Cultivation regimes modulate flavonoid metabolism in rice: Integration of metabolomics and transcriptomics reveals NPK and density effects.

Strawberry (Fragaria × ananassa Duch) are sensitive to salt stress, and breeding salt-tolerant strawberry cultivars is the primary method to develop resistance to increased soil salinization. However, the underlying molecular mechanisms mediating the response of strawberry to salinity stress remain largely unknown. This study evaluated the salinity tolerance of 24 strawberry varieties, and transcriptomic and metabolomic analysis were performed of ‘Sweet Charlie’ (salt-tolerant) and ‘Benihoppe’ (salt-sensitive) to explore salt tolerance mechanisms in strawberry. Compared with the control, we identified 3412 differentially expressed genes (DEGs) and 209 differentially accumulated metabolites (DAMs) in ‘Benihoppe,’ and 5102 DEGs and 230 DAMs in ‘Sweet Charlie.’ DEGs Gene Ontology (GO) enrichment analyses indicated that the DEGs in ‘Benihoppe’ were enriched for ion homeostasis related terms, while in ‘Sweet Charlie,’ terms related to cell wall remodeling were over-represented. DEGs related to ion homeostasis and cell wall remodeling exhibited differential expression patterns in ‘Benihoppe’ and ‘Sweet Charlie.’ In ‘Benihoppe,’ 21 ion homeostasis-related DEGs and 32 cell wall remodeling-related DEGs were upregulated, while 23 ion homeostasis-related DEGs and 138 cell wall remodeling-related DEGs were downregulated. In ‘Sweet Charlie,’ 72 ion homeostasis-related DEGs and 275 cell wall remodeling-related DEGs were upregulated, while 11 ion homeostasis-related DEGs and 20 cell wall remodeling-related DEGs were downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed only four KEGG enriched pathways were shared between ‘Benihoppe’ and ‘Sweet Charlie,’ including flavonoid biosynthesis, phenylalanine metabolism, phenylpropanoid biosynthesis and ubiquinone, and other terpenoid-quinone biosynthesis. Integrating the results of transcriptomic and metabolomics analyses showed that adenosine triphosphate-binding cassette (ABC) transporters and flavonoid pathway genes might play important roles in the salt stress response in strawberry, and DAMs and DEGs related to ABC transporter and flavonoid pathways were differentially expressed or accumulated. The results of this study reveal that cell wall remodeling and ABC transporters contribute to the response to salt stress in strawberry, and that related genes showed differential expression patterns in varieties with different salt tolerances. These findings provide new insights into the underlying molecular mechanism of strawberry response to salt stress and suggest potential targets for the breeding of salt-tolerant strawberry varieties.

Objective: To investigate the role and mechanisms of action of nafamostat mesylate (NM) in rhabdomyolysis-induced acute kidney injury (RIAKI). Methods: RIAKI rats were assigned into control group (CN), RIAKI group (RM), and NM intervention group (NM). Inflammatory cytokines and proenkephalin a 119-159 (PENKID) were assessed. Cell apoptosis and glutathione peroxidase-4 (GPX4) were detected using TUNEL assay and immunohistochemical staining. Mitochondrial membrane potential (MMP) was detected by JC-1 dye. The expression of genes and metabolites after NM intervention was profiled using transcriptomic and metabolomic analysis. The differentially expressed genes (DEGs) were validated using qPCR. The KEGG and conjoint analysis of transcriptome and metabolome were used to analyze the enriched pathways and differential metabolites. The transcription factors were identified based on the animal TFDB 3.0 database. Results: Serum creatinine, blood urea nitrogen, and PENKID were remarkably higher in the RM group and lower in the NM group compared to the CN group. Pro-inflammatory cytokines increased in the RM group and notably decreased following NM treatment compared to the CN group. Tubular pathological damages were markedly attenuated and renal cell apoptosis was reduced significantly in the NM group compared to the RM group. The expression of GPX4 was lower in the RM group compared to the CN group, and it increased significantly after NM treatment. A total of 294 DEGs were identified in the RM group compared with the NM group, of which 192 signaling pathways were enriched, and glutathione metabolism, IL-17 signaling, and ferroptosis-related pathways were the top-ranking pathways. The transcriptional levels of Anpep, Gclc, Ggt1, Mgst2, Cxcl13, Rgn, and Akr1c1 were significantly different between the NM and RM group. Gclc was the key gene contributing to NM-mediated renal protection in RIAKI. Five hundred and five DEGs were annotated. Compared with the RM group, most of the upregulated DEGs in the NM group belonged to Glutathione metabolism, whereas most of the downregulated DEGs were related to the transcription factor Cytokine-cytokine receptor interaction. Conclusion: NM protects the kidneys against RIAKI, which is mainly associated with NM mediated regulation of glutathione metabolism, inflammatory response, ferroptosis-related pathways, and the related key DEGs. Targeting these DEGs might emerge as a potential molecular therapy for RIAKI.

