A novel three-layer module BoMYB1R1-BoMYB4b/BoMIEL1-BoDFR1 regulates anthocyanin accumulation in kale.

Anthocyanin is an important pigment responsible for plant coloration and beneficial to human health. Kale (Brassica oleracea var. acephala), a primary cool-season flowers and vegetables, is an ideal material to study anthocyanin biosynthesis and regulation mechanisms due to its anthocyanin-rich leaves. However, the underlying molecular mechanism of anthocyanin accumulation in kale remains poorly understood. Previously, we demonstrated that BoDFR1 is a key gene controlling anthocyanin biosynthesis in kale. Here, we discovered a 369-bp InDel variation in the BoDFR1 promoter between the two kale inbred lines with different pink coloration, which resulted in reduced transcriptional activity of the BoDFR1 gene in the light-pink line. With the 369-bp insertion as a bait, an R2R3-MYB repressor BoMYB4b was identified using the yeast one-hybrid screening. Knockdown of the BoMYB4b gene led to increased BoDFR1 expression and anthocyanin accumulation. An E3 ubiquitin ligase, BoMIEL1, was found to mediate the degradation of BoMYB4b, thereby promoting anthocyanin biosynthesis. Furthermore, the expression level of BoMYB4b was significantly reduced by light signals, which was attributed to the direct repression of the light-signaling factor BoMYB1R1 on the BoMYB4b promoter. Our study revealed that a novel regulatory module comprising BoMYB1R1, BoMIEL1, BoMYB4b, and BoDFR1 finely regulates anthocyanin accumulation in kale. The findings aim to establish a scientific foundation for genetic improvement of leaf color traits in kale, meanwhile, providing a reference for plant coloration studies.

Inactivation of BoORP3a, an oxysterol-binding protein, causes a low wax phenotype in ornamental kale.

Identifying genes associated with wax deposition may contribute to the genetic improvement of ornamental kale. Here, we characterized a candidate gene for wax contents, BoORP3a, encoding an oxysterol-binding protein. We sequenced the BoORP3a gene and coding sequence from the high-wax line S0835 and the low-wax line F0819, which revealed 12 single nucleotide polymorphisms between the two lines, of which six caused five amino acids substitutions. BoORP3a appeared to be relatively well conserved in Brassicaceae, as determined by a phylogenetic analysis, and localized to the endoplasmic reticulum and the nucleus. To confirm the role of BoORP3a in wax deposition, we generated three orp3a mutants in a high-wax kale background via CRISPR/Cas9-mediated genome editing. Importantly, all three mutants exhibited lower wax contents and glossy leaves. Overall, these data suggest that BoORP3a may participate in cuticular wax deposition in ornamental kale.

BoALG10, an α-1,2 glycosyltransferase, plays an essential role in maintaining leaf margin shape in ornamental kale.

The morphological diversity of leaf margin shapes is an identifying characteristic of many plant species. In our previous work, BoALG10 (α-1,2 glycosyltransferase) was predicted to be a key regulator of leaf margin shape in ornamental kale (Brassica oleracea var. acephala). An alanine and a leucine residue in the conserved domain of the smooth-margined S0835 were replaced by an aspartate and a phenylalanine, respectively, in the corresponding positions of the feathered-margined F0819. However, the expression pattern and function of this gene remain unclear. Here, we examined the expression patterns of BoALG10 using quantitative real-time PCR, and found that statistically significant differences in expression existed between F0819 and S0835 in nine developmental stages. The BoALG10 protein localized to the endoplasmic reticulum. The function of BoALG10 was then examined using complementary mutant assays. The overexpression strains phenocopied the smooth leaf margin after introduction of BoALG10 S0835 into the feathered-margined inbred line F0819. Simultaneously, irregular dissections appeared in the leaf margins of knockout mutants KO-1 and KO-2, which were generated by CRISPR/Cas9 technology from the smooth-margined inbred line S0835. Microscopic observation showed that the leaf margin cells of the smooth-margined plants S0835 and OE-3 were arranged regularly, while the cells of the feathered-margined plants F0819 and KO-1 were of inconsistent size and distributed in an irregular manner, particularly around the indentations of the leaf. This elucidation of BoALG10 function provides a novel insight into the morphological regulation of leaf margin shape.

