Red Chinese Cabbage Transcriptome Analysis Reveals Structural Genes and Multiple Transcription Factors Regulating Reddish Purple Color

Reddish purple Chinese cabbage (RPCC) is a popular variety of Brassica rapa (AA = 20). It is rich in anthocyanins, which have many health benefits. We detected novel anthocyanins including cyanidin 3-(feruloyl) diglucoside-5-(malonoyl) glucoside and pelargonidin 3-(caffeoyl) diglucoside-5-(malonoyl) glucoside in RPCC. Analyses of transcriptome data revealed 32,395 genes including 3345 differentially expressed genes (DEGs) between 3-week-old RPCC and green Chinese cabbage (GCC). The DEGs included 218 transcription factor (TF) genes and some functionally uncharacterized genes. Sixty DEGs identified from the transcriptome data were analyzed in 3-, 6- and 9-week old seedlings by RT-qPCR, and 35 of them had higher transcript levels in RPCC than in GCC. We detected cis-regulatory motifs of MYB, bHLH, WRKY, bZIP and AP2/ERF TFs in anthocyanin biosynthetic gene promoters. A network analysis revealed that MYB75, MYB90, and MYBL2 strongly interact with anthocyanin biosynthetic genes. Our results show that the late biosynthesis genes BrDFR, BrLDOX, BrUF3GT, BrUGT75c1-1, Br5MAT, BrAT-1, BrAT-2, BrTT19-1, and BrTT19-2 and the regulatory MYB genes BrMYB90, BrMYB75, and BrMYBL2-1 are highly expressed in RPCC, indicative of their important roles in anthocyanin biosynthesis, modification, and accumulation. Finally, we propose a model anthocyanin biosynthesis pathway that includes the unique anthocyanin pigments and genes specific to RPCC

Genomic and Post-Translational Modification Analysis of Leucine-Rich-Repeat Receptor-Like Kinases in Brassica rapa.

Among several receptor-like kinases (RLKs), leucine-rich-repeat receptor-like kinases (LRR-RLKs) are a major group of genes that play crucial roles in growth, development and stress responses in plant systems. Given that they have several functional roles, it is important to investigate their roles in Brassica rapa. In the present study, 303 LRR-RLKs were identified in the genome of B. rapa and comparative phylogenetic analysis of 1213 combined LRR-RLKs of B. rapa, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa helped us to categorize the gene family into 15 subfamilies based on their sequence and structural similarities. The chromosome localizations of 293 genes allowed the prediction of duplicates, and motif conservation and intron/exon patterns showed differences among the B. rapa LRR-RLK (BrLRR-RLK) genes. Additionally, computational function annotation and expression analysis was used to predict their possible functional roles in the plant system. Biochemical results for 11 selected genes showed variations in phosphorylation activity. Interestingly, BrBAK1 showed strong auto-phosphorylation and trans-phosphorylation on its tyrosine and threonine residues compared with AtBAK1 in previous studies. The AtBAK1 receptor kinase is involved in plant growth and development, plant innate immunity, and programmed cell death, and our results suggest that BrBAK1 might also be involved in the same functions. Another interesting result was that BrBAK1, BrBRI1, BrPEPR1 and BrPEPR2 showed activity with both anti-phosphotyrosine and anti-phosphothreonine antibodies, indicating that they might have dual-specificity kinase activity. This study provides comprehensive results for the BrLRR-RLKs, revealing expansion of the gene family through gene duplications, structural similarities and variations among the genes, and potential functional roles according to gene ontology, transcriptome profiling and biochemical analysis

Biochemical Analysis of the Role of Leucine-Rich Repeat Receptor-Like Kinases and the Carboxy-Terminus of Receptor Kinases in Regulating Kinase Activity in Arabidopsis thaliana and Brassica oleracea

Protein post-translational modification by phosphorylation is essential for the activity and stability of proteins in higher plants and underlies their responses to diverse stimuli. There are more than 300 leucine-rich repeat receptor-like kinases (LRR-RLKs), a major group of receptor-like kinases (RLKs) that plays an important role in growth, development, and biotic stress responses in higher plants. To analyze auto- and transphosphorylation patterns and kinase activities in vitro, 43 full-length complementary DNA (cDNA) sequences were cloned from genes encoding LRR-RLKs. Autophosphorylation activity was found in the cytoplasmic domains (CDs) of 18 LRR-RLKs; 13 of these LRR-RLKs with autophosphorylation activity showed transphosphorylation in Escherichia coli. BRI1-Associated Receptor Kinase (BAK1), which is critically involved in the brassinosteroid and plant innate immunity signal transduction pathways, showed strong auto- and transphosphorylation with multi-specific kinase activity within 2 h of induction of Brassica oleraceae BAK1-CD (BoBAK1-CD) in E. coli; moreover, the carboxy-terminus of LRR-RLKs regulated phosphorylation and kinase activity in Arabidopsis thaliana and vegetative crops

