Characterization of FLOWERING LOCUS C 5 in Brassica rapa L.

Brassica rapa L., which includes Chinese cabbage, turnip, and pak choi, has more complex flowering time regulation than does Arabidopsis thaliana due to the presence of multiple paralogous flowering time genes. FLOWERING LOCUS C (FLC) is one of the key genes regulating the flowering time, and B. rapa has four FLC paralogs. BrFLC5 on the reference genome is deemed a pseudogene because of a mutation (from G to A) in the splice site of the third intron, but there are some accessions with a G nucleotide in the splice site. In this study, we genotyped 310 B. rapa accessions and found that 19 had homozygous and 81 had heterozygous putative functional BrFLC5 alleles. Accessions of turnip showed the highest proportion with a functional BrFLC5 allele. BrFLC5 acts as a floral repressor when overexpressed in A. thaliana. The BrFLC5 expression level varied in pre-vernalized plants, and this transcriptional variation was not associated with the G/A polymorphism in the third intron. Three accessions having a higher BrFLC5 expression in pre-vernalized plants had a 584-bp insertion in the promoter region. Many regions homologous to this 584-bp sequence are present in the B. rapa genome, and this 584-bp inserted region has tandem duplications of an AT-rich sequence in its central region. The possibility that a high expression of a functional BrFLC5 could contribute to producing premature bolting-resistant lines in B. rapa vegetables is discussed. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01405-0.

A 2-Oxoglutarate-Dependent Dioxygenase Mediates the Biosynthesis of Glucoraphasatin in Radish.

Glucosinolates (GSLs) are secondary metabolites whose degradation products confer intrinsic flavors and aromas to Brassicaceae vegetables. Several structures of GSLs are known in the Brassicaceae, and the biosynthetic pathway and regulatory networks have been elucidated in Arabidopsis (Arabidopsis thaliana). GSLs are precursors of chemical defense substances against herbivorous pests. Specific GSLs can act as feeding blockers or stimulants, depending on the pest species. Natural selection has led to diversity in the GSL composition even within individual species. However, in radish (Raphanus sativus), glucoraphasatin (4-methylthio-3-butenyl glucosinolate) accounts for more than 90% of the total GSLs, and little compositional variation is observed. Because glucoraphasatin is not contained in other members of the Brassicaceae, like Arabidopsis and cabbage (Brassica oleracea), the biosynthetic pathways for glucoraphasatin remain unclear. In this report, we identified and characterized a gene encoding GLUCORAPHASATIN SYNTHASE 1 (GRS1) by genetic mapping using a mutant that genetically lacks glucoraphasatin. Transgenic Arabidopsis, which overexpressed GRS1 cDNA, accumulated glucoraphasatin in the leaves. GRS1 encodes a 2-oxoglutarate-dependent dioxygenase, and it is abundantly expressed in the leaf. To further investigate the biosynthesis and transportation of GSLs in radish, we grafted a grs1 plant onto a wild-type plant. The grafting experiment revealed a leaf-to-root long-distance glucoraphasatin transport system in radish and showed that the composition of GSLs differed among the organs. Based on these observations, we propose a characteristic biosynthesis pathway for glucoraphasatin in radish. Our results should be useful in metabolite engineering for breeding of high-value vegetables.

Ubiquitin-Proteasome Dependent Regulation of the GOLDEN2-LIKE 1 Transcription Factor in Response to Plastid Signals.

Arabidopsis (Arabidopsis thaliana) GOLDEN2-LIKE (GLK) transcription factors promote chloroplast biogenesis by regulating the expression of photosynthesis-related genes. Arabidopsis GLK1 is also known to participate in retrograde signaling from chloroplasts to the nucleus. To elucidate the mechanism by which GLK1 is regulated in response to plastid signals, we biochemically characterized Arabidopsis GLK1 protein. Expression analysis of GLK1 protein indicated that GLK1 accumulates in aerial tissues. Both tissue-specific and Suc-dependent accumulation of GLK1 were regulated primarily at the transcriptional level. In contrast, norflurazon- or lincomycin-treated gun1-101 mutant expressing normal levels of GLK1 mRNA failed to accumulate GLK1 protein, suggesting that plastid signals directly regulate the accumulation of GLK1 protein in a GUN1-independent manner. Treatment of the glk1glk2 mutant expressing functional GFP-GLK1 with a proteasome inhibitor, MG-132, induced the accumulation of polyubiquitinated GFP-GLK1. Furthermore, the level of endogenous GLK1 in plants with damaged plastids was partially restored when those plants were treated with MG-132. Collectively, these data indicate that the ubiquitin-proteasome system participates in the degradation of Arabidopsis GLK1 in response to plastid signals.

Epigenetic regulation of agronomical traits in Brassicaceae.

