Floret color is a crucial phenotypic trait in broccoli, serving as an indicator of maturity and determining its market value. However, the mechanisms underlying color variation remain unclear. In this study, six broccoli varieties with different floret colors at harvest were chosen as materials. The color difference and pigment content of florets were measured, and a combined analysis of anthocyanin-targeted metabolome and transcriptome was conducted. Our findings revealed that chlorophyll a primarily influences green, yellow-green, and light green coloration, while the wax content may contribute to gray-green coloration. The blue-green and dark blue-green coloration are regulated by both chlorophyll a and anthocyanins. Targeted metabolomics identified five anthocyanin compounds, with peonidin-3-O-glucoside as a key metabolite for blue-green coloration and delphinidin-3-O-glucoside-5-O-galactoside and peonidin-3,5-O-diglucoside for dark blue-green coloration. Transcriptomic analysis identified CHLG as a potential key regulator for yellow-green and light-green floret coloration. The blue-green coloration appears to be coregulated by a combination of genes, including the chlorophyll biosynthesis gene HEMF; anthocyanin biosynthesis genes (PAL, FLS, and UGT); and chlorophyll degradation genes (SGR, PPD, and NYC). Furthermore, upstream genes involved in both chlorophyll metabolism (CHLI, CHLD, CHLM, DVR, and CLH) and anthocyanin biosynthesis (PAL, 4CL, CHS, F3′H, and FLS) play crucial roles in determining the dark blue-green coloration of florets. Meanwhile, transcription factors of the WRKY, NAC, and TCP families are involved in chlorophyll metabolism, while those of the bHLH and MYB families participate in anthocyanin synthesis. The WGCNA identified one Hub gene for chlorophyll metabolism and two for anthocyanin synthesis. In conclusion, 35 candidate genes were identified, including 21 involved in chlorophyll metabolism and 14 in anthocyanin biosynthesis. This study provides novel insights into the molecular mechanisms of floret coloration and establishes a foundation for molecular breeding in broccoli.

Pectin methylesterases (PMEs) and their inhibitors (PMEIs) serve as pivotal enzymes in pectin methylation modifications, playing crucial roles in plant morphogenesis, cell adhesion, and maintenance of the cell wall integrity. However, there have been limited studies exploring the functions of the PME/PMEI gene families in the healing process of grafted-cucumber seedlings and their responses to stress conditions. In this study, we identified 52 CsaPME family members and 33 CsaPMEI family members as well as 86 CmoPME family members and 36 CmoPMEI family members. Comprehensive analyses of the PME/PMEI gene families in cucumber and pumpkin were conducted using bioinformatics techniques. Additionally, the PME/PMEI gene families in cucumber and pumpkin exhibited distinct expression modes in different vegetative organs of homologous/heterologous-grafted seedlings and under chilling stress. Notably, the cucumber/pumpkin-grafted seedlings exhibited responses in the roots and leaves involving PMEI and type I proPME genes, facilitating their adaptation to chilling stress. Additionally, an investigation into the responsiveness of cucumber/pumpkin-grafted seedlings during the healing phase to varying light intensity modes revealed that the implementation of a higher light intensity mode resulted in an upregulation of the expression levels of the majority of PME/PMEI family genes, particularly those belonging to the PME family, during the critical stages of isolation layer and callus formation. Based on these findings, six key PME/PMEI family members responsive to different light intensity modes during graft healing were selected. Through the prediction of transcription factor binding sites and an analysis of the response to different light intensity modes during graft healing, four Dof transcription factors with potential regulatory relationships with these six key PME/PMEI genes were identified. This suggests that cucumber/pumpkin-grafted seedlings can regulate key PME/PMEI genes via Dof factors in response to different light intensity modes during the healing process, thereby influencing the progression of graft healing.

As a vital appearance quality trait of broccoli, curd-surface wax powder not only affects its commercial value but also plays a key role in plant resistance to abiotic stresses. However, its formation mechanism remains unclear. Using low-wax variety CK (‘QH18’) and high-wax variety T1 (‘QHMS4’) as materials, this study systematically elucidated the molecular mechanism of wax powder formation via physiological indexes, scanning electron microscopy (SEM), targeted metabolomics, and transcriptomics. Determination of fatty acid (FA) content in broccoli flower bud tissue showed a close association between FA content and wax deposition. SEM observation revealed that T1 had significantly denser wax crystals, mainly granular, than CK. Targeted metabolomics identified 25 fatty acids in the two varieties. And the linolenic and palmitic acids, with high content and significant differences, may be key metabolites regulating wax synthesis. Integrated transcriptomics and metabolomics indicated that BolfabG, BolLACS, BolKCS1, BolKCS2 and BolMAH1 genes are involved in wax biosynthesis. Moreover, AP2/ERF-ERF transcription factor (TF)-encoding genes (BolERF018, BolERF1F.1, BolERF1F.2 and BolERF1C) played the primary role in regulating wax biosynthesis, followed by NAC (BolNAC62.1), MYB (BolMYB44), and MADS-MIKC(BolPISTILLATA). These TFs may regulate BolfabG, BolLACS, BolKCS1, BolACOX2 and BolACAA1 to affect linolenic and palmitic acid balance, altering wax precursor synthesis and accumulation, and finally leading to differences in wax morphology and content. This study reveals a “Transcription Factors–Differentially Expressed Genes–Differentially Accumulated Metabolites–Fatty Acids” (TFs-DEGs-DAMs-FA) network, providing a basis for understanding broccoli wax formation.