Complete Chloroplast Genome Sequence Structure and Phylogenetic Analysis of Kohlrabi (Brassica oleracea var. gongylodes L.).

Kohlrabi is an important swollen-stem cabbage variety belonging to the Brassicaceae family. However, few complete chloroplast genome sequences of this genus have been reported. Here, a complete chloroplast genome with a quadripartite cycle of 153,364 bp was obtained. A total of 132 genes were identified, including 87 protein-coding genes, 37 transfer RNA genes and eight ribosomal RNA genes. The base composition analysis showed that the overall GC content was 36.36% of the complete chloroplast genome sequence. Relative synonymous codon usage frequency (RSCU) analysis showed that most codons with values greater than 1 ended with A or U, while most codons with values less than 1 ended with C or G. Thirty-five scattered repeats were identified and most of them were distributed in the large single-copy (LSC) region. A total of 290 simple sequence repeats (SSRs) were found and 188 of them were distributed in the LSC region. Phylogenetic relationship analysis showed that five Brassica oleracea subspecies were clustered into one group and the kohlrabi chloroplast genome was closely related to that of B. oleracea var. botrytis. Our results provide a basis for understanding chloroplast-dependent metabolic studies and provide new insight for understanding the polyploidization of Brassicaceae species.

BrTTG1 regulates seed coat proanthocyanidin formation through a direct interaction with structural gene promoters of flavonoid pathway and glutathione S-transferases in Brassica rapa L.

Introduction: Seed coat color is a significant agronomic trait in horticultural crops such as Brassica rapa which is characterized by brown or yellow seed coat coloration. Previous Brassica rapa studies have shown that BrTTG1 is responsible for seed coat proanthocyanidin formation, which is dependent on the MYB-bHLH-WD40 complex, whereas some studies have reported that TRANSPARENT TESTA GLABRA 1 (TTG1) directly interacts with the structural gene promoters of the flavonoid pathway. Methods: Herein, the brown-seeded inbred B147 and ttg1 yellow-seeded inbred B80 mutants were used as plant materials for gene expression level analysis, gene promoter clone and transient overexpression. Results: The analysis identified eleven structural genes involved in the flavonoid biosynthesis pathway, which are potentially responsible for BrTTG1- dependent seed coat proanthocyanidin formation. The promoters of these genes were cloned and cis-acting elements were identified. Yeast one-hybrid and dual-luciferase assays confirmed that BrTTG1 directly and independently interacted with proCHS-Bra008792, proDFR-Bra027457, proTT12-Bra003361, proTT19-Bra008570, proTT19-Bra023602 and proAHA10-Bra016610. A TTG1-binding motif (RTWWGTRGM) was also identified. Overexpression of TTG1 in the yellow-seed B. rapa inbred induced proanthocyanidin accumulation by increasing the expression levels of related genes. Discussion: Our study unveiled, for the first time, the direct interaction between TTG1 and the promoters of the flavonoid biosynthesis pathway structural genes and glutathione S-transferases in Brassica rapa. Additionally, we have identified a novel TTG1-binding motif, providing a basis for further exploration into the function of TTG1 and the accumulation of proanthocyanidins in seed coats.

Potential Suitable Habitats of Chili Pepper in China under Climate Change.