Comparative Transcriptome-Based Mining of Senescence-Related MADS, NAC, and WRKY Transcription Factors in the Rapid-Senescence Line DLS-91 of Brassica rapa

Leaf senescence is a developmental process induced by various molecular and environmental stimuli that may affect crop yield. The dark-induced leaf senescence-91 (DLS-91) plants displayed rapid leaf senescence, dramatically decreased chlorophyll contents, low photochemical efficiencies, and upregulation of the senescence-associated marker gene BrSAG12-1. To understand DLS molecular mechanism, we examined transcriptomic changes in DLS-91 and control line DLS-42 following 0, 1, and 4 days of dark treatment (DDT) stages. We identified 501, 446, and 456 DEGs, of which 16.7%, 17.2%, and 14.4% encoded TFs, in samples from the three stages. qRT-PCR validation of 16 genes, namely, 7 MADS, 6 NAC, and 3 WRKY, suggested that BrAGL8-1, BrAGL15-1, and BrWRKY70-1 contribute to the rapid leaf senescence of DLS-91 before (0 DDT) and after (1 and 4 DDT) dark treatment, whereas BrNAC046-2, BrNAC029-2/BrNAP, and BrNAC092-1/ORE1 TFs may regulate this process at a later stage (4 DDT). In-silico analysis of cis-acting regulatory elements of BrAGL8-1, BrAGL42-1, BrNAC029-2, BrNAC092-1, and BrWRKY70-3 of B. rapa provides insight into the regulation of these genes. Our study has uncovered several AGL-MADS, WRKY, and NAC TFs potentially worthy of further study to understand the underlying mechanism of rapid DLS in DLS-91

Immunoblot analysis of FLAG-<i>BrBRI1</i> kinase phosphoprotein production over a time course.

<p>Immunoblot analysis of FLAG-<i>BrBRI1</i> kinase phosphoprotein production over a time course.</p

Auto-phosphorylation of recombinant <i>BrLRR- RLK’s</i> in <i>E</i>. <i>coli</i>.

<p>(A) Anti-phosphotyrosine immunoblots showing auto-phosphorylated proteins. (B) <i>BrLRR-RLK</i> genes showing auto-phosphorylation on anti-phosphothreonine residues. (C) Coomassie Brilliant Blue (CBB) staining of total extracted crude proteins. (D) Recombinant <i>BrLRR-RLK</i> protein expression levels were detected with an anti-FLAG antibody.</p

Intron/exon distribution of the 303 <i>BrLRR-RLK</i>s.

<p>(A) Subfamily (VIII-2 to V), (B) Subfamily (III to X). The genes consist of intronic regions (black line) and/or exonic regions (green line), and blue denotes regions upstream/downstream of the genes and splicing phases: 0 refers to phase 0, 1 to phase 1, and 2 to phase 2.</p

The amino acid patterns and characteristics of the 25 motifs identified in the 303 <i>BrLRR-RLK</i> genes.

<p>aa, amino acids</p><p>The amino acid patterns and characteristics of the 25 motifs identified in the 303 <i>BrLRR-RLK</i> genes.</p

Chromosomal distribution of 293 <i>BrLRR-RLK</i> genes.

<p>Outer circle with colored blocks illustrates different crucifer building blocks on chromosomes. The RD (red color) and non-RD (aqua color) type genes are distributed on chromosomes A01 to A10 in inner circle and paralogous copies are marked with different colored lines in center.</p

Transcript abundance of 303 <i>B</i>. <i>rapa</i> LRR-RLK genes.

<p>Microarray-based expression data for the genes downloaded from the Geo database were examined in six different tissues: callus, root, stem, leaf, flower and silique. The genes were grouped according to subfamily and the color scale at the bottom represents the expression values of the genes in the tissues. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142255#pone.0142255.s006" target="_blank">S6 Fig</a> for the full size version of Fig 5.</p