Tomohiro Kakizaki

Arabidopsis Cor15am Is a Chloroplast Stromal Protein That Has Cryoprotective Activity and Forms Oligomers

Many plants acquire increased freezing tolerance when they are exposed to nonfreezing temperatures of a certain duration. This process is known as cold acclimation and allows plants to protect themselves from freezing injury. A wide variety of polypeptides are induced during cold acclimation, among which is one encoded by COR15A in Arabidopsis (Arabidopsis thaliana). Previous studies showed that the COR15A gene encodes a small, plastid-targeted polypeptide that is processed to a mature form called Cor15am. In this study, we examined the biochemical properties and activities of Cor15am in more detail. We provide evidence that Cor15am localizes almost exclusively to the chloroplast stroma. In addition, the cold-regulated accumulation of Cor15am is affected by chloroplast functionality. Both gel-filtration chromatography and protein cross-linking reveal that Cor15am forms oligomers in the stroma of chloroplasts. Although Cor15am accumulates in response to low temperature, cold acclimation is not a prerequisite for oligomerization of Cor15am. Structural analysis suggests that Cor15am is composed of both ordered and random structures, and can stay soluble with small structural change after boiling and freeze-thaw treatments. Recombinant Cor15am exhibits in vitro cryoprotection of a freeze-labile enzyme, l-lactate dehydrogenase. Furthermore, Cor15am is capable of associating with l-lactate dehydrogenase in vitro and with potential stromal substrates in vivo. On the basis of these results, we propose that Arabidopsis Cor15am is a cryoprotective protein that forms oligomers in the chloroplast stroma, and that direct association of Cor15am with its substrates is part of its cryoprotective mechanism.

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

Self‐incompatibility (SI) enables flowering plants to discriminate between self‐ and non‐self‐pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class‐I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co‐evolution of the genes encoding the male and female determinants.

Coordination of Plastid Protein Import and Nuclear Gene Expression by Plastid-to-Nucleus Retrograde Signaling

Expression of nuclear-encoded plastid proteins and import of those proteins into plastids are indispensable for plastid biogenesis. One possible cellular mechanism that coordinates these two essential processes is retrograde signaling from plastids to the nucleus. However, the molecular details of how this signaling occurs remain elusive. Using the plastid protein import2 mutant of Arabidopsis (Arabidopsis thaliana), which lacks the atToc159 protein import receptor, we demonstrate that the expression of photosynthesis-related nuclear genes is tightly coordinated with their import into plastids. Down-regulation of photosynthesis-related nuclear genes is also observed in mutants lacking other components of the plastid protein import apparatus. Genetic studies indicate that the coordination of plastid protein import and nuclear gene expression is independent of proposed plastid signaling pathways such as the accumulation of Mg-protoporphyrin IX and the activity of ABA INSENSITIVE4 (ABI4). Instead, it may involve GUN1 and the transcription factor AtGLK. The expression level of AtGLK1 is tightly correlated with the expression of photosynthesis-related nuclear genes in mutants defective in plastid protein import. Furthermore, the activity of GUN1 appears to down-regulate the expression of AtGLK1 when plastids are dysfunctional. Based on these data, we suggest that defects in plastid protein import generate a signal that represses photosynthesis-related nuclear genes through repression of AtGLK1 expression but not through activation of ABI4.

Trans -acting small RNA determines dominance relationships in Brassica self-incompatibility

A diploid organism has two copies of each gene, one inherited from each parent. The expression of two inherited alleles is sometimes biased by the effects known as dominant/recessive relationships, which determine the final phenotype of the organism. To explore the mechanisms underlying these relationships, we have examined the monoallelic expression of S-locus protein 11 genes (SP11), which encode the male determinants of self-incompatibility in Brassica. We previously reported that SP11 expression was monoallelic in some S heterozygotes, and that the promoter regions of recessive SP11 alleles were specifically methylated in the anther tapetum. Here we show that this methylation is controlled by trans-acting small non-coding RNA (sRNA). We identified inverted genomic sequences that were similar to the recessive SP11 promoters in the flanking regions of dominant SP11 alleles. These sequences were specifically expressed in the anther tapetum and processed into 24-nucleotide sRNA, named SP11 methylation inducer (Smi). Introduction of the Smi genomic region into the recessive S homozygotes triggered the methylation of the promoter of recessive SP11 alleles and repressed their transcription. This is an example showing sRNA encoded in the flanking region of a dominant allele acts in trans to induce transcriptional silencing of the recessive allele. Our finding may provide new insights into the widespread monoallelic gene expression systems.

Dominance relationships between self-incompatibility alleles controlled by DNA methylation.