Chili pepper (Capsicum annuum L.) is extensively cultivated in China, with its production highly reliant on regional environmental conditions. Given ongoing climate change, it is imperative to assess its impact on chili pepper cultivation and identify suitable habitats for future cultivation. In this study, the MaxEnt model was optimized and utilized to predict suitable habitats for open-field chili pepper cultivation, and changes in these habitats were analyzed using ArcGIS v10.8. Our results showed that the parameter settings of the optimal model were FC = LQPTH and RM = 2.7, and the critical environmental variables influencing chili pepper distribution were annual mean temperature, isothermality, maximum temperature of the warmest month, and precipitation of the warmest quarter. Under current climate conditions, suitable habitats were distributed across all provinces in China, with moderately- and highly-suitable habitats concentrated in the east of the Qinghai-Tibetan Plateau and south of the Inner Mongolia Plateau. Under future climate scenarios, the area of suitable habitats was expected to be larger than the current ones, except for SSP126-2050s, and reached the maximum under SSP126-2090s. The overlapping suitable habitats were concentrated in the east of the Qinghai-Tibetan Plateau and south of the Inner Mongolia Plateau under various climate scenarios. In the 2050s, the centroids of suitable habitats were predicted to shift towards the southwest, except for SSP126, whereas this trend was reversed in the 2090s. Our results suggest that climate warming is conductive to the cultivation of chili pepper, and provide scientific guidance for the introduction and cultivation of chili pepper in the face of climate warming.

Differentially Expressed Genes Identification of Kohlrabi Seedlings (Brassica oleracea var. caulorapa L.) under Polyethylene Glycol Osmotic Stress and AP2/ERF Transcription Factor Family Analysis.

Osmotic stress is a condition in which plants do not get enough water due to changes in environmental factors. Plant response to osmotic stress is a complex process involving the interaction of different stress-sensitive mechanisms. Differentially expressed genes and response mechanisms of kohlrabi have not been reported under osmotic stress. A total of 196,642 unigenes and 33,040 differentially expressed unigenes were identified in kohlrabi seedlings under polyethylene glycol osmotic stress. AP2/ERF, NAC and eight other transcription factor family members with a high degree of interaction with CAT and SOD antioxidant enzyme activity were identified. Subsequently, 151 AP2/ERF genes were identified and analyzed. Twelve conserved motifs were searched and all AP2/ERF genes were clustered into four groups. A total of 149 AP2/ERF genes were randomly distributed on the chromosome, and relative expression level analysis showed that BocAP2/ERF genes of kohlrabi have obvious specificity in different tissues. This study lays a foundation for explaining the osmotic stress resistance mechanism of kohlrabi and provides a theoretical basis for the functional analysis of BocAP2/ERF transcription factor family members.

Cloning and Analysis of Expression of Genes Related to Carotenoid Metabolism in Different Fruit Color Mutants of Pepper (Capsicum annuum L.).

The formation of fruit color in pepper is closely related to the processes of carotenoid metabolism. In this study, red wild-type pepper XHB, SP01, PC01 and their corresponding mutants H0809 (orange), SP02 (yellow), and PC02 (orange) were used as research materials. The Ggps, Psy, Lcyb, Crtz, Zep, and Ccs genes involved in carotenoid biosynthesis were cloned, and bioinformatics and expression analyses were carried out. The results showed that the full lengths of the six genes were 1110 bp, 2844 bp, 1497 bp, 2025 bp, 510 bp, and 1497 bp, and they encoded 369, 419, 498, 315, 169, and 498 amino acids, respectively. Except for the full-length Ccs gene, which could not be amplified in the yellow mutant SP02 and the orange mutant PC02, the complete full-length sequences of the other genes could be amplified in different materials, indicating that the formation of fruit color in the SP02 and PC02 mutants could be closely related to the deletion or mutation of the Ccs gene. The analytical results of real-time quantitative reverse transcription PCR (qRT-PCR) showed that the Ggps, Psy, Lcyb, Crtz, and Zep genes were expressed at different developmental stages of three pairs of mature-fruit-colored materials, but their patterns of expression were not consistent. The orange mutant H0809 could be amplified to the full Ccs gene sequence, but its expression was maintained at a lower level. It showed a significant difference in expression compared with the wild-type XHB, indicating that the formation of orange mutant H0809 fruit color could be closely related to the different regulatory pattern of Ccs expression. The results provide a theoretical basis for in-depth understanding of the molecular regulatory mechanism of the formation of color in pepper fruit.

Effects of morphology and rheology of starch nanoparticles prepared from various coarse cereals on emulsifying ability.

