HLA Genetics for the Human Diseases.

Highly polymorphic human leukocyte antigen (HLA) molecules (alleles) expressed by different classical HLA class I and class II genes have crucial roles in the regulation of innate and adaptive immune responses, transplant rejection and in the pathogenesis of numerous infectious and autoimmune diseases. To date, over 35,000 HLA alleles have been published from the IPD-IMGT/HLA database, and specific HLA alleles and HLA haplotypes have been reported to be associated with more than 100 different diseases and phenotypes. Next generation sequencing (NGS) technology developed in recent years has provided breakthroughs in various HLA genomic/gene studies and transplant medicine. In this chapter, we review the current information on the HLA genomic structure and polymorphisms, as well as the genetic context in which numerous disease associations have been identified in this region.

Haplotype structures and polymorphisms of dog leukocyte antigen (DLA) class I loci shaped by intralocus and interlocus recombination events.

The dog leukocyte antigen (DLA) class I genomic region is located on chromosome 12, and the class I genomic region is composed of at least two distinct haplotypic gene structures, DLA-88-DLA-12 and DLA-88-DLA-88L. However, detailed information of the genomic differences among DLA-88, DLA-12, and DLA-88L are still lacking at the full-length gene level, and therefore, DLA allelic sequences classified for each of these loci are limited in number so far. In this study, we determined the DNA sequence of a 95-kb DLA class I genomic region including DLA-88, DLA-12/88L, and DLA-64 with three DLA homozygous dogs and of 37 full-length allelic gene sequences for DLA-88 and DLA-12/88L loci in 26 DLA class I homozygous dogs. Nucleotide diversity profiles of the 95-kb regions and sequence identity scores of the allelic sequences suggested that DLA-88L is a hybrid gene generated by interlocus and/or intralocus gene conversion between DLA-88 and DLA-12. The putative minimum conversion tract was estimated to be at least an 850-bp segment in length located from the 5´flanking untranslated region to the end of intron 2. In addition, at least one DLA-12 allele (DLA-12*004:01) was newly generated by interlocus gene conversion. In conclusion, the analysis for the occurrence of gene conversion within the dog DLA class I region revealed intralocus gene conversion tracts in 17 of 27 DLA-88 alleles and two of 10 DLA-12 alleles, suggesting that intralocus gene conversion has played an important role in expanding DLA allelic variations.

Reactive oxygen species accumulation is synchronised with growth inhibition of temperature-sensitive recAts polA Escherichia coli.

When combined with recombinase defects, chromosome breakage and double-strand break repair deficiencies render cells inviable. However, cells are viable when an SOS response occurs in recAts polA cells in Escherichia coli. Here, we aimed to elucidate the underlying mechanisms of this process. Transposon mutagenesis revealed that the hslO gene, a redox chaperone Hsp33 involved in reactive oxidative species (ROS) metabolism, was required for the suppression of recAts polA lethality at a restricted temperature. Recently, it has been reported that lethal treatments trigger ROS accumulation. We also found that recAts polA cells accumulated ROS at the restricted temperature. A catalase addition to the medium alleviates the temperature sensitivity of recAts polA cells and decreases ROS accumulation. These results suggest that the SOS response and hslO manage oxidative insult to an acceptable level in cells with oxidative damage and rescue cell growth. Overall, ROS might regulate several cellular processes.

Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

Vitamin C Maintenance against Cell Growth Arrest and Reactive Oxygen Species Accumulation in the Presence of Redox Molecular Chaperone hslO Gene.

Chromosome damage combined with defective recombinase activity renders cells inviable, owing to deficient double-strand break repair. Despite this, recA polA cells grow well under either DNA damage response (SOS) conditions or catalase medium supplementation. Catalase treatments reduce intracellular reactive oxygen species (ROS) levels, suggesting that recA polA cells are susceptible to not only chronic chromosome damage but also ROS. In this study, we used a reducing agent, vitamin C, to confirm whether cell growth could be improved. Vitamin C reduced ROS levels and rescued colony formation in recAts polA cells under restrictive temperatures in the presence of hslO, the gene encoding a redox molecular chaperone. Subsequently, we investigated the role of hslO in the cell growth failure of recAts polA cells. The effects of vitamin C were observed in hslO+ cells; simultaneously, cells converged along several ploidies likely through a completion of replication, with the addition of vitamin C at restrictive temperatures. These results suggest that HslO could manage oxidative stress to an acceptable level, allowing for cell division as well as rescuing cell growth. Overall, ROS may regulate several processes, from damage response to cell division. Our results provide a basis for understanding the unsolved regulatory interplay of cellular processes.

Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis.

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import.

Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.

Identification and Structure of an MHC Class I-Encoded Protein with the Potential to Present N-Myristoylated 4-mer Peptides to T Cells.

Similar to host proteins, N-myristoylation occurs for viral proteins to dictate their pathological function. However, this lipid-modifying reaction creates a novel class of "lipopeptide" Ags targeted by host CTLs. The primate MHC class I-encoded protein, Mamu-B*098, was previously shown to bind N-myristoylated 5-mer peptides. Nevertheless, T cells exist that recognize even shorter lipopeptides, and much remains to be elucidated concerning the molecular mechanisms of lipopeptide presentation. We, in this study, demonstrate that the MHC class I allele, Mamu-B*05104, binds the N-myristoylated 4-mer peptide (C14-Gly-Gly-Ala-Ile) derived from the viral Nef protein for its presentation to CTLs. A phylogenetic tree analysis indicates that these classical MHC class I alleles are not closely associated; however, the high-resolution x-ray crystallographic analyses indicate that both molecules share lipid-binding structures defined by the exceptionally large, hydrophobic B pocket to accommodate the acylated glycine (G1) as an anchor. The C-terminal isoleucine (I4) of C14-Gly-Gly-Ala-Ile anchors at the F pocket, which is distinct from that of Mamu-B*098 and is virtually identical to that of the peptide-presenting MHC class I molecule, HLA-B51. The two central amino acid residues (G2 and A3) are only exposed externally for recognition by T cells, and the methyl side chain on A3 constitutes a major T cell epitope, underscoring that the epitopic diversity is highly limited for lipopeptides as compared with that for MHC class I-presented long peptides. These structural features suggest that lipopeptide-presenting MHC class I alleles comprise a distinct MHC class I subset that mediates an alternative pathway for CTL activation.

Nomenclature report 2019: major histocompatibility complex genes and alleles of Great and Small Ape and Old and New World monkey species.

The major histocompatibility complex (MHC) is central to the innate and adaptive immune responses of jawed vertebrates. Characteristic of the MHC are high gene density, gene copy number variation, and allelic polymorphism. Because apes and monkeys are the closest living relatives of humans, the MHCs of these non-human primates (NHP) are studied in depth in the context of evolution, biomedicine, and conservation biology. The Immuno Polymorphism Database (IPD)-MHC NHP Database (IPD-MHC NHP), which curates MHC data of great and small apes, as well as Old and New World monkeys, has been upgraded. The curators of the database are responsible for providing official designations for newly discovered alleles. This nomenclature report updates the 2012 report, and summarizes important nomenclature issues and relevant novel features of the IPD-MHC NHP Database.

Correction to: Nomenclature report 2019: major histocompatibility complex genes and alleles of great and small ape and old and new world monkey species.

The original version of this article contained a spelling error in the Acknowledgments regarding the name of the funding organisation supporting GM and JAH. UKRI-BBSCR should have been UKRI-BBSRC, as is now indicated correctly below.

Evolutionary basis of HLA-DPB1 alleles affects acute GVHD in unrelated donor stem cell transplantation.

HLA-DPB1 T-cell epitope (TCE) mismatching algorithm and rs9277534 SNP at the 3' untranslated region (3'UTR) in the HLA-DPB1 gene are key factors for transplant-related events in unrelated hematopoietic cell transplantation (UR-HCT). However, the association of these 2 mechanisms has not been elucidated. We analyzed 19 frequent HLA-DPB1 alleles derived from Japanese healthy subjects by next-generation sequencing of the entire HLA-DPB1 gene region and multi-SNP data of the HLA region in 1589 UR-HCT pairs. The risk of acute graft-versus-host disease (aGVHD) was analyzed in 1286 patients with single HLA-DPB1 mismatch UR-HCT. The phylogenetic tree constructed using the entire gene region demonstrated that HLA-DPB1 alleles were divided into 2 groups, HLA-DP2 and HLA-DP5. Although a phylogenetic relationship in the genomic region from exon 3 to 3'UTR (Ex3-3'UTR) obviously supported the division of HLA-DP2 and HLA-DP5 groups, which in exon 2 showed intermingling of HLA-DPB1 alleles in a non-HLA-DP2 and non-HLA-DP5-group manner. Multi-SNP data also showed 2 discriminative HLA-DPB1 groups according to Ex3-3'UTR. Risk of grade 2-4 aGVHD was significantly higher in patient HLA-DP5 group mismatch than patient HLA-DP2 group mismatch (hazard ratio, 1.28; P = .005), regardless of donor mismatch HLA-DP group. Regarding TCE mismatch, increasing risk of aGVHD in patient HLA-DP5 group mismatch and TCE-nonpermissive mismatch were observed only in patients with TCE-permissive mismatch and patient HLA-DP2 group mismatch, respectively. Evolutionary analysis revealed that rs9277534 represented a highly conserved HLA-DPB1 Ex3-3'UTR region and may provoke aGVHD differently to TCE mismatching algorithm, reflecting exon 2 polymorphisms. These findings enrich our understanding of the mechanism of aGVHD in HLA-DPB1 mismatch UR-HCT.

