Histone Deacetylase Inhibitor, Sodium Butyrate-Induced Metabolic Modulation in Platycodon grandiflorus Roots Enhances Anti-Melanogenic Properties.

While the status of histone acetylation is a critical regulator of chromatin's structure with a significant impact on plant physiology, our understanding of epigenetic regulation in the biosynthesis of active compounds in plants is limited. In this study, Platycodon grandiflorus was treated with sodium butyrate (NaB), a histone deacetylase inhibitor, to investigate the influence of histone acetylation on secondary metabolism. Its treatment with NaB increased the acetylation of histone H3 at lysine 9, 14, and 27 and enhanced the anti-melanogenic properties of P. grandiflorus roots. Through transcriptome and differentially expressed gene analyses, we found that NaB influenced the expression of genes that were involved in both primary and secondary metabolic pathways. In addition, NaB treatment caused the accumulation of polyphenolic compounds, including dihydroquercetin, gallic acid, and 2,4-dihydroxybenzoic acid. The NaB-induced transcriptional activation of genes in the phenylpropanoid biosynthetic pathway influenced the anti-melanogenic properties of P. grandiflorus roots. Overall, these findings suggest the potential of an epigenomic approach to enhance the medicinal qualities of medicinal plants.

Physiological and sucrose metabolic responses to waterlogging stress in balloon flower (Platycodon grandiflorus (Jacq.) A. DC).

Waterlogging stress is a major limiting factor resulting in stunted growth and loss of crop productivity, especially for root crops. However, physiological responses to waterlogging have been studied in only a few plant models. To gain insight into how balloon flower (Platycodon grandiflorus (Jacq.) A. DC) responds to waterlogging stress, we investigate changes to sucrose metabolism combined with a physiological analysis. Although waterlogging stress decreased the photosynthetic rate in balloon flower, leaves exhibited an increase in glucose (ninefold), fructose (4.7-fold), and sucrose (2.1-fold), indicating inhibition of sugar transport via the phloem. In addition, roots showed a typical response to hypoxia, such as the accumulation of proline (4.5-fold higher than in control roots) and soluble sugars (2.1-fold higher than in control roots). The activities and expression patterns of sucrose catabolizing enzymes suggest that waterlogging stress leads to a shift in the pathway of sucrose degradation from invertase to sucrose synthase (Susy), which consumes less ATP. Furthermore, we suggest that the waterlogging-stress-induced gene PlgSusy1 encodes the functional Susy enzyme, which may contribute to improving tolerance in balloon flower to waterlogging. As a first step toward understanding the waterlogging-induced regulatory mechanisms in balloon flower, we provide a solid foundation for further understanding waterlogging-induced alterations in source-sink relationships. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01310-y.

Antioxidant and Anti-Melanogenic Activities of Heat-Treated Licorice (Wongam, Glycyrrhiza glabra × G. uralensis) Extract.

Melanin is a brown or black pigment that protects skin from ultraviolet radiation and reactive oxygen species (ROS). However, overproduction of melanin is associated with lentigines, melasma, freckles and skin cancer. Licorice has shown antioxidant, anti-tumor, anti-platelet, anti-inflammatory and immunomodulatory activities and is used as a natural treatment for skin whitening. We aimed to confirm the potential of Wongam, a new cultivar of licorice developed by the Rural Development Administration (RDA), as a whitening agent in cosmetics. In addition, we verified the effect of heat treatment on the bioactivity of licorice by comparing antioxidant and anti-melanogenic activities of licorice extract before and after heating (130 °C). The heat-treated licorice extract (WH-130) showed higher radical-scavenging activities in the ABTS+ (2,2'-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays. In addition, WH-130 inhibited melanogenesis more effectively due to downregulation of tyrosinase in B16F10 melanoma cells than non-heated licorice extract. Moreover, heat treatment increased total phenolic content. In particular, isoliquiritigenin, an antioxidant and anti-melanogenic compound of licorice, was produced by heat treatment. In conclusion, WH-130, with increased levels of bioactive phenolics such as isoliquiritigenin, has potential for development into a novel skin whitening material with applications in cosmetics.

