Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage.

Flavonols are the major class of flavonoids of green Chinese cabbage (Brassica rapa subsp. pekinensis). The B. rapa genome harbors seven flavonol synthase genes (BrFLSs), but they have not been functionally characterized. Here, transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage. Among them, only BrFLS1 showed major FLS activity and additional flavanone 3ß-hydroxylase (F3H) activity, while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities. We generated BrFLS1-knockout (BrFLS1-KO) Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation, which were further advanced to the T2 generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants, while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols. Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants. In accordance, total phenolic contents were slightly enhanced in the BrFLS1-KO plants, which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage. Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes, but subtle morphological changes in their heads were observed. In addition, their seedlings were susceptible to osmotic stress compared to the controls, suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling. In this study, we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

Functional Characterization of BrF3'H, Which Determines the Typical Flavonoid Profile of Purple Chinese Cabbage.

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3'-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3'-hydroxylase BrF3'H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3'H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3'H protein catalyzes the 3'-hydroxylation of a wide range of 4'-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3'H among those tested. Transient expression of BrF3'H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3'H in planta. As the first functional characterization of BrF3'H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

Transcriptome Analysis and Metabolic Profiling of Green and Red Mizuna (Brassica rapa L. var. japonica).

Mizuna (Brassica rapa L. var. japonica), a member of the family Brassicaceae, is rich in various health-beneficial phytochemicals, such as glucosinolates, phenolics, and anthocyanins. However, few studies have been conducted on genes associated with metabolic traits in mizuna. Thus, this study provides a better insight into the metabolic differences between green and red mizuna via the integration of transcriptome and metabolome analyses. A mizuna RNAseq analysis dataset showed 257 differentially expressed unigenes (DEGs) with a false discovery rate (FDR) of <0.05. These DEGs included the biosynthesis genes of secondary metabolites, such as anthocyanins, glucosinolates, and phenolics. Particularly, the expression of aliphatic glucosinolate biosynthetic genes was higher in the green cultivar. In contrast, the expression of most genes related to indolic glucosinolates, phenylpropanoids, and flavonoids was higher in the red cultivar. Furthermore, the metabolic analysis showed that 14 glucosinolates, 12 anthocyanins, five phenolics, and two organic acids were detected in both cultivars. The anthocyanin levels were higher in red than in green mizuna, while the glucosinolate levels were higher in green than in red mizuna. Consistent with the results of phytochemical analyses, the transcriptome data revealed that the expression levels of the phenylpropanoid and flavonoid biosynthesis genes were significantly higher in red mizuna, while those of the glucosinolate biosynthetic genes were significantly upregulated in green mizuna. A total of 43 metabolites, such as amino acids, carbohydrates, tricarboxylic acid (TCA) cycle intermediates, organic acids, and amines, was identified and quantified in both cultivars using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS). Among the identified metabolites, sucrose was positively correlated with anthocyanins, as previously reported.

Saccharibacillus brassicae sp. nov., an endophytic bacterium isolated from kimchi cabbage (Brassica rapa subsp. pekinensis) seeds.

Strain ATSA2T was isolated from surface-sterilized kimchi cabbage (Brassica rapa subsp. pekinensis) seeds and represents a novel bacterium based on the polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ATSA2T formed a lineage within genus Saccharibacillus and was most closely to Saccharibacillus deserti WLG055T (98.1%) and Saccharibacillus qing-shengii H6T (97.9%). The whole-genome of ATSA2T comprised a 5,619,468 bp of circular chromosome with 58.4% G + C content. The DNA-DNA relatedness values between strain ATSA2T and its closely related type strains S. deserti WLJ055 and S. qingshengii H6T were 26.0% and 24.0%, respectively. Multiple gene clusters associated with plant growth promotion activities (stress response, nitrogen and phosphorus metabolism, and auxin biosynthesis) were annotated in the genome. Strain ATSA2T was Gram-positive, endospore-forming, facultatively anaerobic, and rod-shaped It grew at 15-37°C (optimum 25°C), pH 6.0-10.0 (optimum pH 8.0), and in the presence of 0-5% (w/v) NaCl (optimum 1%). The major cellular fatty acids (> 10%) of strain ATSA2T were anteiso-C15:0 and C16:0. MK-7 was the major isoprenoid quinone. The major polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, and three unknown glycolipids. Based on its phylogenetic, genomic, phenotypic, and chemotaxo-nomic features, strain ATSA2T is proposed to represent a novel species of genus Saccharibacillus, for which the name is Saccharibacillus brassicae sp. nov. The type strain is ATSA2T (KCTC 43072T = CCTCC AB 2019223T).