Epigenetic regulation, covalent modification of DNA and changes in histone proteins are closely linked to plant development and stress response through flexibly altering the chromatin structure to regulate gene expression. In this review, we will illustrate the importance of epigenetic influences by discussing three agriculturally important traits of Brassicaceae. (1) Vernalization, an acceleration of flowering by prolonged cold exposure regulated through epigenetic silencing of a central floral repressor, FLOWERING LOCUS C. This is associated with cold-dependent repressive histone mark accumulation, which confers competency of consequence vegetative-to-reproductive phase transition. (2) Hybrid vigor, in which an F1 hybrid shows superior performance to the parental lines. Combination of distinct epigenomes with different DNA methylation states between parental lines is important for increase in growth rate in a hybrid progeny. This is independent of siRNA-directed DNA methylation but dependent on the chromatin remodeler DDM1. (3) Self-incompatibility, a reproductive mating system to prevent self-fertilization. This is controlled by the S-locus consisting of SP11 and SRK which are responsible for self/non-self recognition. Because self-incompatibility in Brassicaceae is sporophytically controlled, there are dominance relationships between S haplotypes in the stigma and pollen. The dominance relationships in the pollen rely on de novo DNA methylation at the promoter region of a recessive allele, which is triggered by siRNA production from a flanking region of a dominant allele.

The production and characterization of a BoFLC2 introgressed Brassica rapa by repeated backcrossing to an F1.

Flowering time is an important agronomic trait for Brassica rapa crops, and previous breeding work in Brassica has successfully transmitted other important agronomic traits from donor species. However, there has been no previous attempts to produce hybrids replacing the original Brassica FLC alleles with alien FLC alleles. In this paper, we introduce the creation of a chromosome substitution line (CSSL) containing a homozygous introgression of Flowering Locus C from Brassica oleracea (BoFLC2) into a B. rapa genomic background, and characterize the CSSL line with respect to the parental cultivars. The preferential transmission of alien chromosome inheritance and the pattern of transmission observed during the production of the CSSLs are also discussed.

The tandem repeated organization of NB-LRR genes in the clubroot-resistant CRb locus in Brassica rapa L.

To facilitate prevention of clubroot disease, a major threat to the successful cultivation of Chinese cabbage (Brassica rapa L.), we bred clubroot-resistant (CR) cultivars by introducing resistance genes from CR turnips via conventional breeding. Among 11 CR loci found in B. rapa, we identified CRb in Chinese cabbage cultivar 'CR Shinki' as a single dominant gene for resistance against Plasmodiophora brassicae pathotype group 3, against which the stacking of Crr1 and Crr2 loci was not effective. However, the precise location and pathotype specificity of CRb have been controversial, because CRa and Rcr1 also map near this locus. Previously, our fine-mapping study revealed that CRb is located in a 140-kb genomic region on chromosome A03. Here, we determined the nucleotide sequence of an approximately 64-kb candidate region in the resistant line; this region contains six open reading frames (ORFs) similar to NB-LRR encoding genes that are predicted to occur in tandem with the same orientation. Among the six ORFs present, only four on the genome of the resistant line showed a strong DNA sequence identity with each other, and only one of those four could confer resistance to P. brassicae isolate No. 14 of the pathotype group 3. These results suggest that these genes evolved through recent gene duplication and uneven crossover events that could lead to the acquisition of clubroot resistance. The DNA sequence of the functional ORF was identical to that of the previously cloned CRa gene; thus, we showed that the independently identified CRb and CRa are one and the same clubroot-resistance gene.

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.

Novel glucosinolate composition lacking 4-methylthio-3-butenyl glucosinolate in Japanese white radish (Raphanus sativus L.).

KEY MESSAGE: Genetic analysis and gene mapping of the 4-methylthio-3-butenyl glucosinolate-less trait of white radish were performed and a white radish cultivar with new glucosinolate composition was developed. A spontaneous mutant having significantly low 4-methylthio-3-butenyl glucosinolate (4MTB-GSL) content was identified from a landrace of Japanese white radish (Raphanus sativus L.) through intensive evaluation of glucosinolate profiles of 632 lines including genetic resources and commercial cultivars using high-performance liquid chromatography (HPLC) analysis. A line lacking 4MTB-GSL was developed using the selected mutant as a gene source. Genetic analyses of F1, F2, and BC1F1 populations of this line suggested that the 4MTB-GSL-less trait is controlled by a single recessive allele. Using SNP and SCAR markers, 96 F2 plants were genotyped, and a linkage map having nine linkage groups with a total map distance of 808.3 cM was constructed. A gene responsible for the 4MTB-GSL-less trait was mapped between CL1753 and CL5895 at the end of linkage group 1. The genetic distance between these markers was 4.2 cM. By selfing and selection of plants lacking 4MTB-GSL, a new cultivar, 'Daikon parental line No. 5', was successfully developed. This cultivar was characterized by glucoerucin, which accounted for more than 90% of the total glucosinolates (GSLs). The total GSL content in roots was ca. 12 µmol/g DW, significantly lower than those in common white radish cultivars. Significance of this line in radish breeding is discussed.

