Series-Spatial Transcriptome Profiling of Leafy Head Reveals the Key Transition Leaves for Head Formation in Chinese Cabbage

Chinese cabbage is an important leaf heading vegetable crop. At the heading stage, its leaves across inner to outer show significant morphological differentiation. However, the genetic control of this complex leaf morphological differentiation remains unclear. Here, we reported the transcriptome profiling of Chinese cabbage plant at the heading stage using 24 spatially dissected tissues representing different regions of the inner to outer leaves. Genome-wide transcriptome analysis clearly separated the inner leaf tissues from the outer leaf tissues. In particular, we identified the key transition leaf by the spatial expression analysis of key genes for leaf development and sugar metabolism. We observed that the key transition leaves were the first inwardly curved ones. Surprisingly, most of the heading candidate genes identified by domestication selection analysis obviously showed a corresponding expression transition, supporting that key transition leaves are related to leafy head formation. The key transition leaves were controlled by a complex signal network, including not only internal hormones and protein kinases but also external light and other stimuli. Our findings provide new insights and the rich resource to unravel the genetic control of heading traits.

Impacts of allopolyploidization and structural variation on intraspecific diversification in Brassica rapa

Background Despite the prevalence and recurrence of polyploidization in the speciation of flowering plants, its impacts on crop intraspecific genome diversification are largely unknown. Brassica rapa is a mesopolyploid species that is domesticated into many subspecies with distinctive morphotypes. Results Herein, we report the consequences of the whole-genome triplication (WGT) on intraspecific diversification using a pan-genome analysis of 16 de novo assembled and two reported genomes. Among the genes that derive from WGT, 13.42% of polyploidy-derived genes accumulate more transposable elements and non-synonymous mutations than other genes during individual genome evolution. We denote such genes as being “flexible.” We construct the Brassica rapa ancestral genome and observe the continuing influence of the dominant subgenome on intraspecific diversification in B. rapa. The gene flexibility is biased to the more fractionated subgenomes (MFs), in contrast to the more intact gene content of the dominant LF (least fractionated) subgenome. Furthermore, polyploidy-derived flexible syntenic genes are implicated in the response to stimulus and the phytohormone auxin; this may reflect adaptation to the environment. Using an integrated graph-based genome, we investigate the structural variation (SV) landscapes in 524 B. rapa genomes. We observe that SVs track morphotype domestication. Four out of 266 candidate genes for Chinese cabbage domestication are speculated to be involved in the leafy head formation. Conclusions This pan-genome uncovers the possible contributions of allopolyploidization on intraspecific diversification and the possible and underexplored role of SVs in favorable trait domestication. Collectively, our work serves as a rich resource for genome-based B. rapa improvement.

Divergence of three BRX homoeologs in Brassica rapa and its effect on leaf morphology

The leafy head characteristic is a special phenotype of Chinese cabbage resulting from artificial selection during domestication and breeding. BREVIS RADIX (BRX) has been suggested to control root elongation, shoot growth, and tiller angle in Arabidopsis and rice. In Brassica rapa, three BrBRX homoeologs have been identified, but only BrBRX.1 and BrBRX.2 were found to be under selection in leaf-heading accessions, indicating their functional diversification in leafy head formation. Here, we show that these three BrBRX genes belong to a plant-specific BRX gene family but that they have significantly diverged from other BRX-like members on the basis of different phylogenetic classifications, motif compositions and expression patterns. Moreover, although the expression of these three BrBRX genes differed, compared with BrBRX.3, BrBRX.1, and BrBRX.2 displayed similar expression patterns. Arabidopsis mutant complementation studies showed that only BrBRX.1 could rescue the brx root phenotype, whereas BrBRX.2 and BrBRX.3 could not. However, overexpression of each of the three BrBRX genes in Arabidopsis resulted in similar pleiotropic leaf phenotypes, including epinastic leaf morphology, with an increase in leaf number and leaf petiole length and a reduction in leaf angle. These leaf traits are associated with leafy head formation. Further testing of a SNP (T/C) in BrBRX.2 confirmed that this allele in the heading accessions was strongly associated with the leaf-heading trait of B. rapa. Our results revealed that all three BrBRX genes may be involved in the leaf-heading trait, but they may have functionally diverged on the basis of their differential expression.

Upregulation of a KN1 homolog by transposon insertion promotes leafy head development in lettuce

Leafy head is a unique type of plant architecture found in some vegetable crops, with leaves bending inward to form a compact head. The genetic and molecular mechanisms underlying leafy head in vegetables remain poorly understood. We genetically fine-mapped and cloned a major quantitative trait locus controlling heading in lettuce. The candidate gene (LsKN1) is a homolog of knotted 1 (KN1) from Zea mays. Complementation and CRISPR/Cas9 knockout experiments confirmed the role of LsKN1 in heading. In heading lettuce, there is a CACTA-like transposon inserted into the first exon of LsKN1 (LsKN1▽). The transposon sequences act as a promoter rather than an enhancer and drive high expression of LsKN1▽. The enhanced expression of LsKN1▽ is necessary but not sufficient for heading in lettuce. Data from ChIP-sequencing, electrophoretic mobility shift assays, and dual luciferase assays indicate that the LsKN1▽ protein binds the promoter of LsAS1 and down-regulates its expression to alter leaf dorsoventrality. This study provides insight into plant leaf development and will be useful for studies on heading in other vegetable crops.