Experimental techniques for the development of a uterus transplantation model in cynomolgus macaques.

Uterus transplantation (UTx) is now a treatment for women with uterine factor infertility to have a child. However, UTx is still largely at the experimental stage, and many medical issues remain unsolved. Therefore, adequate studies in large animals including non-human primates are required for validation of these issues. UTx research, especially in non-human primates, can provide important information for its full establishment in humans due to the anatomical and physiological similarities between the two. We accumulated data from UTx studies using cynomolgus macaques since 2009 and established autologous and allogeneic UTx models which led to deliveries after performing the procedure. In this paper, we summarized key points to develop UTx models in cynomolgus macaques based on our experience. UTx models in non-human primates can surely contribute new and beneficial knowledge in this field and can be useful for the further development of UTx in humans.

Evaluation of alloreactive T cells based on the degree of MHC incompatibility using flow cytometric mixed lymphocyte reaction assay in dogs.

It has become anticipated that regenerative medicine will extend into the field of veterinary medicine as new treatments for various disorders. Although the use of allogeneic stem cells for tissue regeneration is more attractive than that of autologous cells in emergencies, the therapeutic potential of allogeneic transplantation is often limited by allo-immune responses inducing graft rejection. Therefore, a methodology for quantifying and monitoring alloreactive T cells is necessary for evaluating allo-immune responses. The mixed lymphocyte reaction (MLR) is widely used to evaluate T cell alloreactivity. In human, flow cytometric MLR with carboxyfluorescein diacetate succinimidyl ester has been established and used as a more useful assay than conventional MLR with radioisotope labeling. However, the available information about alloreactivity based on the differences of dog major histocompatibility complex (MHC) (dog leukocyte antigen, DLA) is quite limited in dog. In this paper, we describe our established flow cytometric MLR method that can quantify the T cell alloreactivity while distinguishing cell phenotypes in dog, and T cell alloreactivity among DLA-type matched pairs was significantly lower than DLA-mismatched pairs, suggesting that our developed flow cytometric MLR method is useful for quantifying T cell alloreactivity. In addition, we demonstrated the advantage of DLA homozygous cells as a donor (stimulator) for allogeneic transplantation. We also elucidated that the frequency of alloreactive T cell precursors was almost the same as that of mouse and human (1-10%). To our knowledge, this is the first report to focus on the degree of allo-immune responses in dog based on the differences of DLA polymorphisms.

Inflammatory myopathy associated with PD-1 inhibitors.

OBJECTIVE: To characterize the inflammatory myopathy associated with programmed cell death 1 inhibitors (PD-1 myopathy). METHODS: We studied 19 Japanese patients with PD-1 myopathy (13 men and 6 women, mean age 70 years), who were referred to Keio University. As control groups, we used 68 patients with anti-signal recognition particle antibodies, 51 patients with anti-aminoacyl transfer RNA synthetase antibodies and 460 healthy subjects. RESULTS: In regard to muscle-disease severity, 10 patients showed a mild form of disease and 9 patients showed a severe form. Non-small cell lung cancer was the most common underlying cancer. PD-1 inhibitor consisted of 11 nivolumab and 8 pembrolizumab. PD-1 myopathy occurred 29 days on average after the first administration of PD-1 inhibitor. The initial manifestation of muscle weakness was ptosis in 10 patients, 15 patients had ptosis, 13 diplopia, 8 facial muscle weakness, 10 bulbar symptoms, 13 limb weakness, 14 neck weakness, 4 cardiac involvement, 6 respiratory involvement and 16 myalgia. Ocular, facial, cardiac and respiratory involvement and myalgia were more frequently observed than controls. Serum creatine kinase was increased to 5247 IU/L on average. Autoantibodies related to inflammatory myopathy were negative, while anti-striational antibodies were found in 13 (68%) patients. HLA-C*12:02 alleles were more frequently detected than healthy controls. Muscle pathology was characterized by multifocal necrotic myofibers with endomysial inflammation and expression of MHC class I. Immunosuppressive therapy with corticosteroids was generally effective for muscle weakness. CONCLUSIONS: Based on our clinical, histological and immunological findings, PD-1 myopathy is a discrete subset of inflammatory myopathy.