Whole-genome sequence data and analysis of Saccharibacillus sp. ATSA2 isolated from Kimchi cabbage seeds.

Saccharibacillus sp. ATSA2 was isolated from Kimchi cabbage seeds grown in Gyeongbuk province in the Republic of Korea. Whole-genome sequencing of Saccharibacillus sp. ATSA2 was performed using the PacBio RSII and Illumina HiSeq platforms, and it features a 5,619,468 bp circular chromosome with 58.4% G + C content. The genome includes 4543 protein-coding genes, 104 RNA genes (70 transfer RNA genes, 30 ribosomal RNA genes, and 4 non-coding RNA), and 73 pseudogenes. Multiple gene clusters associated with stress responses, nitrogen and phosphorus metabolism, and plant hormone biosynthesis were annotated in the genome. The genome information will provide fundamental knowledge of this organism as well as insight for understanding microbial activity in the agricultural application. The whole-genome sequence of Saccharibacillus sp. ATSA2 is available at GenBank/EMBL/DDBJ under accession number CP041217.

Comparative Metabolic Profiling of Green and Purple Pakchoi (Brassica Rapa Subsp. Chinensis).

Pakchoi (Brassica rapa subsp. chinensis) is cultivated for its nutritional value, particularly with regard to vitamins, minerals and dietary fibers. However, limited metabolic information is available on the phyto-nutritional traits of pakchoi. Our GC-TOF MS analysis showed that green pakchoi has higher contents of carbon metabolism-associated metabolites such as sugars, sugar derivatives and inositol, while purple pakchoi has higher levels of nitrogen metabolism-associated metabolites such as amino acids and amino acid derivatives. To compare the content and composition of secondary metabolites in green and purple pakchoi, we analyzed phenylpropanoid-derived compounds and anthocyanins in mature leaves using an HPLC-UV system. This analysis identified 9 phenylpropanoid-derived compounds and 12 anthocyanins in the mature leaves of green and purple pakchoi. The level of rutin was significantly higher in purple pakchoi compared with green pakchoi, consistent with the expression of phenylpropanoid biosynthetic genes in the two pakchoi cultivars. The data obtained from this comprehensive metabolic profiling would be helpful to improve our understanding of the nutritional values of pakchoi cultivars as food sources.

High-throughput sequencing and de novo assembly of Brassica oleracea var. Capitata L. for transcriptome analysis.

BACKGROUND: The cabbage, Brassica oleracea var. capitata L., has a distinguishable phenotype within the genus Brassica. Despite the economic and genetic importance of cabbage, there is little genomic data for cabbage, and most studies of Brassica are focused on other species or other B. oleracea subspecies. The lack of genomic data for cabbage, a non-model organism, hinders research on its molecular biology. Hence, the construction of reliable transcriptomic data based on high-throughput sequencing technologies is needed to enhance our understanding of cabbage and provide genomic information for future work. METHODOLOGY/PRINCIPAL FINDINGS: We constructed cDNAs from total RNA isolated from the roots, leaves, flowers, seedlings, and calcium-limited seedling tissues of two cabbage genotypes: 102043 and 107140. We sequenced a total of six different samples using the Illumina HiSeq platform, producing 40.5 Gbp of sequence data comprising 401,454,986 short reads. We assembled 205,046 transcripts (≥ 200 bp) using the Velvet and Oases assembler and predicted 53,562 loci from the transcripts. We annotated 35,274 of the loci with 55,916 plant peptides in the Phytozome database. The average length of the annotated loci was 1,419 bp. We confirmed the reliability of the sequencing assembly using reverse-transcriptase PCR to identify tissue-specific gene candidates among the annotated loci. CONCLUSION: Our study provides valuable transcriptome sequence data for B. oleracea var. capitata L., offering a new resource for studying B. oleracea and closely related species. Our transcriptomic sequences will enhance the quality of gene annotation and functional analysis of the cabbage genome and serve as a material basis for future genomic research on cabbage. The sequencing data from this study can be used to develop molecular markers and to identify the extreme differences among the phenotypes of different species in the genus Brassica.

Small RNA and transcriptome deep sequencing proffers insight into floral gene regulation in Rosa cultivars.