As a major abiotic stress, soil salinity limits seed germination and plant growth, development and production. Seed germination is highly related not only to the seedlings survival rate but also subsequent vegetative growth. Populus euphratica and P. pruinosa are closely related species that show a distinguished adaptability to salinity stress. In this study, we performed an integrative transcriptome analyses of three seed germination phases from P. euphratica and P. pruinosa under salt stress. A two-dimensional data set of this study provides a comprehensive view of the dynamic biochemical processes that underpin seed germination and salt tolerance. Our analysis identified 12831 differentially expressed genes (DEGs) for seed germination processes and 8071 DEGs for salt tolerance in the two species. Furthermore, we identified the expression profiles and main pathways in each growth phase. For seed germination, a large number of DEGs, including those involved in energy production and hormonal regulation pathways, were transiently and specifically induced in the late phase. In the comparison of salt tolerance between the two species, the flavonoid and brassinosteroid pathways were significantly enriched. More specifically, in the flavonoid pathway, FLS and F3′5′H exhibited significant differential expression. In the brassinosteroid pathway, the expression levels of DWF4, BR6OX2 and ROT3 were notably higher in P. pruinosa than in P. euphratica. Our results describe transcript dynamics and highlight secondary metabolite pathways involved in the response to salt stress during the seed germination of two desert poplars.

Bifidobacterium bifidum strains, an important component of probiotic foods, can form biofilms on abiotic surfaces, leading to increased self-resistance. However, little is known about the molecular mechanism of B. bifidum biofilm formation. A time series transcriptome sequencing and untargeted metabolomics analysis of both B. bifidum biofilm and planktonic cells was performed to identify key genes and metabolites involved in biofilm formation. Two hundred thirty-five nonredundant differentially expressed genes (DEGs) (including vanY, pstS, degP, groS, infC, groL, yajC, tadB and sigA) and 219 nonredundant differentially expressed metabolites (including L-threonine, L-cystine, L-tyrosine, ascorbic acid, niacinamide, butyric acid and sphinganine) were identified. Thirteen pathways were identified during the integration of both transcriptomics and metabolomics data, including ABC transporters; quorum sensing; two-component system; oxidative phosphorylation; cysteine and methionine metabolism; glutathione metabolism; glycine, serine and threonine metabolism; and valine, leucine and isoleucine biosynthesis. The DEGs that relate to the integration pathways included asd, atpB, degP, folC, ilvE, metC, pheA, pstS, pyrE, serB, ulaE, yajC and zwf. The differentially accumulated metabolites included L-cystine, L-serine, L-threonine, L-tyrosine, methylmalonate, monodehydroascorbate, nicotinamide, orthophosphate, spermine and tocopherol. These results indicate that quorum sensing, two-component system and amino acid metabolism are essential during B. bifidum biofilm formation.