Starch nanoparticles (SNPs) were prepared by rapid nanoprecipitation using coarse cereal starches, including quinoa, waxy proso millet, non-waxy proso millet, and maize starches (amylose content, 2.20 %-13.96 %). Morphology and dynamic light scattering analyses showed that the particle size of SNPs ranged from 190.35 nm to 310.25 nm. Ultrasonic pretreatment reduced the particle size. Quinoa and waxy proso millet SNPs swelled to flocculent, whereas non-waxy proso millet and maize SNPs showed spherical particles. Quinoa and waxy proso millet SNPs did not completely nucleate after nanoprecipitation. Quinoa SNPs formed a stable network in water, showing great viscoelastic behavior with higher G' and G''. Subsequently, the SNPs suspensions were used to prepare emulsions. The quinoa SNPs-based Pickering emulsion was the smallest and most uniform (12.96 µm), with great stability. These results suggested that the quinoa SNPs suspension with a stable network could be used as an efficient emulsifier for stabilizing Pickering emulsions.

Transcriptomics Integrated with Metabolomics Unveil Carotenoids Accumulation and Correlated Gene Regulation in White and Yellow-Fleshed Turnip (Brassica rapa ssp. rapa).

Turnip (Brassica rapa ssp. rapa) is considered to be a highly nutritious and health-promoting vegetable crop, whose flesh color can be divided into yellow and white. It is widely accepted that yellow-fleshed turnips have higher nutritional value. However, reports about flesh color formation is lacking. Here, the white-fleshed inbred line, W21, and yellow-fleshed inbred line, W25, were profiled from the swollen root of the turnip at three developmental periods to elucidate the yellow color formation. Transcriptomics integrated with metabolomics analysis showed that the PSY gene was the key gene affecting the carotenoids formation in W25. The coding sequence of BrrPSY-W25 was 1278 bp and that of BrrPSY-W21 was 1275 bp, and BrrPSY was more highly expressed in swollen roots in W25 than in W21. Transient transgenic tobacco leaf over-expressing BrrPSY-W and BrrPSY-Y showed higher transcript levels and carotenoids contents. Results revealed that yellow turnip formation is due to high expression of the PSY gene rather than mutations in the PSY gene, indicating that a post-transcriptional regulatory mechanism may affect carotenoids formation. Results obtained in this study will be helpful for explaining the carotenoids accumulation of turnips.

The sequence structure and phylogenetic analysis by complete mitochondrial genome of kohlrabi (Brassica oleracea var. gongylodes L.).

Kohlrabi (Brassica oleracea var. gongylodes L.) is an important dietary rhizome vegetable in the Brassicaceae family. However, to date, few mitochondrial genomic resources have been reported for kohlrabi. In this study, we obtained the complete mitochondrial DNA sequence of 219,964 bp from an individual green kohlrabi. A total of 61 genes were annotated, including 33 protein-coding genes, 23 transfer RNA genes, three ribosomal RNA genes, and two pseudo genes. In addition, 1,001 open reading frames and five RNA editing sites were annotated. Relative synonymous codon usage analysis revealed significant difference in usage frequency of synonymous codon. Phylogenetic inference showed that kohlrabi is closely related to B. oleracea var. botrytis. This study provides a good foundation for further understanding the relationship and evolutionary origins among Brassicaceae crops.

Metabolite Analysis of Jerusalem Artichoke (Helianthus tuberosus L.) Seedlings in Response to Polyethylene Glycol-Simulated Drought Stress.