CmWRKY15-1 Promotes Resistance to Chrysanthemum White Rust by Regulating CmNPR1 Expression.

Chrysanthemum white rust (CWR), a disease caused by the fungus Puccinia horiana Henn., seriously impairs the production and ornamental value of chrysanthemums. We previously isolated the disease-resistance gene CmWRKY15-1 from the chrysanthemum and generated CmWRKY15-1 transgenic plants. Here, we determined that CmWRKY15-1-overexpressing lines of the susceptible cultivar 'Jinba' show higher defensive enzyme activity and lower H2O2 levels than a wild type after inoculation with P. horiana, indicating that CmWRKY15-1 positively regulates plant responses to P. horiana. To further explore the mechanism underlying this effect, we performed RNA sequencing using the leaves of wild-type and CmWRKY15-1-RNA interference lines of the resistant cultivar 'C029' after treatment with P. horiana. We identified seven differentially expressed genes in the salicylic acid (SA) pathway, including CmNPR1 (Non-expressor of pathogenesis-related genes 1), encoding an important regulator of this pathway. We isolated the CmNPR1 promoter by hiTAIL-PCR and predicted that it contains pathogen-induced W-box elements. The promoter region of CmNPR1 was activated by P. horiana in a ß-glucuronidase activity assay. Yeast one-hybrid assays showed that CmWRKY15-1 binds to the CmNPR1 promoter region to regulate its expression. Finally, we confirmed the interaction between CmWRKY15-1 and CmNPR1 in a bimolecular fluorescence complementation assay. We propose that CmWRKY15-1 interacts with CmNPR1 to activate the expression of downstream pathogenesis-related genes that enhance resistance to P. horiana through the SA pathway. These findings shed light on the mechanism underlying resistance to CWR.

Transcriptome Analysis of Green and White Leaf Ornamental Kale Reveals Coloration-Related Genes and Pathways.

Leaf color is a crucial agronomic trait in ornamental kale. However, the molecular mechanism regulating leaf pigmentation patterns in green and white ornamental kale is not completely understood. To address this, we performed transcriptome and pigment content analyses of green and white kale leaf tissues. A total of 5,404 and 3,605 different expressed genes (DEGs) were identified in the green vs. white leaf and the green margin vs. white center samples. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis showed that 24 and 15 common DEGs in two pairwise comparisons were involved in chlorophyll metabolism and carotenoid biosynthesis, respectively. Seventeen genes related to chlorophyll biosynthesis were significantly upregulated in green leaf tissue, especially chlH and por. Of the 15 carotenoid biosynthesis genes, all except CYP707A and BG1 were lower expressed in white leaf tissue. Green leaf tissue exhibited higher levels of chlorophyll and carotenoids than white leaf tissue. In addition, the DEGs involved in photosystem and chlorophyll-binding proteins had higher expression in green leaf tissue. The PSBQ, LHCB1.3, LHCB2.4, and HSP70 may be key genes of photosynthesis and chloroplast formation. These results demonstrated that green and white coloration in ornamental kale leaves was caused by the combined effects of chlorophyll and carotenoid biosynthesis, chloroplast development, as well as photosynthesis. These findings enhance our understanding of the molecular mechanisms underlying leaf color development in ornamental kale.

A single-base insertion in BoDFR1 results in loss of anthocyanins in green-leaved ornamental kale.