Ethyl Acetate Fraction of Aqueous Extract of Lentinula edodes Inhibits Osteoclastogenesis by Suppressing NFATc1 Expression.

Bone tissue is continuously remodeled by the coordinated action of osteoclasts and osteoblasts. Nuclear factor-activated T cells c1 (NFATc1) is a well-known transcription factor for osteoclastogenesis and transcriptionally activated by the c-Fos and nuclear factor-kappa B (NF-κB) signaling pathways in response to receptor activation of NF-κB ligand (RANKL). Since excessive RANKL signaling causes an increase of osteoclast formation and bone resorption, inhibition of RANKL or its signaling pathway is an attractive therapeutic approach to the treatment of pathologic bone loss. In this study, we show that an ethyl acetate fraction (LEA) from the shiitake mushroom, Lentinula edodes, inhibited RANKL-induced osteoclast differentiation by blocking the NFATc1 signaling pathway. We found that the water extract and its subsequent ethyl acetate fraction of L. edodes significantly suppressed osteoclast formation. Comparative transcriptome analysis revealed that LEA specifically downregulated a set of RANKL target genes, including Nfatc1. Next, we found that LEA suppresses Nfatc1 expression mainly through the inhibition of the transactivity of p65 and NFATc1. Moreover, treatment of LEA rescued an osteoporotic phenotype in a zebrafish model of glucocorticoid-induced osteoporosis. Collectively, our findings define an undocumented role of the shiitake mushroom extract in regulating bone development.

Establishment of a UPLC-PDA/ESI-Q-TOF/MS-Based Approach for the Simultaneous Analysis of Multiple Phenolic Compounds in Amaranth (A. cruentus and A. tricolor).

We used ultraperformance liquid chromatography coupled with a photodiode-array detector and electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-PDA/ESI-Q-TOF/MS) to rapidly and accurately quantify 17 phenolic compounds. Then, we applied this method to the seed and leaf extracts of two Amaranthus species to identify and quantify phenolic compounds other than the 17 compounds mentioned above. Compounds were eluted within 30 min on a C18 column using a mobile phase (water and acetonitrile) containing 0.1% formic acid, and the specific wavelength and ion information of the compounds obtained by PDA and ESI-Q-TOF/MS were confirmed. The proposed method showed good linearity (r2 > 0.990). Limits of detection and quantification were less than 0.1 and 0.1 µg/mL, respectively. Intra- and interday precision were less than 2.4% and 1.8%, respectively. Analysis of amaranth seed and leaf extracts using the established method showed that the seeds contained high amounts of 2,4-dihydroxybenzoic acid and kaempferol, and leaves contained diverse phenolic compounds. In addition, six tentatively new phenolic compounds were identified. Moreover, seeds potentially contained 2,3-dihydroxybenzaldehyde, a beneficial bioactive compound. Thus, our method was an efficient approach for the qualitative and quantitative analysis of phenolic compounds, and could be used to investigate phenolic compounds in plants.

Brassinosteroids facilitate xylem differentiation and wood formation in tomato.