The mutation of ent-kaurene synthase, a key enzyme involved in gibberellin biosynthesis, confers a non-heading phenotype to Chinese cabbage (Brassica rapa L. ssp. pekinensis)

The presence of a leafy head is a vital agronomic trait that facilitates the evaluation of the yield and quality of Chinese cabbage. A non-heading mutant (nhm1) was identified in an ethyl methanesulfonate mutagenesis population of the heading Chinese cabbage double haploid line FT. Segregation analysis revealed that a single recessive gene, Brnhm1, controlled the mutant phenotype. Using MutMap, Kompetitive allele-specific PCR, and cloning analyses, we demonstrated that BraA07g042410.3C, which encodes an ent-kaurene synthase involved in the gibberellin biosynthesis pathway, is the nhm1 mutant candidate gene. A single-nucleotide mutation (C to T) in the fourth exon of BraA07g042410.3C caused an amino acid substitution from histidine to tyrosine. Compared to that of the wild-type FT, BraA07g042410.3C in the leaves of the nhm1 mutant had lower levels of expression. In addition, gibberellin contents were lower in the mutant than in the wild type, and the mutant plant phenotype could be restored to that of the wild type after exogenous GA3 treatment. These results indicate that BraA07g042410.3C caused the non-heading mutation. This is the first study to demonstrate a relationship between gibberellin content in the leaves and leafy head formation in Chinese cabbage. These findings facilitate the understanding of the mechanisms underlying leafy head development in Chinese cabbage.

BcpLH organizes a specific subset of microRNAs to form a leafy head in Chinese cabbage (Brassica rapa ssp. pekinensis)

HYL1 (HYPONASTIC LEAVES 1) in Arabidopsis thaliana encodes a double-stranded RNA-binding protein needed for proper miRNA maturation, and its null mutant hyl1 shows a typical leaf-incurvature phenotype. In Chinese cabbage, BcpLH (Brassica rapa ssp. pekinensis LEAFY HEADS), a close homolog of HYL1, is differentially expressed in juvenile leaves, which are flat, and in adult leaves, which display extreme incurvature. BcpLH lacks protein–protein interaction domains and is much shorter than HYL1. To test whether BcpLH is associated with defects in microRNA (miRNA) biogenesis and leaf flatness, we enhanced and repressed the activity of BcpLH by transgenics and investigated BcpLH-dependent miRNAs and plant morphology. BcpLH promoted miRNA biogenesis by the proper processing of primary miRNAs. BcpLH downregulation via antisense decreased a specific subset of miRNAs and increased the activities of their target genes, causing upward curvature of rosette leaves and early leaf incurvature, concurrent with the enlargement, earliness, and round-to-oval shape transition of leafy heads. Moreover, BcpLH-dependent miRNAs in Chinese cabbage are not the same as HYL1-dependent miRNAs in Arabidopsis. We suggest that BcpLH controls a specific subset of miRNAs in Chinese cabbage and coordinates the direction, extent, and timing of leaf curvature during head formation in Brassica rapa.

Conjunctive Analyses of BSA-Seq and BSR-Seq to Reveal the Molecular Pathway of Leafy Head Formation in Chinese Cabbage

As the storage organ of Chinese cabbage, the leafy head was harvested as a commercial product due to its edible value. In this study, the bulked segregant analysis (BSA) and bulked segregant RNA-Seq (BSR) were performed with F2 separation progeny to study the molecular mechanism of leafy head formation in Chinese cabbage. BSA-Seq analysis located four candidate regions containing 40 candidate genes, while BSR-Seq analysis revealed eight candidate regions containing 607 candidate genes. The conjunctive analyses of these two methods identified that Casein kinase gene BrCKL8 (Bra035974) is the common candidate gene related with leafy head formation in Chinese cabbage, and it showed high expression levels at the three segments of heading type plant leaves. The differentially expressed genes (DEGs) between two set pairs of cDNA sequencing bulks were divided into two categories: one category was related with five hormone pathways (Auxin, Ethylene, Abscisic acid, Jasmonic acid and Gibberellin), the other category was composed of genes that associate with the calcium signaling pathway. Moreover, a series of upregulated transcriptional factors (TFs) were also identified by the association analysis of BSR-Seq analysis. The leafy head development was regulated by various biological processes and effected by diverse external environment factors, so our research will contribute to the breeding of perfect leaf-heading types of Chinese cabbage.

Genome-wide transcriptome analysis reveals molecular pathways involved in leafy head formation of Chinese cabbage (Brassica rapa)

Chinese cabbage plants go through seedling and rosette stages before forming their leafy head. Chinese cabbage plants resemble pak-choi plants at their seedling stage, but in their rosette stage the leaves of Chinese cabbage differentiate, as they increase in size with shorter petioles. In order to understand the molecular pathways that play a role in leafy head formation, transcript abundance of young emerging leaves was profiled during development of two Chinese cabbage genotypes and a single pak-choi genotype. The two Chinese cabbages differed in many aspects, among others earliness, leaf size and shape, leaf numbers, and leafy head shape. Genome-wide transcriptome analysis clearly separated the seedling stages of all three genotypes together with the later stages from pak-choi, from the later developmental stages of both Chinese cabbages (rosette, folding, and heading). Weighted correlation network analysis and hierarchical clustering using Euclidean distances resulted in gene clusters with transcript abundance patterns distinguishing the two Chinese cabbages from pak-choi. Three clusters included genes with transcript abundance affected by both genotype and developmental stage, whereas two clusters showed only genotype effects. This included a genotype by developmental stage cluster highly enriched with the MapMan category photosynthesis, with high expression during rosette and folding in Chinese cabbages and low expression in the heading inner leaves that are not exposed to light. The other clusters contained many genes in the MapMan categories Cell, showing again differences between pak-choi and both Chinese cabbages. We discuss how this relates to the differences in leaf blade growth between Chinese cabbage and pak-choi, especially at the rosette stage. Overall, comparison of the transcriptome between leaves of two very different Chinese cabbages with pak-choi during plant development allowed the identification of specific gene categories associated with leafy head formation.