BackgroundChelidonium majus is a well-known traditional Chinese medicine, and has been reported of the effect in relieving cough and asthma. However, the mechanism of action is still unknown.MethodsAsthmatic SD rats were first sensitized and established through ovalbumin (OVA) motivation. Subsequently, Hematoxylin and eosin (H&E) staining, Masson’s trichrome (Masson) staining, Periodic acid-Schiff (PAS) staining and inflammatory cytokines assay of interleukin (IL)-4, IL-6, IL-17 were implemented to evaluate the protective effects of Chelidonium majus on asthma. Then, the effects of Chelidonium majus and their molecular mechanisms of action on asthma were detected based on the integration of transcriptomics and metabolomics analyses.ResultsAfter administration with Chelidonium majus, the histological injuries of inflammation, collagen deposition and mucus secretion in lungs were attenuated and the serum inflammatory cytokines perturbations were also converted. Furthermore, integrated analysis revealed that after Chelidonium majus treatment, 7 different expression genes (DEGs) (Alox15, P4ha1, Pla2g16, Pde3a, Nme1, Entpd8 and Adcy9) and 9 metabolic biomarkers (ADP, Xanthosine, Hypoxanthine, Inosine, prostaglandin E2 (PGE2), prostaglandin F2a (PGF2a), phosphatidylserine, Creatine and LysoPC (10:0)) were discovered to be connected with the enrichment metabolic pathways, including Purine metabolism, Arachidonic acid metabolism, Arginine and proline metabolism and Glycerophospholipid metabolism. The obtained metabolic biomarkers and DEGs were mainly related to energy metabolism and inflammation, and may be potential therapeutic targets.ConclusionChelidonium majus relieved OVA-induced asthma in rats by regulating the Alox15, P4ha1, Pla2g16, Pde3a, Nme1, Entpd8 and Adcy9 genes expression to restore the disorders in energy metabolism and inflammation.

Seed germination is a crucial developmental event in the plant life cycle. Proper germination significantly impacts the yield and quality. This study focuses on macadamia nuts, exploring the physiological, metabolic, and molecular biological characteristics during seed germination. The water content of macadamia seeds reached a peak in the seed imbibition stage, followed by a gradual decline. Key components such as fats, proteins, and soluble sugars decreased consistently. The enzyme activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione peroxidase (GPX) increased throughout seed germination, reaching peak levels in leaf growth stages. A total of 1523 metabolites and 13,035 differentially expressed genes (DEGs) were detected by transcriptomic and metabolomic analyses, among which 1320 were transcription factors. The transcriptome and metabolome integration analysis showed significant overlaps between DEGs and differential metabolites in pathways such as phenylpropanoid biosynthesis. Quercetin, trifolin, rutin, myricetin, and quercetin 3-beta-D-sophoroside may play important roles in seed germination. FG2 and CYP75A/CYP75B1 were key genes in the flavonoid pathway. Hormones such as auxin (IAA), cytokinin (CTK), gibberellin (GA), and abscisic acid (ABA) exhibited stage-specific changes during germination. This study provided important theoretical basis and practical guidance for optimizing seed germination rates and enhancing stress resistance capabilities.