MAIN CONCLUSION: BR signaling pathways facilitate xylem differentiation and wood formation by fine tuning SlBZR1/SlBZR2-mediated gene expression networks involved in plant secondary growth. Brassinosteroid (BR) signaling and BR crosstalk with diverse signaling cues are involved in the pleiotropic regulation of plant growth and development. Recent studies reported the critical roles of BR biosynthesis and signaling in vascular bundle development and plant secondary growth; however, the molecular bases of these roles are unclear. Here, we performed comparative physiological and anatomical analyses of shoot morphological growth in a cultivated wild-type tomato (Solanum lycopersicum cv. BGA) and a BR biosynthetic mutant [Micro Tom (MT)]. We observed that the canonical BR signaling pathway was essential for xylem differentiation and sequential wood formation by facilitating plant secondary growth. The gradual retardation of xylem development phenotypes during shoot vegetative growth in the BR-deficient MT tomato mutant recovered completely in response to exogenous BR treatment or genetic complementation of the BR biosynthetic DWARF (D) gene. By contrast, overexpression of the tomato Glycogen synthase kinase 3 (SlGSK3) or CRISPR-Cas9 (CR)-mediated knockout of the tomato Brassinosteroid-insensitive 1 (SlBRI1) impaired BR signaling and resulted in severely defective xylem differentiation and secondary growth. Genetic modulation of the transcriptional activity of the tomato Brassinazole-resistant 1/2 (SlBZR1/SlBZR2) confirmed the positive roles of BR signaling pathways for xylem differentiation and secondary growth. Our data indicate that BR signaling pathways directly promote xylem differentiation and wood formation by canonical BR-activated SlBZR1/SlBZR2.

Metabolic Control of Tobacco Pollination by Sugars and Invertases.

Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrates.

Transcriptome Analysis in Chinese Cabbage (Brassica rapa ssp. pekinensis) Provides the Role of Glucosinolate Metabolism in Response to Drought Stress.

Although drought stress is one of the most limiting factors in growth and production of Chinese cabbage (Brassica rapa L. ssp. pekinensis), the underlying biochemical and molecular causes are poorly understood. In the present study, to address the mechanisms underlying the drought responses, we analyzed the transcriptome profile of Chinese cabbage grown under drought conditions. Drought stress transcriptionally activated several transcription factor genes, including AP2/ERFs, bHLHs, NACs and bZIPs, and was found to possibly result in transcriptional variation in genes involved in organic substance metabolic processes. In addition, comparative expression analysis of selected BrbZIPs under different stress conditions suggested that drought-induced BrbZIPs are important for improving drought tolerance. Further, drought stress in Chinese cabbage caused differential acclimation responses in glucosinolate metabolism in leaves and roots. Analysis of stomatal aperture indicated that drought-induced accumulation of glucosinolates in leaves directly or indirectly controlled stomatal closure to prevent water loss, suggesting that organ-specific responses are essential for plant survival under drought stress condition. Taken together, our results provide information important for further studies on molecular mechanisms of drought tolerance in Chinese cabbage.

The Arabidopsis PLAT domain protein1 promotes abiotic stress tolerance and growth in tobacco.

Plant growth and consequently crop yield can be severely compromised by abiotic and biotic stress conditions. Transgenic approaches that resulted in increased tolerance against abiotic stresses often were typically accompanied by adverse effects on plant growth and fitness under optimal growing conditions. Proteins that belong to the PLAT-plant-stress protein family harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and are ubiquitously present in monocot and dicot plant species. Until now, only limited data is available for PLAT-plant-stress family members, which suggested that these proteins in general could promote tolerance towards stress responses. We studied the function of the Arabidopsis PLAT-plant-stress protein AtPLAT1 employing heterologous gain-of-function analysis in tobacco. AtPLAT1 conferred increased abiotic stress tolerance in tobacco, evident by improved tolerance towards cold, drought and salt stresses, and promoted growth, reflected by a faster development under non-stressed conditions. However, the overexpression of AtPLAT1 in tobacco reduced the tolerance towards biotic stress conditions and, therefore, could be involved in regulating the crosstalk between abiotic and biotic stress responses. Thus, we showed that heterologously expressed AtPLAT1 functions as positive regulator of abiotic stress tolerance and plant growth, which could be an important new asset for strategies to develop plants with improved abiotic stress tolerance, without growth and subsequent yield penalties under optimal growth conditions.

Cell-to-cell movement of viruses via plasmodesmata.