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.

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.

Large insertion in radish GRS1 enhances glucoraphanin content in intergeneric hybrids, Raphanobrassica (Raphanus sativus L. x Brassica oleracea var. acephala).

Glucosinolates (GSLs), precursors of isothiocyanates (ITCs), are present in Brassicaceae plants have been found to have health benefits. Sulforaphane (4-(methylsulfinyl)butyl ITC) is an ITC stored in the form of 4-(methylsulfinyl)butyl GSL (glucoraphanin, 4MSOB) in Brassica vegetables, such as broccoli and kale. Sulforaphane activates Nrf2 expression, a transcription factor responsible for inducing physiological activities such as detoxification in the human body, and it represents a functional component unique to cruciferous vegetables. Raphanobrassica is an inter-generic hybrid between radish and kale, and it contains a high amount of 4MSOB. However, Raphanobrassica contains as much 4-methylsulfinyl-3-butenyl GSL (glucoraphenin, 4MSO3B) as it does 4MSOB. GLUCORAPHASATIN SYNTHASE 1 (GRS1) is an enzyme present in radish that synthesizes 4-methylthio-3-butenyl GSL (glucoraphasatin, 4MT3B), a precursor of 4MSO3B, using 4-(methylthio)butyl GSL (glucoerucin, 4MTB) as a substrate. Since the precursor of 4MSOB is also 4MTB, it was considered that both 4MSOB and 4MSO3B accumulate owing to competition in Raphanobrassica. We hypothesized that owing to the impaired function of GRS1 in Raphanobrassica, it may be possible to breed Raphanobrassica cultivars containing a high 4MSOB content. In this study, we generated Raphanobrassica populations with functional and defective GRS1 and compared the GSL composition in the two populations using high-performance liquid chromatography. The mean 4MSOB content in leaves of the defective-type populations was higher than that in the functional-type population, and the defective/functional ratio ranged from 2.02 to 2.51-fold, supporting this hypothesis. Furthermore, leaves, flower buds, stems, and roots contained higher amounts of 4MSOB in the defective population than in the functional population. The leaf 4MSOB content of defective Raphanobrassica grown in this study was comparable to that of previously studied vegetables (such as broccoli sprouts) with high 4MSOB content. Raphanobrassica with defective GRS1 represents a new leafy vegetable with high 4MSOB content which exhibits anti-cancerous and anti-inflammatory potentials.

Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids.

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.

Small variation of glucosinolate composition in Japanese cultivars of radish (Raphanus sativus L.) requires simple quantitative analysis for breeding of glucosinolate component.

To reveal varietally differing glucosinolate (GSL) contents in radish (Raphanus sativus L.) cultivated in Japan, the total and individual GSLs of 28 cultivars were analyzed using high-performance liquid chromatography. In these cultivars, GSL types including three aliphatic GSLs (glucoraphenin, glucoerucin, and 4-methylthio-3-butenyl GSL (4MTB-GSL)) and three indolyl GSLs (4-hydroxyglucobrassicin, glucobrassicin, and 4-methoxy-glucobrassicin) were detected. No cultivar-specific type of GSL was identified. The dominant GSL was 4MTB-GSL, but its contents differed remarkably: 8.6 µmol/g in 'Koushin' to 135.7 µmol/g in 'Karami 199'. Over about 90% of all GSLs in Japanese radish type are 4MTB-GSL, a higher percentage than in Chinese or European garden radish cultivars. A simple, rapid method for estimating total GSL contents in crude extracts was established because of the small variation of glucosinolate composition in Japanese cultivars. The total GSL content can be estimated using an equation for prediction with absorbance at 425 nm in a mixture of GSL crude extract and palladium (II) chloride solution: Total GSL (µmol/g) = 305.47 × A(425) - 29.66. Its coefficient of determination (R(2)) and standard error of prediction (SEP) are 0.968 and 8.052. This method enables total GSL content estimation from more than 200 samples per person per day.

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.

Genome Triplication Leads to Transcriptional Divergence of FLOWERING LOCUS C Genes During Vernalization in the Genus Brassica.