Jerusalem artichokes are a perennial crop with high drought tolerance and high value as a raw material to produce biofuels, functional feed, and food. However, there are few comprehensive metabolomic studies on Jerusalem artichokes under drought conditions. METHODS: Ultra-performance liquid chromatography and tandem mass spectrometry were used to identify differential metabolites in Jerusalem artichoke seedling leaves under polyethylene glycol (PEG) 6000-simulated drought stress at 0, 18, 24, and 36 h. RESULTS: A total of 661 metabolites and 236 differential metabolites were identified at 0 vs. 18, 18 vs. 24, and 24 vs. 36 h. 146 differential metabolites and 56 common were identified and at 0 vs. 18, 24, and 36 h. Kyoto Encyclopedia of Genes and Genomes enrichment identified 236 differential metabolites involved in the biosynthesis of secondary metabolites and amino acids. Metabolites involved in glycolysis, phenolic metabolism, tricarboxylic cycle, glutamate-mediated proline biosynthesis, urea cycle, amino acid metabolism, unsaturated fatty acid biosynthesis, and the met salvage pathway responded to drought stress. CONCLUSION: A metabolic network in the leaves of Jerusalem artichokes under drought stress is proposed. These results will improve understanding of the metabolite response to drought stress in Jerusalem artichokes and develop a foundation for breeding drought-resistant varieties.

Comparative transcriptome and flavonoids components analysis reveal the structural genes responsible for the yellow seed coat color of Brassica rapa L.

BACKGROUND: Seed coat color is an important horticultural trait in Brassica crops, which is divided into two categories: brown/black and yellow. Seeds with yellow seed coat color have higher oil quality, higher protein content and lower fiber content. Yellow seed coat color is therefore considered a desirable trait in hybrid breeding of Brassica rapa, Brassica juncea and Brassica napus. METHODS: Comprehensive analysis of the abundance transcripts for seed coat color at three development stages by RNA-sequencing (RNA-seq) and corresponding flavonoids compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out in B. rapa. RESULTS: We identified 41,286 unigenes with 4,989 differentially expressed genes between brown seeds (B147) and yellow seeds (B80) at the same development stage. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified 19 unigenes associated with the phenylpropanoid, flavonoid, flavone and flavonol biosynthetic pathways as involved in seed coat color formation. Interestingly, expression levels of early biosynthetic genes (BrCHS, BrCHI, BrF3H, BrF3'H and BrFLS) in the flavonoid biosynthetic pathway were down-regulated while late biosynthetic genes (BrDFR, BrLDOX and BrBAN) were hardly or not expressed in seeds of B80. At the same time, BrTT8 and BrMYB5 were down-regulated in B80. Results of LC-MS also showed that epicatechin was not detected in seeds of B80. We validated the accuracy of our RNA-seq data by RT-qPCR of nine critical genes. Epicatechin was not detected in seeds of B80 by LC-MS/MS. CONCLUSIONS: The expression levels of flavonoid biosynthetic pathway genes and the relative content of flavonoid biosynthetic pathway metabolites clearly explained yellow seed color formation in B. rapa. This study provides a foundation for further research on the molecular mechanism of seed coat color formation.

Low Temperature Promotes Anthocyanin Biosynthesis and Related Gene Expression in the Seedlings of Purple Head Chinese Cabbage (Brassica rapa L.).

To elucidate the effect of low temperature on anthocyanin biosynthesis in purple head Chinese cabbage, we analyzed anthocyanin accumulation and related gene expression in the seedlings of purple head Chinese cabbage, white head parent Chinese cabbage, and its purple male parent under a normal 25 °C temperature and a low 12 °C temperature. Anthocyanin accumulation in purple lines was strongly induced by low temperature, and the total anthocyanin content of seedlings was significantly enhanced. In addition, nearly all phenylpropanoid metabolic pathway genes (PMPGs) were down-regulated, some early biosynthesis genes (EBGs) were up-regulated, and nearly all late biosynthesis genes (LBGs) directly involved in anthocyanin biosynthesis showed higher expression levels in purple lines after low-temperature induction. Interestingly, a R2R3-MYB transcription factor (TF) gene 'BrMYB2' and a basic-helix-loop-helix (bHLH) regulatory gene 'BrTT8' were highly up-regulated in purple lines after low temperature induction, and two negative regulatory genes 'BrMYBL2.1' and 'BrLBD38.2' were up-regulated in the white line. BrMYB2 and BrTT8 may play important roles in co-activating the anthocyanin structural genes in purple head Chinese cabbage after low-temperature induction, whereas down-regulation of BrMYB2 and up-regulation of some negative regulators might be responsible for white head phenotype formation. Data presented here provide new understanding into the anthocyanin biosynthesis mechanism during low temperature exposure in Brassica crops.