Plant viruses utilize plasmodesmata (PD), unique membrane-lined cytoplasmic nanobridges in plants, to spread infection cell-to-cell and long-distance. Such invasion involves a range of regulatory mechanisms to target and modify PD. Exciting discoveries in this field suggest that these mechanisms are executed by the interaction between plant cellular components and viral movement proteins (MPs) or other virus-encoded factors. Striking working analogies exist among endogenous non-cell-autonomous proteins and viral MPs, in which not only do they all use PD to traffic, but also they exploit same regulatory components to exert their functions. Thus, this review discusses on the viral strategies to move via PD and the PD-regulatory mechanisms involved in viral pathogenesis.

RNA-seq analysis of Rubus idaeus cv. Nova: transcriptome sequencing and de novo assembly for subsequent functional genomics approaches.

KEY MESSAGE: Using Illumina sequencing technology, we have generated the large-scale transcriptome sequencing data containing abundant information on genes involved in the metabolic pathways in R. idaeus cv. Nova fruits. Rubus idaeus (Red raspberry) is one of the important economical crops that possess numerous nutrients, micronutrients and phytochemicals with essential health benefits to human. The molecular mechanism underlying the ripening process and phytochemical biosynthesis in red raspberry is attributed to the changes in gene expression, but very limited transcriptomic and genomic information in public databases is available. To address this issue, we generated more than 51 million sequencing reads from R. idaeus cv. Nova fruit using Illumina RNA-Seq technology. After de novo assembly, we obtained 42,604 unigenes with an average length of 812 bp. At the protein level, Nova fruit transcriptome showed 77 and 68 % sequence similarities with Rubus coreanus and Fragaria versa, respectively, indicating the evolutionary relationship between them. In addition, 69 % of assembled unigenes were annotated using public databases including NCBI non-redundant, Cluster of Orthologous Groups and Gene ontology database, suggesting that our transcriptome dataset provides a valuable resource for investigating metabolic processes in red raspberry. To analyze the relationship between several novel transcripts and the amounts of metabolites such as γ-aminobutyric acid and anthocyanins, real-time PCR and target metabolite analysis were performed on two different ripening stages of Nova. This is the first attempt using Illumina sequencing platform for RNA sequencing and de novo assembly of Nova fruit without reference genome. Our data provide the most comprehensive transcriptome resource available for Rubus fruits, and will be useful for understanding the ripening process and for breeding R. idaeus cultivars with improved fruit quality.

Comparative Analysis of Polyphenolic Compounds in Different Amaranthus Species: Influence of Genotypes and Harvesting Year.

Amaranth is a nutritionally valuable crop, as it contains phenolic acids and flavonoids, yielding diverse plant secondary metabolites (PSMs) like phytosterol, tocopherols, and carotenoids. This study explored the variations in the contents of seventeen polyphenolic compounds within the leaves of one hundred twenty Amaranthus accessions representing nine Amaranthus species. The investigation entailed the analysis of phenolic content across nine Amaranthus species, specifically A. hypochondriacus, A. cruentus, A. caudatus, A. tricolor, A. dubius, A. blitum, A. crispus, A. hybridus, and A. viridis, utilizing ultra performance liquid chromatography with photodiode array detection (UPLC-PDA). The results revealed significant differences in polyphenolic compounds among accessions in which rutin content was predominant in all Amaranthus species in both 2018 and 2019. Among the nine Amaranthus species, the rutin content ranged from 95.72 ± 199.17 µg g-1 (A. dubius) to 1485.09 ± 679.51 µg g-1 (A. viridis) in 2018 and from 821.59 ± 709.95 µg g-1 (A. tricolor) to 3166.52 ± 1317.38 µg g-1 (A. hypochondriacus) in 2019. Correlation analysis revealed, significant positive correlations between rutin and kaempferol-3-O-ß-rutinoside (r = 0.93), benzoic acid and ferulic acid (r = 0.76), and benzoic acid and kaempferol-3-O-ß-rutinoside (r = 0.76), whereas gallic acid showed consistently negative correlations with each of the 16 phenolic compounds. Wide variations were identified among accessions and between plants grown in the two years. The nine species and one hundred twenty Amaranthus accessions were clustered into six groups based on their seventeen phenolic compounds in each year. These findings contribute to expanding our understanding of the phytochemical traits of accessions within nine Amaranthus species, which serve as valuable resources for Amaranthus component breeding and functional material development.