The genus Brassica includes oil crops, vegetables, condiments, fodder crops, and ornamental plants. Brassica species underwent a whole genome triplication event after speciation between ancestral species of Brassica and closely related genera including Arabidopsis thaliana. Diploid species such as Brassica rapa and Brassica oleracea have three copies of genes orthologous to each A. thaliana gene, although deletion in one or two of the three homologs has occurred in some genes. The floral transition is one of the crucial events in a plant's life history, and time of flowering is an important agricultural trait. There is a variation in flowering time within species of the genus Brassica, and this variation is largely dependent on a difference in vernalization requirements. In Brassica, like in A. thaliana, the key gene of vernalization is FLOWERING LOCUS C (FLC). In Brassica species, the vernalization response including the repression of FLC expression by cold treatment and the enrichment of the repressive histone modification tri-methylated histone H3 lysine 27 (H3K27me3) at the FLC locus is similar to A. thaliana. B. rapa and B. oleracea each have four paralogs of FLC, and the allotetraploid species, Brassica napus, has nine paralogs. The increased number of paralogs makes the role of FLC in vernalization more complicated; in a single plant, paralogs vary in the expression level of FLC before and after vernalization. There is also variation in FLC expression levels between accessions. In this review, we focus on the regulatory circuits of the vernalization response of FLC expression in the genus Brassica.

Induction of TOC and TIC genes during photomorphogenesis is mediated primarily by cryptochrome 1 in Arabidopsis.

The majority of genes encoding photosynthesis-associated proteins in the nucleus are induced by light during photomorphogenesis, allowing plants to establish photoautotrophic growth. Therefore, optimizing the protein import apparatus of plastids, designated as the translocon at the outer and inner envelope membranes of chloroplast (TOC-TIC) complex, upon light exposure is a prerequisite to the import of abundant nuclear-encoded photosynthesis-associated proteins. However, the mechanism that coordinates the optimization of the TOC-TIC complex with the expression of nuclear-encoded photosynthesis-associated genes remains to be characterized in detail. To address this question, we investigated the mechanism by which plastid protein import is regulated by light during photomorphogenesis in Arabidopsis. We found that the albino plastid protein import2 (ppi2) mutant lacking Toc159 protein import receptors have active photoreceptors, even though the mutant fails to induce the expression of photosynthesis-associated nuclear genes upon light illumination. In contrast, many TOC and TIC genes are rapidly induced by blue light in both WT and the ppi2 mutant. We uncovered that this regulation is mediated primarily by cryptochrome 1 (CRY1). Furthermore, deficiency of CRY1 resulted in the decrease of some TOC proteins in vivo. Our results suggest that CRY1 plays key roles in optimizing the content of the TOC-TIC apparatus to accommodate the import of abundant photosynthesis-associated proteins during photomorphogenesis.

The role of FRIGIDA and FLOWERING LOCUS C genes in flowering time of Brassica rapa leafy vegetables.

There is a wide variation of flowering time among lines of Brassica rapa L. Most B. rapa leafy (Chinese cabbage etc.) or root (turnip) vegetables require prolonged cold exposure for flowering, known as vernalization. Premature bolting caused by low temperature leads to a reduction in the yield/quality of these B. rapa vegetables. Therefore, high bolting resistance is an important breeding trait, and understanding the molecular mechanism of vernalization is necessary to achieve this goal. In this study, we demonstrated that BrFRIb functions as an activator of BrFLC in B. rapa. We showed a positive correlation between the steady state expression levels of the sum of the BrFLC paralogs and the days to flowering after four weeks of cold treatment, suggesting that this is an indicator of the vernalization requirement. We indicate that BrFLCs are repressed by the accumulation of H3K27me3 and that the spreading of H3K27me3 promotes stable FLC repression. However, there was no clear relationship between the level of H3K27me3 in the BrFLC and the vernalization requirement. We also showed that if there was a high vernalization requirement, the rate of repression of BrFLC1 expression following prolonged cold treatments was lower.

Long noncoding RNAs in Brassica rapa L. following vernalization.

Brassica rapa L. is an important agricultural crop that requires a period of prolonged cold for flowering. This process is known as vernalization. Studies have shown that long noncoding RNAs (lncRNAs) play important roles in abiotic stress responses and several cold-responsive noncoding RNAs have been suggested to be involved in vernalization. We examined the transcriptome of the Chinese cabbage inbred line (B. rapa L. var. pekinensis) RJKB-T24, and identified 1,444 long intergenic noncoding RNAs (lincRNAs), 551 natural antisense transcripts (NATs), and 93 intronic noncoding RNAs (incRNAs); 549 of the 2,088 lncRNAs significantly altered their expression in response to four weeks of cold treatment. Most differentially expressed lncRNAs did not lead to a change of expression levels in mRNAs covering or near lncRNAs, suggesting that the transcriptional responses to four weeks of cold treatment in lncRNA and mRNA are independent. However, some differentially expressed mRNAs had NATs with expression altered in the same direction. These genes were categorized as having an abiotic stress response, suggesting that the paired-expression may play a role in the transcriptional response to vernalization or cold treatment. We also identified short-term cold treatment induced NATs in BrFLC and BrMAF genes, which are involved in vernalization. The lncRNAs we identified differed from those reported in Arabidopsis thaliana, suggesting the role of lncRNAs in vernalization differ between these two species.

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.

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.