KEY MESSAGE: A CRISPR/Cas9-based knockout assay verified that BoDFR1 drives anthocyanin accumulation in ornamental kale and that BoDFR2, an ortholog of BoDFR1, is redundant. Anthocyanins are widely distributed in nature and give plants their brilliant colors. Leaf color is an important trait for ornamental kale. In this study, we measured anthocyanin contents and performed transcriptome deep sequencing (RNA-seq) of leaves from pink and green ornamental kale. We observed substantial differences in the expression levels of the two DIHYDROFLAVONOL 4-REDUCTASE-encoding genes BoDFR1 (Bo9g058630) and its ortholog BoDFR2 (Bo2g116380) between green-leaved and pink-leaved kale by RNA-seq and RT-qPCR. We cloned and sequenced BoDFR1 and BoDFR2 from both types of kale. We identified a 1-bp insertion in BoDFR1 and a 2-bp insertion in BoDFR2 in green-leaved kale compared to the sequences obtained from pink-leaved kale, both mapping to the second exon of their corresponding gene and leading to premature termination of translation. To confirm the genetic basis of the absence of anthocyanins in green kale, we used CRISPR/Cas9 genome editing to separately knock out BoDFR1 or BoDFR2 in the pink-leaved ornamental kale inbred line P23. We detected very low accumulation of anthocyanins in the resulting mutants Bodfr1-1 and Bodfr1-2, while Bodfr2-1 and Bodfr2-2 had anthocyanin levels comparable to those of the wild-type. We conclude that the insertion in BoDFR1, rather than that in BoDFR2, underlies the lack of anthocyanins in green-leaved ornamental kale. This work provides insight into the function of DFR and will contribute to germplasm improvement of ornamental plants.

Simultaneous changes in anthocyanin, chlorophyll, and carotenoid contents produce green variegation in pink-leaved ornamental kale.

BACKGROUND: Anthocyanin, chlorophyll, and carotenoid pigments are widely distributed in plants, producing various colors. Ornamental kale (Brassica oleracea var. acephala DC) which has colorful inner leaves is an ideal plant to explore how these three pigments contribute to leaf color. The molecular mechanisms of the coloration in ornamental kale could provide reference for exploring the mechanisms of pigmentation in other plants. RESULTS: In this study, we sequenced the transcriptome and determined the pigment contents of an unusual cultivar of ornamental kale with three different types of leaf coloration: pink (C3), light pink (C2), and variegated pink-green (C1). A total of 23,965 differentially expressed genes were detected in pairwise comparisons among the three types of leaves. The results indicate that Bo9g058630 coding dihydroflavonol 4-reductase (DFR) and Bo3g019080 coding shikimate O-hydroxycinnamoyltransferase (HCT) acted in anthocyanin biosynthesis in pink leaves. Bo1g053420 coding pheophorbidase (PPD) and Bo3g012430 coding 15-cis-phytoene synthase (crtB) were identified as candidate genes for chlorophyll metabolism and carotenoid biosynthesis, respectively. The transcription factors TT8, MYBL2, GATA21, GLK2, and RR1 might participate in triggering the leaf color change in ornamental kale. Anthocyanin content was highest in C3 and lowest in C1. Chlorophyll and carotenoid contents were lowest in C2 and highest in C1. CONCLUSIONS: Based on these findings, we suspected that the decrease in anthocyanin biosynthesis and the increase in chlorophyll and carotenoid biosynthesis might be the reason for the leaf changing from pink to variegate pink-green in this unusual cultivar. Our research provides insight into the molecular mechanisms of leaf coloration in ornamental kale, contributing to a theoretical foundation for breeding new varieties.

Chrysanthemum WRKY15-1 promotes resistance to Puccinia horiana Henn. via the salicylic acid signaling pathway.

Chrysanthemum white rust disease, which is caused by the fungus Puccinia horiana Henn., severely reduces the ornamental quality and yield chrysanthemum. WRKY transcription factors function in the disease-resistance response in a variety of plants; however, it is unclear whether members of this family improve resistance to white rust disease in chrysanthemum. In this study, using PCR, we isolated a WRKY15 homologous gene, CmWRKY15-1, from the resistant chrysanthemum cultivar C029. Real-time quantitative PCR (RT-qPCR) revealed that CmWRKY15-1 exhibited differential expression patterns between the immune cultivar C029 and the susceptible cultivar Jinba upon P. horiana infection. In addition, salicylic acid (SA) treatment strongly induced CmWRKY15-1 expression. Overexpression of CmWRKY15-1 in the chrysanthemum-susceptible cultivar Jinba increased tolerance to P. horiana infection. Conversely, silencing CmWRKY15-1 via RNA interference (RNAi) in C029 increased sensitivity to P. horiana infection. We also determined that P. horiana infection increased both the endogenous SA content and the expression of salicylic acid biosynthesis genes in CmWRKY15-1-overexpressing plants, whereas CmWRKY15-1 RNAi plants exhibited the opposite effects under the same conditions. Finally, the transcript levels of pathogenesis-related (PR) genes involved in the SA pathway were positively associated with CmWRKY15-1 expression levels. Our results demonstrated that CmWRKY15-1 plays an important role in the resistance of chrysanthemum to P. horiana by influencing SA signaling.