PlgMYBR1, an R2R3-MYB transcription factor, plays as a negative regulator of anthocyanin biosynthesis in Platycodon grandiflorus.

Floral color plays a major role in pollinator specificity, and changes in color may result in pollinator shifts and pollinator-mediated speciation. In the purple flowers of Platycodon grandiflorus, anthocyanins are the major pigment metabolites, whereas white flowers result due to the absence of anthocyanins. The lack of anthocyanins may be due to the inhibition of the anthocyanin biosynthesis pathway. However, the molecular mechanism of anthocyanin biosynthesis in P. grandiflorus is not fully understood. Hence, we identified R2R3-MYB transcription factor, PlgMYBR1, as a negative regulator for anthocyanin biosynthesis using sequence homology and tissue-specific expression pattern analyses. A heterologous co-expression assay suggested that PlgMYBR1 inhibited the function of AtPAP1 (Arabidopsis thaliana production of anthocyanin pigment 1), indicating that PlgMYBR1 plays as a repressor of anthocyanin biosynthesis in P. grandiflorus. Our results provide a foundation for future efforts to understand the anthocyanin biosynthesis in P. grandiflorus and, thereby, to improve flower color through genetic engineering. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03490-6.

Overexpression of the Brassica rapa bZIP Transcription Factor, BrbZIP-S, Increases the Stress Tolerance in Nicotiana benthamiana.

In higher plants, S1-basic region-leucine zipper (S1-bZIP) transcription factors fulfill crucial roles in the physiological homeostasis of carbon and amino acid metabolisms and stress responses. However, very little is known about the physiological role of S1-bZIP in cruciferous vegetables. Here, we analyzed the physiological function of S1-bZIP from Brassica rapa (BrbZIP-S) in modulating proline and sugar metabolism. Overexpression of BrbZIP-S in Nicotiana benthamiana resulted in delayed chlorophyll degradation during the response to dark conditions. Under heat stress or recovery conditions, the transgenic lines exhibited a lower accumulation of H2O2, malondialdehyde, and protein carbonyls compared to the levels in transgenic control plants. These results strongly indicate that BrbZIP-S regulates plant tolerance against dark and heat stress. We propose that BrbZIP-S is a modulator of proline and sugar metabolism, which are required for energy homeostasis in response to environmental stress conditions.

Gibberellic acid 3 enhanced the anticancer activity of Abeliophyllum distichum adventitious roots by activating the diterpenoid biosynthesis pathway.

The industrial value of various plants has been improved through the of plant cell culture systems with elicitation. In this study, the adventitious root of Abeliophyllum distichum (AdAR) was treated with gibberellic acid 3 (GA3) to improve its anticancer property. The hexane fraction of the GA3-treated A. distichum adventitious root exhibited a stronger cytotoxic activity against A549 cells than the hexane fraction of AdAR. Through GC/MS and principal component analysis, we identified ferruginol and sugiol as anticancer compounds, which were induced by GA3 treatment in AdAR. Gene expression analysis combined with functional characterisation suggests that the GA3 treatment increased the transcription of geranylgeranyl pyrophosphate synthases and copalyl diphosphate synthase, which led to the accumulation of diterpenoids, including ferruginol and sugiol. Overall, these findings can contribute to the advancement of metabolic engineering for enhancing the biosynthesis of active diterpenoids, and facilitate the large-scale production of bioactive compounds sourced from A. distichum.