In crucifers, the pollen S-determinant gene, SP11, is sporophytically expressed in the anther tapetum, and the pollen self-incompatibility phenotype is determined by the dominance relationships between the two S-haplotypes it carries. We report here that 5′ promoter sequences of recessive SP11 alleles are specifically methylated in the tapetum before the initiation of SP11 transcription. These results suggest that tissue-specific monoallelic de novo DNA methylation is involved in determining the dominance interactions that determine the cruciferous self-incompatibility phenotype.

Draft Sequences of the Radish (Raphanus sativus L.) Genome

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.

Glucosinolate metabolism, functionality and breeding for the improvement of Brassicaceae vegetables

Unique secondary metabolites, glucosinolates (S-glucopyranosyl thiohydroximates), are naturally occurring S-linked glucosides found mainly in Brassicaceae plants. They are enzymatically hydrolyzed to produce sulfate ions, D-glucose, and characteristic degradation products such as isothiocyanates. The functions of glucosinolates in the plants remain unclear, but isothiocyanates possessing a pungent or irritating taste and odor might be associated with plant defense from microbes. Isothiocyanates have been studied extensively in experimental in vitro and in vivo carcinogenesis models for their cancer chemopreventive properties. The beneficial isothiocyanates, glucosinolates that are functional for supporting human health, have received attention from many scientists studying plant breeding, plant physiology, plant genetics, and food functionality. This review presents a summary of recent topics related with glucosinolates in the Brassica family, along with a summary of the chemicals, metabolism, and genes of glucosinolates in Brassicaceae. The bioavailabilities of isothiocyanates from certain functional glucosinolates and the importance of breeding will be described with emphasis on glucosinolates.

Evaluation of the Protective Activities of a Late Embryogenesis Abundant (LEA) Related Protein, Cor15am, during Various Stresses in Vitro

Arabidopsis Cor15am is a late embryogenesis abundant (LEA) related protein that has been shown to exhibit cryoprotective activity in vitro. In this study, we further investigated the mechanisms by which Cor15am protects substrates from inactivation. Although Cor15am did not exhibit refolding activity, it showed protective activity against various stresses in vitro. This might be attributable to the activity of Cor15am in attenuating the aggregation of the substrates. Our data indicate that Cor15am functions as a protectant against various stresses by preventing protein aggregation.

Identification and characterization of Cor413im proteins as novel components of the chloroplast inner envelope

Plastids are surrounded by two membrane layers, the outer and inner envelope membranes, which have various transport and metabolic activities. A number of envelope membrane proteins have been identified by biochemical approaches and have been assigned to specific functions. Despite those efforts, the chloroplast envelope membrane is expected to contain a number of as yet unidentified proteins that may affect specific aspects of plant growth and development. In this report, we identify and characterize a novel class of inner envelope membrane proteins, designated as Cor413 chloroplast inner envelope membrane group (Cor413im). Both in vivo and in vitro studies indicate that Cor413im proteins are targeted to the chloroplast envelope. Biochemical analyses of Cor413im1 demonstrate that it is an integral membrane protein in the inner envelope of chloroplasts. Quantitative real-time PCR analysis reveals that COR413IM1 is more abundant than COR413IM2 in cold-acclimated Arabidopsis leaves. The analyses of T-DNA insertion mutants indicate that a single copy of COR413IM genes is sufficient to provide normal freezing tolerance to Arabidopsis. Based on these data, we propose that Cor413im proteins are novel components that are targeted to the chloroplast inner envelope in response to low temperature.

QTL analysis using SNP markers developed by next-generation sequencing for identification of candidate genes controlling 4-methylthio-3-butenyl glucosinolate contents in roots of radish, Raphanus sativus L.

SNP markers for QTL analysis of 4-MTB-GSL contents in radish roots were developed by determining nucleotide sequences of bulked PCR products using a next-generation sequencer. DNA fragments were amplified from two radish lines by multiplex PCR with six primer pairs, and those amplified by 2,880 primer pairs were mixed and sequenced. By assembling sequence data, 1,953 SNPs in 750 DNA fragments, 437 of which have been previously mapped in a linkage map, were identified. A linkage map of nine linkage groups was constructed with 188 markers, and five QTLs were detected in two F2 populations, three of them accounting for more than 50% of the total phenotypic variance being repeatedly detected. In the identified QTL regions, nine SNP markers were newly produced. By synteny analysis of the QTLs regions with Arabidopsis thaliana and Brassica rapa genome sequences, three candidate genes were selected, i.e., RsMAM3 for production of aliphatic glucosinolates linked to GSL-QTL-4, RsIPMDH1 for leucine biosynthesis showing strong co-expression with glucosinolate biosynthesis genes linked to GSL-QTL-2, and RsBCAT4 for branched-chain amino acid aminotransferase linked to GSL-QTL-1. Nucleotide sequences and expression of these genes suggested their possible function in 4MTB-GSL biosynthesis in radish roots.