Complete chloroplast genome sequence of turnip (Brassica rapa. ssp. rapa): genome structure and phylogenetic analysis.

Turnip (Brassica rapa. ssp. rapa) is considered worldwide to be one of the most important leaf and root vegetable crops in the Brassicaceae family. However, to date, few chloroplast (cp) genomic resources have been reported for this genus. Here, we determined the complete cp genome sequences of Brassica rapa ssp. rapa. A 153,621 bp quadripartite cycle without any gap was obtained with a large single-copy region (LSC) of 83,512 bp, a small single-copy region (SSC) of 17,683 bp, and two inverted repeat (IR), IRa and IRb of 26,213 bp. A total of 132 genes were identified, including 87 protein-coding genes (PCG), 37 transfer RNA (tRNA), and 8 ribosomal RNA (rRNA). The phylogenetic analysis of ten other crops selected showed that the turnip was most closely related to the Brassica rapa.

Physicochemical and functional properties of protein isolate obtained from cottonseed meal.

To investigate the effect of preparation methods of cottonseed meals on protein properties, the physicochemical and functional properties of proteins isolated from hot-pressed solvent extraction cottonseed meal (HCM), cold-pressed solvent extraction cottonseed meal (CCM) and subcritical fluid extraction cottonseed meal (SCM) were investigated. Cottonseed proteins had two major bands (at about 45 and 50kD), two X-ray diffraction peaks (8.5° and 19.5°) and one endothermic peak (94.31°C-97.72°C). Proteins of HCM showed relatively more ß-sheet (38.3%-40.5%), and less ß-turn (22.2%-25.8%) and α-helix (15.8%-19.5%), indicating the presence of highly denatured protein molecules. Proteins of CCM and SCM exhibited high water/oil absorption capacity, emulsifying abilities, surface hydrophobicity and fluorescence intensity, suggesting that the proteins have potential as functional ingredients in the food industry.

Characteristics of Color Development in Seeds of Brown- and Yellow-Seeded Heading Chinese Cabbage and Molecular Analysis of Brsc, the Candidate Gene Controlling Seed Coat Color.

The proanthocyanidin (PA) is the main flavonoids which affect the seed coat color in Brassica species. In this paper, characteristics of color development and accumulation of flavonoids were analyzed in the seeds of brown-seeded (B147) and yellow-seeded (B80) heading Chinese cabbage (Brassica rapa L. ssp. Pekinensis). It is found that the content of phenolic compounds in B147 were significantly more than that of B80 by using dimethylaminocinnamaldehyde (DMACA) staining and toluidine blue O (TBO) staining. In previous studies, the locus associated with seed coat color has been mapped. The results of whole genome re-sequencing showed that there are large fragment deletions variation in the mapping region between the brown-seeded parent '92S105' and the yellow-seeded parent '91-125.' Based on the B. rapa genome annotation information, the TRANSPARENT TESTA GLABRA 1 (TTG1), is likely to be the candidate gene controlling seed coat color. A 94-base deletion was found in the 96th base downstream of the initiation codon in the TTG1 of yellow seed, thus, the termination codon TGA was occurred in the 297th base which makes the full length of TTG1 of yellow seed is 300 bp. Based on the differential sequences of TTG1 of brown and yellow seed, a functional marker, Brsc-yettg1, was developed to detect the variation of TTG1. Quantitative real-time PCR analysis of BrTTG1 in different tissues showed that expression levels of BrTTG1 was not tissue-specific. During the whole seed development period, the expression of BrTTG1 in B147 was higher than that of B80. The expression levels of four structural genes, BrDFR, BrANS, BrANR1, and BrANR2 in B147 were also higher than those in B80. The co-segregation molecular markers obtained in this report and TTG1 related information provide a basis for further understanding of the molecular mechanism of seed coat color in heading Chinese cabbage.