Information regarding transcriptome and metabolome has significantly contributed to the identification of potential therapeutic targets for the management of a variety of cancers. Obesity has been shown to have profound effects on both cancer cell transcriptome and metabolome and to affect the outcome of cancer therapy. The information regarding the potential effects of obesity on breast cancer (BC) transcriptome, metabolome, and its integration to identify novel pathways related to disease progression are still elusive. We assessed the whole blood transcriptome and serum metabolome as circulating metabolites, of obese BC patients and compared them with non-obese BC patients. In these patients, 186 differentially expressed genes (DEGs) were observed, with 156 upregulated and 30 downregulated, significantly. The DEGs were enriched in different cellular pathways: cell cycle, one carbon pathway, homologous recombination cellular senescence, and notch signaling pathway. Our results confirmed the altered expression of several DEGs by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, 96 deregulated metabolites were identified when untargeted metabolomics was performed in obese BC patients when compared to non-obese BC patients, these were enriched in 71 pathways, most of them involved in ATP generation and cell proliferation. Finally, to provide a more comprehensive understanding of the association between obesity and BC, integration analysis between transcriptome and metabolomics data at the pathway level, revealed seven enriched pathways in obese BC vs. non-obese BC patients, that includes glutathione metabolism, glycine and serine metabolism, valine, leucine, and isoleucine degradation, purine metabolism, pyrimidine metabolism, thyroid hormone synthesis, and vitamin B6 metabolism; which may provide resistance for BC cell to dodge the circulating immune cells in whole blood. In conclusion, this study provides information on the unique pathways alteration at transcriptome and metabolome levels in obese BC patients, which may become an important tool for researchers and contribute to a rise in the knowledge on the molecular interaction between obesity and BC. Further studies are needed to confirm this and to elucidate the exact underlying mechanism for the effects of obesity on the BC initiation or/and progression. Citation Format: Mohammed Abdullah Hassan, Kaltoom Al-Sakkaf, Mohammed Razeeth Shait Mohammed, Ashraf Dallol, Jaudah Al-Maghrabi, Alia Aldahlawi, Sawsan Ashoor, Mabrouka Maamra, Jiannis Ragoussis, Wei Wu, Mohammad Imran Khan, Abdulrahman Al-Malki, Hani Choudhry. Integration of transcriptome and metabolome provides unique insights to pathways associated with obese breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 243.

Integration of Transcriptome and Metabolome Analyses Reveals the Mechanistic Basis for Cadmium Accumulation in Maize.

Objectives To delineate the interaction network between gene transcription and DNA methylation in PBMC of systemic sclerosis (SSc) patients and to identify methylation-regulated genes which are involved in the pathogenesis of SSc. Methods Genome-wide transcriptome and DNA methylome were profiled on PBMC from 30 SSc patients and 30 matched normal controls (NC). Differential expressed genes (DEGs) and differential methylated positions (DMPs) were integratively analyzed to identify methylation-regulated genes and associated molecular pathways. Results Transcriptome profiling distinguished 453 DEGs in SSc. Global DNA methylation analysis identified 925 DMPs located on 618 genes. Integration of DEGs and DMPs revealed 20 DEGs containing inversely associated DMPs. These 20 potential methylation-regulated DEGs (MeDEGs), including 12 up-regulated genes (ELANE, CTSG, LTBR, C3AR1, CSTA, SPI1, ODF3B, SAMD4A, PLAUR, NFE2, ZYX and CTSZ) and 8 down-regulated genes (RUNX3, PRF1, PRKCH, PAG1, RASSF5, FYN, CXCR6 and F2R), are predominantly involved in the migration, proliferation, activation and inflammation of immune cells. Unsupervised cluster analysis of the top MeDEGs distinguished SSc from NC with 90% sensitivity and 87% specificity. 4 MeDEGs, F2R, FYN, PAG1 and PRKCH, exhibited significantly progressive decrease in SSc with interstitial lung disease (ILD) compared with SSc without ILD. Conclusion By integration of genome-wide transcriptome and DNA methylome, we identified differential expressed genes associated with aberrant DNA methylation in the PBMC of SSc. The epigenetically dysregulated genes may lead to the abnormal activation of immune regulatory pathways which may contribute to the pathogenesis of SSc.