De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis.

The ripe fruit of Momordica cochinchinensis Spreng, known as gac, is featured by very high carotenoid content. Although this plant might be a good resource for carotenoid metabolic engineering, so far, the genes involved in the carotenoid metabolic pathways in gac were unidentified due to lack of genomic information in the public database. In order to expedite the process of gene discovery, we have undertaken Illumina deep sequencing of mRNA prepared from aril of gac fruit. From 51,446,670 high-quality reads, we obtained 81,404 assembled unigenes with average length of 388 base pairs. At the protein level, gac aril transcripts showed about 81.5% similarity with cucumber proteomes. In addition 17,104 unigenes have been assigned to specific metabolic pathways in Kyoto Encyclopedia of Genes and Genomes, and all of known enzymes involved in terpenoid backbones biosynthetic and carotenoid biosynthetic pathways were also identified in our library. To analyze the relationship between putative carotenoid biosynthesis genes and alteration of carotenoid content during fruit ripening, digital gene expression analysis was performed on three different ripening stages of aril. This study has revealed putative phytoene synthase, 15-cis-phytone desaturase, zeta-carotene desaturase, carotenoid isomerase and lycopene epsilon cyclase might be key factors for controlling carotenoid contents during aril ripening. Taken together, this study has also made availability of a large gene database. This unique information for gac gene discovery would be helpful to facilitate functional studies for improving carotenoid quantities.

Identification and Functional Characterization of FLOWERING LOCUS T in Platycodon grandiflorus.

Platycodon grandiflorus roots have been used as a foodstuff and traditional medicine for thousands of years in East Asia. In order to increase the root development of P. grandiflorus, cultivators removed the inflorescences, suggesting the possible negative effect of flowering on root development. This indicates that the genetic improvement of P. grandiflorus by late flowering is a potential approach to increase productivity. However, nothing is known about key genes integrating multiple flowering pathways in P. grandiflorus. In order to fill this gap, we identified potential homologs of the FLOWERING LOCUS T (FT) gene in P. grandiflorus. The alignment with other FT members and phylogenetic analysis revealed that the P. grandiflorus FT (PlgFT) protein contains highly conserved functional domains and belongs to the FT-like clade. The expression analysis revealed spatial variations in the transcription of PlgFT in different organs. In addition, the expression level of PlgFT was increased by high temperature but not by photoperiodic light input signals, presumably due to lacking the CONSTANS binding motif in its promoter region. Furthermore, PlgFT induced early flowering upon its overexpression in P. grandiflorus, suggesting the functional role of PlgFT in flowering. Taken together, we functionally characterized PlgFT as a master regulator of P. grandiflorus flowering under inductive high temperature, which will serve as an important target gene for improving the root productivity.

Complete Chloroplast Genome of the Inverted Repeat-Lacking Species Vicia bungei and Development of Polymorphic Simple Sequence Repeat Markers.

Background: Vicia bungei is an economically important forage crop in South Korea and China. Although detailed genetic and genomic data can improve population genetic studies, conservation efforts, and improved breeding of crops, few such data are available for Vicia species in general and none at all for V. bungei. Therefore, the main objectives of this study were to sequence, assemble, and annotate V. bungei chloroplast genome and to identify simple sequence repeats (SSRs) as polymorphic genetic markers. Results: The whole-genome sequence of V. bungei was generated using an Illumina MiSeq platform. De novo assembly of complete chloroplast genome sequences was performed for the low-coverage sequence using CLC Genome Assembler with a 200-600-bp overlap size. Vicia bungei chloroplast genome was 130,796-bp long. The genome lacked an inverted repeat unit and thus resembled those of species in the inverted repeat-lacking clade within Fabaceae. Genome annotation using Dual OrganellarGenoMe Annotator (DOGMA) identified 107 genes, comprising 75 protein-coding, 28 transfer RNA, and 4 ribosomal RNA genes. In total, 432 SSRs were detected in V. bungei chloroplast genome, including 64 mononucleotides, 14 dinucleotides, 5 trinucleotides, 4 tetranucleotides, 233 pentanucleotides, 90 hexanucleotides, and 14 complex repeated motifs. These were used to develop 232 novel chloroplast SSR markers, 39 of which were chosen at random to test amplification and genetic diversity in Vicia species (20 accessions from seven species). The unweighted pair group method with arithmetic mean cluster analysis identified seven clusters at the interspecies level and intraspecific differences within clusters. Conclusion: The complete chloroplast genome sequence of V. bungei was determined. This reference genome should facilitate chloroplast resequencing and future searches for additional genetic markers using population samples. The novel chloroplast genome resources and SSR markers will greatly contribute to the conservation of the genus Vicia and facilitate genetic and evolutionary studies of this genus and of other higher plants.