BACKGROUND: Roses (Rosa sp.), which belong to the family Rosaceae, are the most economically important ornamental plants--making up 30% of the floriculture market. However, given high demand for roses, rose breeding programs are limited in molecular resources which can greatly enhance and speed breeding efforts. A better understanding of important genes that contribute to important floral development and desired phenotypes will lead to improved rose cultivars. For this study, we analyzed rose miRNAs and the rose flower transcriptome in order to generate a database to expound upon current knowledge regarding regulation of important floral characteristics. A rose genetic database will enable comprehensive analysis of gene expression and regulation via miRNA among different Rosa cultivars. RESULTS: We produced more than 0.5 million reads from expressed sequences, totalling more than 110 million bp. From these, we generated 35,657, 31,434, 34,725, and 39,722 flower unigenes from Rosa hybrid: 'Vital', 'Maroussia', and 'Sympathy' and Rosa rugosa Thunb., respectively. The unigenes were assigned functional annotations, domains, metabolic pathways, Gene Ontology (GO) terms, Plant Ontology (PO) terms, and MIPS Functional Catalogue (FunCat) terms. Rose flower transcripts were compared with genes from whole genome sequences of Rosaceae members (apple, strawberry, and peach) and grape. We also produced approximately 40 million small RNA reads from flower tissue for Rosa, representing 267 unique miRNA tags. Among identified miRNAs, 25 of them were novel and 242 of them were conserved miRNAs. Statistical analyses of miRNA profiles revealed both shared and species-specific miRNAs, which presumably effect flower development and phenotypes. CONCLUSIONS: In this study, we constructed a Rose miRNA and transcriptome database, and we analyzed the miRNAs and transcriptome generated from the flower tissues of four Rosa cultivars. The database provides a comprehensive genetic resource which can be used to better understand rose flower development and to identify candidate genes for important phenotypes.

Screening of tissue-specific genes and promoters in tomato by comparing genome wide expression profiles of Arabidopsis orthologues.

Constitutive overexpression of transgenes occasionally interferes with normal growth and developmental processes in plants. Thus, the development of tissue-specific promoters that drive transgene expression has become agriculturally important. To identify tomato tissue-specific promoters, tissue-specific genes were screened using a series of in silico-based and experimental procedures, including genome-wide orthologue searches of tomato and Arabidopsis databases, isolation of tissue-specific candidates using an Arabidopsis microarray database, and validation of tissue specificity by reverse transcription-polymerase chain reaction (RT-PCR) analysis and promoter assay. Using these procedures, we found 311 tissue-specific candidate genes and validated 10 tissue-specific genes by RT-PCR. Among these identified genes, histochemical analysis of five isolated promoter::GUS transgenic tomato and Arabidopsis plants revealed that their promoters have different but distinct tissue-specific activities in anther, fruit, and root, respectively. Therefore, it appears these in silico-based screening approaches in addition to the identification of new tissue-specific genes and promoters will be helpful for the further development of tailored crop development.

A genome-wide comparison of NB-LRR type of resistance gene analogs (RGA) in the plant kingdom.

Plants express resistance (R) genes to recognize invaders and prevent the spread of pathogens. To analyze nucleotide binding site, leucine-rich repeat (NB-LRR) genes, we constructed a fast pipeline to predict and classify the R gene analogs (RGAs) by applying in-house matrices. With predicted ~37,000 RGAs, we can directly compare RGA contents across entire plant lineages, from green algae to flowering plants. We focused on the highly divergent NBLRRs in land plants following the emergence of mosses. We identified entire loss of Toll/Interleukin-1 receptor, NBLRR (TNL) in Poaceae family of monocots and interestingly from Mimulus guttatus (a dicot), which leads to the possibility of species-specific TNL loss in other sequenced flowering plants. Using RGA maps, we have elucidated a positive correlation between the cluster sizes of NB-LRRs and their numbers. The cluster members were observed to consist of the same class of NB-LRRs or their variants, which were probably generated from a single locus for an R gene. Our website ( http://sol.kribb.re.kr/PRGA/ ), called plant resistance gene analog (PRGA), provides useful information, such as RGA annotations, tools for predicting RGAs, and analyzing domain profiles. Therefore, PRGA provides new insights into R-gene evolution and is useful in applying RGA as markers in breeding and or systematic studies.

Overexpression of Arabidopsis dehydration- responsive element-binding protein 2C confers tolerance to oxidative stress.