Polyhexamethylene guanidine phosphate (PHMG-p) was used as a humidifier disinfectant in Korea. PHMG induced severe pulmonary fibrosis in Koreans. The objective of this study was to elucidate mechanism of pulmonary toxicity caused by PHMG-p in rats using multi-omics analysis. Wistar rats were intratracheally instilled with PHMG-p by single (1.5mg/kg) administration or 4-week (0.1mg/kg, 2 times/week) repeated administration. Histopathologic examination was performed with hematoxylin and eosin staining. Alveolar macrophage aggregation and granulomatous inflammation were observed in rats treated with single dose of PHMG-p. Pulmonary fibrosis, chronic inflammation, bronchiol-alveolar fibrosis, and metaplasia of squamous cell were observed in repeated dose group. Next generation sequencing (NGS) was performed for transcriptome profiling after mRNA isolation from bronchiol-alveoli. Bronchiol-alveoli proteomic profiling was performed using an Orbitrap Q-exactive mass spectrometer. Serum and urinary metabolites were determined using 1H-NMR. Among 418 differentially expressed genes (DEGs) and 67 differentially expressed proteins (DEPs), changes of 16 mRNA levels were significantly correlated with changes of their protein levels in both single and repeated dose groups. Remarkable biological processes represented by both DEGs and DEPs were defense response, inflammatory response, response to stress, and immune response. Arginase 1 (Arg1) and lipocalin 2 (Lcn2) were identified to be major regulators for PHMG-p-induced pulmonary toxicity based on merged analysis using DEGs and DEPs. In metabolomics study, 52 metabolites (VIP > 0.5) were determined in serum and urine of single and repeated-dose groups. Glutamate and choline were selected as major metabolites. They were found to be major factors affecting inflammatory response in association with DEGs and DEPs. Arg1 and Lcn2 were suggested to be major gene and protein related to pulmonary damage by PHMG-p while serum or urinary glutamate and choline were endogenous metabolites related to pulmonary damage by PHMG-p.

IntroductionIndigofera stachyodes Lindl. is a perennial shrub belonging to the Fabaceae family that has been traditionally utilized as a medicinal plant by ethnic minority groups in Guizhou Province, China. This species exhibits significant ethnopharmacological value in local traditional medicine systems. The plant predominantly inhabits karst mountainous regions characterized by frequent drought stress, which represents a typical harsh habitat for plant growth. Notably, drought conditions particularly impair the establishment and development of I. stachyodes seedlings. However, the molecular mechanisms underlying its drought tolerance and adaptive responses remain largely unexplored, warranting further investigation at the molecular level.MethodsWe conducted pot-based water control experiments to subject I. stachyodes seedlings to drought stress treatments (CK, T0, T2). Root tissues from each treatment group were analyzed using transcriptomics (RNA-seq) and metabolomics (LC-MS/GC-MS) approaches to identify differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs). Through integrated analysis of DEGs and DEMs, we performed KEGG pathway enrichment and constructed co-expression networks to elucidate the molecular mechanisms underlying drought stress responses in the roots of I. stachyodes seedlings.ResultsA total of 11,509 DEGs were detected in the transcriptome. Among them, the CK vs T0 group shared 7,191 DEGs, the CK vs T2 group shared 1,264 DEGs, and the T2 vs T0 group shared 3,054 DEGs. In the metabolome, a total of 622 metabolites were detected. Among them, the CK vs T0 group shared 187 DEMs, the CK vs T2 group shared 127 DEMs, and the T2 vs T0 group shared 86 DEMs. The transcriptome-metabolome analysis revealed that the roots of I. stachyodes seedlings regulate metabolic balance through the phenylpropanoid biosynthesis pathway and the flavonoid biosynthesis pathway when subjected to varying degrees of drought stress. Metabolites such as p-coumaric acid, sinapine malate, eugenol, coumestrol, medicarpin, prunin, isosakuranetin, vitexin, gallocatechin, catechin, garbunzol and dihydromyricetin, along with genes including PAL, C4H, COMT, 4CL, CHS, DFR, HIDH, I2’H, IF7GT, IF7MAT, IFR, VR, PTS and IFS are potential key substances that enable the roots of I. stachyodes seedlings to resist drought stress.DiscussionThese results elucidate that the roots of I. stachyodes seedlings can resist drought stress and adapt to drought environments by regulating the expression of genes and the synthesis of metabolites in the flavonoid and phenylpropanoid metabolic pathways, providing a foundation to facilitate the domestication of wild I. stachyodes.

