Camalexin, a major phytoalexin in Arabidopsis thaliana, consists of an indole ring and a thiazole ring. The indole ring is produced from Trp, which is converted to indole-3-acetonitrile (IAN) by CYP79B2/CYP79B3 and CYP71A13. Conversion of Cys(IAN) to dihydrocamalexic acid and subsequently to camalexin is catalyzed by CYP71B15. Recent studies proposed that Cys derivative, not Cys itself, is the precursor of the thiazole ring that conjugates with IAN. The nature of the Cys derivative and how it conjugates to IAN and subsequently forms Cys(IAN) remain obscure. We found that protein accumulation of multiple glutathione S-transferases (GSTs), elevation of GST activity, and consumption of glutathione (GSH) coincided with camalexin production. GSTF6 overexpression increased and GSTF6-knockout reduced camalexin production. Arabidopsis GSTF6 expressed in yeast cells catalyzed GSH(IAN) formation. GSH(IAN), (IAN)CysGly, and gGluCys(IAN) were determined to be intermediates within the camalexin biosynthetic pathway. Inhibitor treatments and mutant analyses revealed the involvement of g-glutamyl transpeptidases (GGTs) and phytochelatin synthase (PCS) in the catabolism of GSH(IAN). The expression of GSTF6, GGT1, GGT2, and PCS1 was coordinately upregulated during camalexin biosynthesis. These results suggest that GSH is the Cys derivative used during camalexin biosynthesis, that the conjugation of GSH with IAN is catalyzed by GSTF6, and that GGTs and PCS are involved in camalexin biosynthesis.

Chinese cabbage is the most consumed leafy crop in East Asian countries. However, premature bolting induced by continuous low temperatures severely decreases the yield and quality of the Chinese cabbage, and therefore restricts its planting season and geographic distribution. In the past 40 years, spring Chinese cabbage with strong winterness has been selected to meet the market demand. Here, we report a genome variation map of Chinese cabbage generated from the resequencing data of 194 geographically diverse accessions of three ecotypes. In-depth analyses of the selection sweeps and genome-wide patterns revealed that spring Chinese cabbage was selected from a specific population of autumn Chinese cabbage around the area of Shandong peninsula in northern China. We identified 23 genomic loci that underwent intensive selection, and further demonstrated by gene expression and haplotype analyses that the incorporation of elite alleles of VERNALISATION INSENTIVE 3.1 (BrVIN3.1) and FLOWER LOCUS C 1 (BrFLC1) is a determinant genetic source of variation during selection. Moreover, we showed that the quantitative response of BrVIN3.1 to cold due to the sequence variations in the cis elements of the BrVIN3.1 promoter significantly contributes to bolting-time variation in Chinese cabbage. Collectively, our study provides valuable insights into the genetic basis of spring Chinese cabbage selection and will facilitate the breeding of bolting-resistant varieties by molecular-marker-assisted selection, transgenic or gene editing approaches.

Chinese cabbage varieties with orange inner leaves are of interest due to their attractive appearance and high carotenoid content. Previously, a recessive locus, Br-or, that confers this trait was mapped to linkage group A09 in Brassica rapa, and Bra031539 was identified as the candidate gene. We used a backcross population consisting of 1,392 individuals for fine mapping; three linked markers delimited the Br-or locus to a region of 9.47 kb, which contained only one predicted functional gene, Bra031539 (the carotenoid isomerase gene BrCRTISO). We identified two bacterial artificial chromosome (BAC) clones carrying Br-or and confirmed that this allele of BrCRTISO confers the mutant phenotype by sequencing. Many DNA sequence variations are present in Br-or, including a 90-bp DNA deletion in the promoter and a 501-bp insertion at the 3′ end of the gene. GUS staining showed that the spatial expression of BrCRTISO was perfectly coincident with the site of carotenoid synthesis and that the 90-bp deletion in the promoter slightly downregulated expression of the gene. Using an in vitro enzyme assay, we showed that the mutant Br-or protein cannot convert prolycopene to all-trans-lycopene. Light has a positive effect on the expression of carotenoid biosynthesis genes, except for beta-ring carotene hydroxylase (CHXB), and this coincided with the increase in total lycopene and β-carotene in the Br-or mutant. We also demonstrated that loss of BrCRTISO function, upregulation of the upstream genes, and downregulation of downstream genes lead to the accumulation of prolycopene and confer an orange color to the inner head leaves in Chinese cabbage. Keywords Brassica rapa L. ssp. pekinensis . Orange leaf . Br-or . BrCRTISO . Carotenoid biosynthesis Abbreviations CHXB Beta-ring carotene hydroxylase DH Doubled haploid GAPDH Glyceraldehyde-3-phosphate dehydrogenase IPI Isopentenyl pyrophosphate isomerase LCYB Lycopene beta-cyclase LCYE Lycopene epsilon-cyclase PDS Phytoene desaturase PSY Phytoene synthase VDE Violaxanthin de-epoxidase ZDS Zeta-carotene desaturase ZEP Zeaxanthin epoxidase

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of plant growth, development and responses to a wide variety of stimuli. In a conditional gain-of-function transgenic system, the activation of AtMEK5, a MAPK kinase, can in turn activate endogenous AtMAPK3 and AtMAPK6, and can lead to a striking increase in ethylene production and induce hypersensitive response (HR)-like cell death in Arabidopsis. However, the role of the increased ethylene production in regulating this HR-like cell death remains unknown. Using Arabidopsis transgenic plants that express AtMEK5 DD , an active mutant of AtMEK5 that is under the control of a steroid-inducible promoter, we tested the contribution of ethylene to cell death. We found that ethylene biosynthesis occurs before cell death. Cell death was delayed by inhibiting AtMEK5-induced ethylene production using inhibitors of ACC-synthases, ACCoxidases or ethylene receptors. In the mutants AtMEK5 DD /etr1-1 and AtMEK5 DD /ein2-1, both of which showed insensitivity to ethylene, the expression of AtMEK5 DD protein, activity of AtMAPK3 and AtMAPK6, and ethylene production were the same as those seen in AtMEK5 DD transgenic plants, but cell death was also delayed. These data suggest that ethylene signaling perception is required to accelerate cell death that is induced by AtMEK5 activation.

Brassica rapa displays a wide range of morphological diversity which is exploited for a variety of food crops. Here we present a high-quality genome assembly for pak choi (Brassica rapa L. subsp. chinensis), an important non-heading leafy vegetable, and comparison with the genomes of heading type Chinese cabbage and the oilseed form, yellow sarson. Gene presence-absence variation (PAV) and genomic structural variations (SV) were identified, together with single nucleotide polymorphisms (SNPs). The structure and expression of genes for leaf morphology and flowering were compared between the three morphotypes revealing candidate genes for these traits in B. rapa. The pak choi genome assembly and its comparison with other B. rapa genome assemblies provides a valuable resource for the genetic improvement of this important vegetable crop and as a model to understand the diversity of morphological variation across Brassica species.