Dehydration-responsive element-binding proteins (DREBs)regulate plant responses to environmental stresses. In the current study, transcription of DREB2C, a class 2 Arabidopsis DREB, was induced by a superoxide anion propagator, methyl viologen (MV). The oxidative stress tolerance of DREB2C-overexpressing transgenic plants was significantly greater than that of wild-type plants, as measured by ion leakage and chlorophyll fluorescence under light conditions. The transcriptional activity of several ascorbate peroxidase (APX) genes as well as APX protein activity was induced in DREB2C overexpressors. Additionally, the level of H2O2 in the overexpressors was lower than in wt plants under similar oxidative stress conditions. An electrophoretic mobility shift assay and transient activator reporter assay showed that APX2 expression was regulated by heat shock factor A3 (HsfA3) and that HsfA3 is regulated at the transcriptional level by DREB2C. These results suggest that DREB2C plays an important role in promoting oxidative stress tolerance in Arabidopsis.

Arabidopsis DREB2C functions as a transcriptional activator of HsfA3 during the heat stress response.

The dehydration-responsive element binding protein (DREB) family is important in regulating plant responses to abiotic stresses. DREB2C is one of the Arabidopsis class 2 DREBs and is induced by heat stress (HS). Here, we present data concerning the interaction of DREB2C with heat shock factor A3 (HsfA3) in the HS signal transduction cascade. RT-PCR showed that HsfA3 is the most up-regulated gene among the 21 Arabidopsis Hsfs in transgenic plants over-expressing DREB2C. DREB2C and HsfA3 displayed similar transcription patterns in response to HS and DREB2C specifically transactivated the DRE-dependent transcription of HsfA3 in Arabidopsis mesophyll protoplasts. Yeast one-hybrid assays and invitro electrophoretic mobility shift assays further showed that DREB2C interacts with two DREs located in the HsfA3 promoter with a binding preference for the distal DRE2. Deletion mutants of DREB2C indicated that transactivation activity was located in the C-terminal region. In addition, dual activator-reporter assays showed that the induction of heat shock protein (Hsp) genes in transgenic plants could be attributed to the transcriptional activity of HsfA3. Taken together, these results indicate that DREB2C and HsfA3 are key players in regulating the heat tolerance of Arabidopsis.

Silencing of SlFTR-c, the catalytic subunit of ferredoxin:thioredoxin reductase, induces pathogenesis-related genes and pathogen resistance in tomato plants.

As a heterodimeric protein, ferredoxin:thioredoxin reductase (FTR) catalyses the light-dependant activation of several photosynthetic enzymes. The active site of the catalytic subunit of FTR contains a redox-active disulfide and a [4Fe-4S] center. We isolated the catalytic subunit gene of FTR, designated SlFTR-c, from tomato (Solanum lycopersicum L.). SlFTR-c transcripts were detected in all tissues examined, including roots, leaves, flowers, fruits, and seeds. Interestingly, virus-induced gene silencing (VIGS) of SlFTR-c resulted in necrotic lesions with typical cell death symptoms and reactive oxygen species (ROS) production in tomato leaves. Moreover, these SlFTR-c-silenced plants displayed enhanced disease resistance against bacterial pathogens, specifically Pseudomonas syringae pv. tomato DC3000, by the induction of defense-related genes (SlPR-1, SlPR-2, SlPR-5, SlGlucA, SlChi3, and SlChi9). Taken together, it seems that SlFTR-c works as a regulator of programmed cell death (PCD) and pathogen resistance in tomato plants.

Distinct expression patterns of two Arabidopsis phytocystatin genes, AtCYS1 and AtCYS2, during development and abiotic stresses.

The phytocystatins of plants are members of the cystatin superfamily of proteins, which are potent inhibitors of cysteine proteases. The Arabidopsis genome encodes seven phytocystatin isoforms (AtCYSs) in two distantly related AtCYS gene clusters. We selected AtCYS1 and AtCYS2 as representatives for each cluster and then generated transgenic plants expressing the GUS reporter gene under the control of each gene promoter. These plants were used to examine AtCYS expression at various stages of plant development and in response to abiotic stresses. Histochemical analysis of AtCYS1 promoter- and AtCYS2 promoter-GUS transgenic plants revealed that these genes have similar but distinct spatial and temporal expression patterns during normal development. In particular, AtCYS1 was preferentially expressed in the vascular tissue of all organs, whereas AtCYS2 was expressed in trichomes and guard cells in young leaves, caps of roots, and in connecting regions of the immature anthers and filaments and the style and stigma in flowers. In addition, each AtCYS gene has a unique expression profile during abiotic stresses. High temperature and wounding stress enhanced the expression of both AtCYS1 and AtCYS2, but the temporal and spatial patterns of induction differed. From these data, we propose that these two AtCYS genes play important, but distinct, roles in plant development and stress responses.