BackgroundOsmanthus fragrans (Oleaceae) is one of the most important ornamental plant species in China. Many cultivars with different leaf color phenotypes and good ornamental value have recently been developed. For example, a new cultivar ‘Qiannan Guifei’, presents a rich variety of leaf colors, which change from red to yellow-green and ultimately to green as leaves develop, making this cultivar valuable for landscaping. However, the biochemical characteristics and molecular mechanisms underlying leaf color changes of these phenotypes have not been elucidated. It has been hypothesized that the biosynthesis of different pigments in O. fragrans might change during leaf coloration. Here, we analyzed transcriptional changes in genes involved in chlorophyll (Chl), flavonoid, and carotenoid metabolic pathways and identified candidate genes responsible for leaf coloration in the new cultivar ‘Qiannan Guifei’.MethodsLeaf samples were collected from ‘Qiannan Guifei’ plants at the red (R), yellow-green (YG) and green (G) leaf stages. We compared the different-colored leaves via leaf pigment concentrations, chloroplast ultrastructure, and transcriptomic data. We further analyzed differentially expressed genes (DEGs) involved in the Chl, flavonoid, and carotenoid metabolic pathways. In addition, we used qRT-PCR to validate expression patterns of the DEGs at the three stages.ResultsWe found that, compared with those at the G stage, chloroplasts at the R and YG stages were less abundant and presented abnormal morphologies. Pigment analyses revealed that the leaves had higher flavonoid and anthocyanin levels at the R stage but lower Chl and carotenoid concentrations. Similarly, Chl and carotenoid concentrations were lower at the YG stage than at the G stage. By using transcriptomic sequencing, we further identified 61 DEGs involved in the three pigment metabolic pathways. Among these DEGs, seven structural genes (OfCHS, OfCHI, OfF3H, OfDFR, OfANS, OfUGT andOf3AT) involved in the flavonoid biosynthesis pathway were expressed at the highest level at the R stage, thereby increasing the biosynthesis of flavonoids, especially anthocyanins. Six putativeOfMYB genes, including three flavonoid-related activators and three repressors, were also highly expressed at the R stage, suggesting that they might coordinately regulate the accumulation of flavonoids, including anthocyanins. Additionally, expressions of the Chl biosynthesis-related genes OfHEMA, OfCHLG and OfCAO and the carotenoid biosynthesis-related genes OfHYB and OfZEP were upregulated from the R stage to the G stage, which increased the accumulation of Chl and carotenoids throughout leaf development. In summary, we screened the candidate genes responsible for the leaf color changes of ‘Qiannan Guifei’, improved current understanding of the regulatory mechanisms underlying leaf coloration and provided potential targets for future leaf color improvement in O. fragrans.

Certain plant genotypes can achieve optimal growth under appropriate environmental conditions. Under high planting density conditions, plants undergo competition for uptake and utilization of light and nutrients. However, the relationship between whole-genome expression patterns and the planting density in perennial woody plants remains unknown. In this study, whole-genome RNA sequencing of poplar (Populus × euramericana) was carried out at three different sampling heights to determine gene expression patterns under high (HD) and low (LD) planting densities. As a result, 4,004 differentially expressed genes (DEGs) were detected between HD and LD, of which 2,300, 701, and 1,003 were detected at the three positions, upper, middle and bottom, respectively. Function annotation results further revealed that a large number of the DEGs were involved in distinct biological functions. There were significant changes in the expression of metabolism-related and stimulus-related genes in response to planting density. There were 37 DEGs that were found at all three positions and were subsequently screened. Several DEGs related to plant light responses and photosynthesis were observed at different positions. Meanwhile, numbers of genes related to auxin/indole-3-acetic acid, and carbon and nitrogen metabolism were also revealed, displaying overall trends of upregulation under HD. These findings provide a basis for identifying candidate genes related to planting density and could increase our molecular understanding of the effect of planting density on gene expression.