Identification of SSR markers closely linked to the yellow seed coat color gene in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Research on the yellow-seeded variety of heading Chinese cabbage will aid in broadening its germplasm resources and lay a foundation for AA genome research in Brassica crops. Here, an F2 segregating population of 1575 individuals was constructed from two inbred lines (brown-seeded '92S105' and yellow-seeded '91-125'). This population was used to identify the linkage molecular markers of the yellow seed coat trait using simple sequence repeat (SSR) techniques combined with a bulk segregant analysis (BSA). Of the 144 SSR primer pairs on the A01-A10 chromosomes from the Brassica database (http://brassicadb.org/brad/), two pairs located on the A06 chromosome showed polymorphic bands between the bulk DNA pools of eight brown-seeded and eight yellow-seeded F2 progeny. Based on the genome sequence, 454 SSR markers were designed to A06 to detect these polymorphic bands and were synthesized. Six SSR markers linked to the seed coat color gene were successfully selected for fine linkage genetic map construction, in which the two closest flanking markers, SSR449a and SSR317, mapped the Brsc-ye gene to a 40.2 kb region with distances of 0.07 and 0.06 cM, respectively. The molecular markers obtained in this report will assist in the marker-assisted selection and breeding of yellow-seeded lines in Brassica rapa L. and other close species.

Allelism analysis of BrRfp locus in different restorer lines and map-based cloning of a fertility restorer gene, BrRfp1, for pol CMS in Chinese cabbage (Brassica rapa L.).

KEY MESSAGE: In Chinese cabbage, there are two Rf loci for pol CMS and one of them was mapped to a 12.6-kb region containing a potential candidate gene encoding PPR protein. In Chinese cabbage (Brassica rapa), polima cytoplasmic male sterility (pol CMS) is an important CMS type and is widely used for hybrid breeding. By extensive test crossing in Chinese cabbage, four restorer lines (92s105, 01s325, 00s109, and 88s148) for pol CMS were screened. By analyzing the allelism of the four restorer lines, it was found that 92s105, 01s325, and 00s109 had the same "restorers of fertility" (Rf) locus (designated as BrRfp1), but 88s148 had a different Rf locus (designated as BrRfp2). For fine mapping the BrRfp1 locus of 92s105, a BC1F1 population with 487 individuals and a BC1F2 population with 2485 individuals were successively constructed. Using simple sequence repeat (SSR) markers developed from Brassica rapa reference genome and InDel markers derived from whole-genome resequencing data of 94c9 and 92s105, BrRfp1 was mapped to a 12.6-kb region containing a potential candidate gene encoding pentatricopeptide repeat-containing protein. Based on the nucleotide polymorphisms of the candidate gene sequence between the restoring and nonrestoring alleles, a co-segregating marker SC718 was developed, which would be helpful for hybrid breeding by marker-assisted screening and for detecting new restorer lines.

Carbon dots: surface engineering and applications.

Carbon dots have attracted a great deal of attention because of their high performance, cheap and facile preparation, and potential applications in a wide area. In order to broaden their applications, especially to meet specific requirements, surface engineering, including tailoring surface functional group coating and subsequent chemical modification as required, is an effective strategy for further functionalization of carbon dots. In this article, representative approaches to coating the surface with various functional groups, and strategies for conjugating specific materials onto the surface of carbon dots for functional modification via covalent bonds, electrostatic interactions and hydrogen bonds are highlighted, as well as the results from explorations of their various applications in target modulated sensing, accurate drug delivery and bioimaging at high resolution.