Identification of novel polymorphisms and two distinct haplotype structures in dog leukocyte antigen class I genes: DLA-88, DLA-12 and DLA-64.

The current information on the polymorphism variation and haplotype structure of the domestic dog leukocyte antigen (DLA) genes is limited in comparison to other experimental animals. In this paper, to better elucidate the degree and types of polymorphisms and genetic differences for DLA-88, DLA-12 and DLA-64, we genotyped four families of 38 beagles and another 404 unrelated dogs representing 49 breeds by RT-PCR based Sanger sequencing. We also sequenced and analyzed the genomic organization of the DLA-88 and DLA-12 gene segments to better define these two-gene DLA haplotypes more precisely. We identified 45 alleles for DLA-88, 15 for DLA-12 and six for DLA-64, of which 20, 14 and six, respectively, were newly described alleles. Therefore, this study shows that the DLA-12 and DLA-64 loci are far more polymorphic than previously reported. Phylogenetic analysis strongly supported that the DLA-88, DLA-12 and DLA-64 alleles were independently generated after the original divergence of the DLA-79 alleles. Two distinct DLA-88 and DLA-12 haplotype structures, tentatively named DLA-88-DLA-12 and DLA-88-DLA-88L, were identified, and the novel haplotype DLA-88-DLA-88L contributed to 32.7% of the unrelated dogs. Quantitative real-time PCR analysis showed that the gene expression levels of DLA-88L and DLA-88 were similar, and that the gene expression level of DLA-12 was significantly lower. In addition, haplotype frequency estimations using frequently occurring alleles revealed 45 different DLA-class I haplotypes (88-88L/12-64) overall, and 22 different DLA-class I haplotypes in homozygous dogs for 18 breeds and mongrels.

Genetic association of swine leukocyte antigen class II haplotypes and body weight in Microminipigs.

OBJECTIVE: Microminipigs are a novel animal model with extensive applications in laboratory studies owing, in part, to their extremely small body sizes. In this study, the relationship between swine leukocyte antigen (SLA) class II haplotype and body weight was evaluated in the Microminipig population. METHODS: A total of 1,900 haplotypes, covering SLA class II haplotypes Lr-0.7, Lr-0.23, Lr-0.17, Lr-0.37, Lr-0.16, Lr-0.11, Lr-0.13, and Lr-0.18, were analyzed in 950 piglets. Birth weights and weights on postnatal day 50 were examined in piglets with eight different SLA class II haplotypes. RESULTS: The mean birth weight of piglets with the Lr-0.23 haplotype (0.415 kg, n = 702) was significantly lower than that of piglets with Lr-0.17 (0.445 kg, n = 328) and Lr-0.37 (0.438 kg, n = 383) haplotypes. At postnatal day 50, the mean body weight of piglets with the Lr-0.23 haplotype (3.14 kg) was significantly lower than that of piglets with the Lr-0.13 haplotype (3.46 kg, p<0.01). There were no significant differences in daily gains (DGs) among the eight haplotypes. However, piglets with the Lr-0.11 and -0.18 haplotype combination or any heterozygous haplotype combinations containing Lr-0.23 had significantly lower DGs than those of piglets with the Lr-0.18, 0.37 haplotype combination. CONCLUSION: Piglets with the Lr-0.23 haplotype had relatively low body weights at birth and on postnatal day 50 and slightly lower DGs than those of piglets with other haplotypes. Therefore, the Lr-0.23 SLA class II haplotype may be a suitable marker for the selective breeding of Microminipigs with small body sizes.

Characterization of thylakoid membrane in a heterocystous cyanobacterium and green alga with dual-detector fluorescence lifetime imaging microscopy with a systematic change of incident laser power.

