.

Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach.

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.