Constitutive activation of stress-inducible genes in a brassinosteroid-insensitive 1 (bri1) mutant results in higher tolerance to cold.

Many plant hormones are involved in coordinating the growth responses of plants under stress. However, not many mechanistic studies have explored how plants maintain the balance between growth and stress responses. Brassinosteroids (BRs), plant-specific steroid hormones, affect many aspects of plant growth and development over a plant's lifetime. In this study we determined that exogenous treatment of BR helped the plant overcome the cold condition only when pretreated with less than 1 nM, and the brassinosteroid-insensitive 1 (bri1) mutation, which results in defective BR signaling and subsequent dwarfism, generates an increased tolerance to cold. In contrast, BRI1-overexpressing plants were more sensitive to the same stress than wild-type. We found that the bri1 mutant and BRI1-overexpressing transgenic plants contain higher basal level of expression of CBFs/DREB1s than wild-type. However, representative cold stress-related genes were regulated with the same pattern to cold in wild-type, bri1-9 and BRI1 overexpressing plants. To examine the global gene expression and compare the genes that show differential expression pattern in bri1-9 and BRI1-GFP plants other than CBFs/DREB1s, we analyzed differential mRNA expression using the cDNA microarray analysis in the absence of stress. Endogenous expression of both stress-inducible genes as well as genes encoding transcription factors that drive the expression of stress-inducible genes were maintained at higher levels in bri1-9 than either in wild-type or in BRI1 overexpressing plants. This suggests that the bri1-9 mutant could always be alert to stresses that might be exerted at any times by constitutive activation of subsets of defense.

Over-expression of the Arabidopsis DRE/CRT-binding transcription factor DREB2C enhances thermotolerance.

The dehydration responsive element binding protein 2 (DREB2) subgroup belongs to the plant-specific APETALA2/ethylene-responsive element binding factor (AP2/ERF) family of transcription factors. We have characterized cDNA encoding Arabidopsis thaliana DREB2C, which is induced by mild heat stress. Both an electrophoretic mobility shift assay (EMSA) and a yeast one-hybrid assay revealed that DREB2C(145-528) was able to form a complex with the dehydration responsive element/C-repeat (DRE/CRT; A/GCCGAC) motif. A trans-activating ability test in yeast demonstrated that DREB2C could effectively function as a trans-activator. Constitutive expression of DREB2C under the control of the cauliflower mosaic virus (CaMV) 35S promoter led to enhanced thermotolerance in transgenic lines of Arabidopsis. Microarray and RT-PCR analyses of transgenic plants revealed that DREB2C regulates expression of several heat stress-inducible genes that contain DRE/CRT elements in their promoters. From these data, we deduced that DREB2C is a regulator of heat stress tolerance in Arabidopsis.

Gene expression profiles during heat acclimation in Arabidopsis thaliana suspension-culture cells.

Thermotolerance is induced by moderated heat acclimation. Suspension cultures of heat-acclimated Arabidopsis thaliana L. (Heynh.), ecotype Columbia, show thermotolerance against lethal heat shock (9 min, 50 degrees C), as evidenced by a chlorophyll assay and fluorescein diacetate staining. To monitor the genome-wide transcriptome changes induced by heat acclimation at 37 degrees C, we constructed an A. thaliana cDNA microarray containing 7,989 unique genes, and applied it to A. thaliana suspension-culture cells harvested at various times (0.5, 1, 2.5, 6, and 16 h) during heat acclimation. Data analysis revealed 165 differentially expressed genes that were grouped into ten clusters. We compared these genes with published and publicly available microarray heat-stress-related data sets in AtGenExpress. Heat-shock proteins were strongly expressed, as previously reported, and we found several of the up-regulated genes encoded detoxification and regulatory proteins. Moreover, the transcriptional induction of DREB2 (dehydration responsive element-binding factor 2) subfamily genes and COR47/rd17 under heat stress suggested cross-talk between the signaling pathways for heat and dehydration responses.