Plant Response to Cold Stress: Cold Stress Changes Antioxidant Metabolism in Heading Type Kimchi Cabbage (Brassica rapa L. ssp. Pekinensis).

Cold stress is known as the important yield-limiting factor of heading type Kimchi cabbage (HtKc, Brassica rapa L. ssp. pekinensis), which is an economically important crop worldwide. However, the biochemical and molecular responses to cold stress in HtKc are largely unknown. In this study, we conducted transcriptome analyses on HtKc grown under normal versus cold conditions to investigate the molecular mechanism underlying HtKc responses to cold stress. A total of 2131 genes (936 up-regulated and 1195 down-regulated) were identified as differentially expressed genes and were significantly annotated in the category of "response to stimulus". In addition, cold stress caused the accumulation of polyphenolic compounds, including p-coumaric, ferulic, and sinapic acids, in HtKc by inducing the phenylpropanoid pathway. The results of the chemical-based antioxidant assay indicated that the cold-induced polyphenolic compounds improved the free-radical scavenging activity and antioxidant capacity, suggesting that the phenylpropanoid pathway induced by cold stress contributes to resistance to cold-induced reactive oxygen species in HtKc. Taken together, our results will serve as an important base to improve the cold tolerance in plants via enhancing the antioxidant machinery.

ROS-mediated plasmodesmal regulation requires a network of an Arabidopsis receptor-like kinase, calmodulin-like proteins, and callose synthases.

Plasmodesmata (PD) play a critical role in symplasmic communication, coordinating plant activities related to growth & development, and environmental stress responses. Most developmental and environmental stress signals induce reactive oxygen species (ROS)-mediated signaling in the apoplast that causes PD closure by callose deposition. Although the apoplastic ROS signals are primarily perceived at the plasma membrane (PM) by receptor-like kinases (RLKs), such components involved in PD regulation are not yet known. Here, we show that an Arabidopsis NOVEL CYS-RICH RECEPTOR KINASE (NCRK), a PD-localized protein, is required for plasmodesmal callose deposition in response to ROS stress. We identified the involvement of NCRK in callose accumulation at PD channels in either basal level or ROS-dependent manner. Loss-of-function mutant (ncrk) of NCRK induces impaired callose accumulation at the PD under the ROS stress resembling a phenotype of the PD-regulating GLUCAN SYNTHASE-LIKE 4 (gsl4) knock-out plant. The overexpression of transgenic NCRK can complement the callose and the PD permeability phenotypes of ncrk mutants but not kinase-inactive NCRK variants or Cys-mutant NCRK, in which Cys residues were mutated in Cys-rich repeat ectodomain. Interestingly, NCRK mediates plasmodesmal permeability in mechanical injury-mediated signaling pathways regulated by GSL4. Furthermore, we show that NCRK interacts with calmodulin-like protein 41 (CML41) and GSL4 in response to ROS stress. Altogether, our data indicate that NCRK functions as an upstream regulator of PD callose accumulation in response to ROS-mediated stress signaling pathways.