Yellow seed breeding is an effective method to improve oil yield and quality in rapeseed (Brassica napus L.). However, naturally occurring yellow-seeded genotypes have not been identified in B. napus. Mustard (Brassica juncea L.) has some natural, yellow-seeded germplasms, yet the molecular mechanism underlying this trait remains unclear. In this study, a BC9 population derived from the cross of yellow seed mustard "Wuqi" and brown seed mustard "Wugong" was used to analyze the candidate genes controlling the yellow seed color of B. juncea. Subsequently, yellow-seeded (BY) and brown-seeded (BB) bulks were constructed in the BC9 population and subjected to bulked segregant RNA sequencing (BSR-Seq). A total of 511 differentially expressed genes (DEGs) were identified between the brown and yellow seed bulks. Enrichment analysis revealed that these DEGs were involved in the phenylpropanoid biosynthetic process and flavonoid biosynthetic process, including key genes such as 4CL, C4H, LDOX/TT18, PAL1, PAL2, PAL4, TT10, TT12, TT4, TT8, BAN, DFR/TT3, F3H/TT6, TT19, and CHI/TT5. In addition, 111,540 credible single-nucleotide polymorphisms (SNPs) and 86,319 INDELs were obtained and used for quantitative trait locus (QTL) identification. Subsequently, two significant QTLs on chromosome A09, namely, qSCA09-3 and qSCA09-7, were identified by G' analysis, and five DEGs (BjuA09PAL2, BjuA09TT5, BjuA09TT6, BjuA09TT4, BjuA09TT3) involved in the flavonoid pathway were identified as hub genes based on the protein-to-protein network. Among these five genes, only BjuA09PAL2 and BjuA09F3H had SNPs between BY and BB bulks. Interestingly, the majority of SNPs in BjuA09PAL2 were consistent with the SNPs identified between the high-quality assembled B. juncea reference genome "T84-66" (brown-seed) and "AU213" (yellow-seed). Therefore, BjuA09PAL2, which encodes phenylalanine lyase, was considered as the candidate gene associated with yellow seed color of B. juncea. The identification of a novel gene associated with the yellow seed coloration of B. juncea through this study may play a significant role in enhancing yellow seed breeding in rapeseed.

Chinese cabbage that prefers cold conditions is also affected by low-temperature stress, such as the accumulation of leaf anthocyanins. Research on anthocyanin biosynthesis and regulation mechanisms has made great progress. However, research on anthocyanin accumulation for resistance to biological and non-biological stress is still lacking. To study the relationship between anthocyanin accumulation of Chinese cabbage and resistance under low-temperature conditions, RNA sequencing (RNA-seq) was performed on Chinese cabbage ‘Xiao Baojian’ grown at a low temperature for four time periods and at a control temperature for five time periods. In Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, 7954 differentially expressed genes (DEGs) were enriched, of which 587 DEGs belonged to "biosynthesis of other secondary metabolites." Gene temporal expression patterns were used to discover enriched genes related to phenylpropanoid biosynthesis; flavonoid biosynthesis and anthocyanin biosynthesis pathways were found in cluster 1. The interaction networks were constructed, and hub genes were selected, showing that flavonoid biosynthesis pathway genes (DFR, ANS, F3H, FLS1, CHS1, CHS3, and TT8) and defense mechanisms-related genes (DFR, SNL6, and TKPR1) interact with each other. Anthocyanin biosynthesis DEGs in Chinese cabbage were evaluated under low-temperature conditions to map the relevant pathways, and expression maps of transcription factors in the flavonoid pathway were created at various periods. Low temperature upregulated the expression of genes related to anthocyanin biosynthesis. Taken together, our results provide further analysis of the relationship between plant anthocyanin synthesis and stress resistance and may also provide further insights for the future development of high-quality color and cold-tolerant Chinese cabbage germplasm resources.