Fluorescence Lifetime Imaging Microscopy (FLIM) has been applied to plants, algae and cyanobacteria, in which excitation laser conditions affect the chlorophyll fluorescence lifetime due to several mechanisms. However, the dependence of FLIM data on input laser power has not been quantitatively explained by absolute excitation probabilities under actual imaging conditions. In an effort to distinguish between photosystem I and photosystem II (PSI and PSII) in microscopic images, we have obtained dependence of FLIM data on input laser power from a filamentous cyanobacterium Anabaena variabilis and single cellular green alga Parachlorella kessleri. Nitrogen-fixing cells in A. variabilis, heterocysts, are mostly visualized as cells in which short-lived fluorescence (≤0.1 ns) characteristic of PSI is predominant. The other cells in A. variabilis (vegetative cells) and P. kessleri cells show a transition in the status of PSII from an open state with the maximal charge separation rate at a weak excitation limit to a closed state in which charge separation is temporarily prohibited by previous excitation(s) at a relatively high laser power. This transition is successfully reproduced by a computer simulation with a high fidelity to the actual imaging conditions. More details in the fluorescence from heterocysts were examined to assess possible functions of PSII in the anaerobic environment inside the heterocysts for the nitrogen-fixing enzyme, nitrogenase. Photochemically active PSII:PSI ratio in heterocysts is tentatively estimated to be typically below our detection limit or at most about 5% in limited heterocysts in comparison with that in vegetative cells.

Comparative genomics of the human, macaque and mouse major histocompatibility complex.

The MHC is a highly polymorphic genomic region that encodes the transplantation and immune regulatory molecules. It receives special attention for genetic investigation because of its important role in the regulation of innate and adaptive immune responses and its strong association with numerous infectious and/or autoimmune diseases. The MHC locus was first discovered in the mouse and for the past 50 years it has been studied most intensively in both mice and humans. However, in recent years the macaque species have emerged as some of the more important and advanced experimental animal models for biomedical research into MHC with important human immunodeficiency virus/simian immunodeficiency virus and transplantation studies undertaken in association with precise MHC genotyping and haplotyping methods using Sanger sequencing and next-generation sequencing. Here, in this special issue on 'Macaque Immunology' we provide a short review of the genomic similarities and differences among the human, macaque and mouse MHC class I and class II regions, with an emphasis on the association of the macaque class I region with MHC polymorphism, haplotype structure and function.

A Scalable Open-Source Pipeline for Large-Scale Root Phenotyping of Arabidopsis.

Large-scale phenotyping of multicellular organisms is one of the current challenges in biology. We present a comprehensive and scalable pipeline that allows for the efficient phenotyping of root growth traits on a large scale. This includes a high-resolution, low-cost acquisition setup as well as the automated image processing software BRAT. We assess the performance of this pipeline in Arabidopsis thaliana under multiple growth conditions and show its utility by performing genome-wide association studies on 16 root growth traits quantified by BRAT each day during a 5-d time-course experiment. The most significantly associated genome region for root growth rate is a locus encoding a calcium sensing receptor. We find that loss of function and overexpression of this gene can significantly alter root growth in a growth condition dependent manner and that the minor natural allele of the Calcium Sensor Receptor locus is highly significantly enriched in populations in coastal areas, demonstrating the power of our approach to identify regulators of root growth that might have adaptive relevance.

Ferritin 2 domain-containing protein found in lacquer tree (Toxicodendron vernicifluum) sap has negative effects on laccase and peroxidase reactions.

Lacquer tree sap, a raw material of traditional paints in East Asia, is hardened through laccase-catalyzed oxidation and the following polymerization of phenolic compound urushiol. In the sap's water-insoluble fraction, we found two plantacyanins and a ferritin 2 domain-containing protein (TvFe2D, a homolog of Arabidopsis AT1G47980 and AT3G62730). The recombinant TvFe2D protein suppressed the accumulation of laccase-catalyzed oxidation products of a model substrate syringaldazine without decreasing oxygen consumption, the second substrate of laccase. The suppression was also observed when another substrate guaiacol or another oxidizing enzyme peroxidase was used. The functional domain of the suppression was the C-terminal half, downstream of the ferritin 2 domain. The results suggest that this protein may be involved in regulating the sap polymerization/hardening. We also discuss the possibility that homologous proteins of TvFe2D in other plants might be involved in the laccase- or peroxidase-mediated polymerization of phenolic compounds, such as lignin and flavonoids.