The dihydroflavonol 4-reductase BoDFR1 drives anthocyanin accumulation in pink-leaved ornamental kale.

KEY MESSAGE: Overexpression and virus-induced gene silencing verified BoDFR1 conferred the anthocyanin accumulation in pink-leaved ornamental kale. Leaf color is an essential trait in the important horticultural biennial plant ornamental kale (Brassica oleracea var. acephala). The identity of the gene conferring this striking trait and its mode of inheritance are topics of debate. Based on an analysis of F1, F2, BC1P1, and BC1P2 ornamental kale populations derived from a cross between a pink-leaved P28 and white-leaved D10 line, we determined that the pink leaf trait is controlled by a semi-dominant gene. We cloned two genes potentially involved in anthocyanin biosynthesis in ornamental kale: Bo9g058630 and Bo6g100940. Based on their variation in sequence, we speculated that Bo9g058630, encoding the kale dihydroflavonol-4 reductase (BoDFR1) enzyme, plays a critical role in the development of the pink leaf trait. Indeed, an InDel marker specific for BoDFR1 completely co-segregated with the pink leaf trait in our F2 population. We then generated the 35Spro: DFR-GUS overexpression vector, which we transformed into D10. Overexpression of BoDFR1 indeed restored some anthocyanin accumulation in this white-leaved parental line. In addition, we targeted BoDFR1 in P28 using virus-induced gene silencing. Again, silencing of BoDFR1 resulted in a substantial decrease in anthocyanin accumulation. This work lays the foundation for further exploration of the mechanism underlying anthocyanin accumulation in pink-leaved ornamental kale.

Fine mapping and identification of the leaf shape gene BoFL in ornamental kale.

KEY MESSAGE: BoFL, a candidate gene conferring the feathered-leaved trait in ornamental kale, is located in a 374.532-kb region on chromosome C9. Leaf shape is an important economic trait in ornamental kale (Brassica oleracea var. acephala). Identifying the genes that influence leaf shape would provide insight into the mechanism underlying leaf development. In this study, we constructed F1, F2, BC1P1, BC1P2, and F2:3 populations from a cross between a feathered-leaved inbred line, F0819, and a smooth-leaved inbred line, S0835, of ornamental kale. Genetic analysis showed that the feathered-leaved trait was controlled by a semi-dominant gene, BoFL. Using bulked segregant analysis sequencing, we mapped the BoFL gene to a 4.17-Mb interval on chromosome C9. Next, we narrowed down the candidate region to 374.532-kb by fine-scale mapping between the two flanking markers INDEL940 and INDEL5443. We identified 38 genes in the candidate region, among which only Bo9g184610 showed significant variation in expression level between the two parental lines. Sequencing of the gene in the parental lines identified three single-nucleotide polymorphism (SNP) differences, containing two nonsynonymous and one synonymous SNPs, which resulted in coding variations of an aspartate and a phenylalanine residue in F0819, but an alanine and a leucine residue in S0835. A cleaved amplified polymorphic sequence (CAPS) marker, CAPS4610, corresponding to the first nonsynonymous SNP co-segregated with the leaf shape trait. We thus speculated that the gene conferring the feathered-leaved trait is BoALG10, a homolog of ALG10, which encodes an alpha-1,2-glucosyltransferase in Arabidopsis thaliana. This work will be useful for understanding the mechanism of leaf development and provides important information for the breeding of kale with novel leaf shapes.