Solanaceae have played an important role in elucidating how flower color is specified by the flavonoid biosynthesis pathway (FBP), which produces anthocyanins and other secondary metabolites. With well-established reverse genetics tools and rich genomic resources, Solanaceae provide a robust framework to examine the diversification of this well-studied pathway over short evolutionary timescales and to evaluate the predictability of genetic perturbation on pathway flux. Genomes of eight Solanaceae species, nine related asterids, and four rosids were mined to evaluate variation in copy number of the suite of FBP enzymes involved in anthocyanin biosynthesis. Comparison of annotation sources indicated that the NCBI annotation pipeline generated more and longer FBP annotations on average than genome-specific annotation pipelines. The pattern of diversification of each enzyme among asterids was assessed by phylogenetic analysis, showing that the CHS superfamily encompasses a large paralogous family of ancient and recent duplicates, whereas other FBP enzymes have diversified via recent duplications in particular lineages. Heterologous expression of a pansy F3′5′H gene in tobacco changed flower color from pink to dark purple, demonstrating that anthocyanin production can be predictably modified using reverse genetics. These results suggest that the Solanaceae FBP could be an ideal system to model genotype-to-phenotype interactions for secondary metabolism.

The basic helix-loop-helix (bHLH) transcription factors are widely distributed across eukaryotic kingdoms and participate in various physiological processes. To date, the bHLH family has been identified and functionally analyzed in many plants. However, systematic identification of bHLH transcription factors has yet to be reported in orchids. Here, 94 bHLH transcription factors were identified from the Cymbidium ensifolium genome and divided into 18 subfamilies. Most CebHLHs contain numerous cis-acting elements associated with abiotic stress responses and phytohormone responses. A total of 19 pairs of duplicated genes were found in the CebHLHs, of which 13 pairs were segmentally duplicated genes and six pairs were tandemly duplicated genes. Expression pattern analysis based on transcriptome data revealed that 84 CebHLHs were differentially expressed in four different color sepals, especially CebHLH13 and CebHLH75 of the S7 subfamily. The expression profiles of CebHLH13 and CebHLH75 in sepals, which are considered potential genes regulating anthocyanin biosynthesis, were confirmed through the qRT-PCR technique. Furthermore, subcellular localization results showed that CebHLH13 and CebHLH75 were located in the nucleus. This research lays a foundation for further exploration of the mechanism of CebHLHs in flower color formation.

Phalaenopsis orchids are popular worldwide due to their high ornamental and economic value; the spike and inflorescence formation of their flowers could be efficiently controlled under proper conditions. In this study, transcriptomic profiles and endogenous hormone changes were investigated to better understand the spike formation of Phalaenopsis. Morphological observations revealed four spike initiation statuses (i.e., S0: the status refers to axillary buds remaining dormant in the leaf axils; S1: the status refers to the 0.5 cm-long initial spike; S2: the status refers to the 1 cm-long spike; S3: the status refers to the 3 cm-long spike) during the process of spike development, while anatomical observations revealed four related statuses of inflorescence primordium differentiation. A total of 4080 differentially expressed genes were identified based on pairwise comparisons of the transcriptomic data obtained from the S0 to S3 samples; high levels of differential gene expression were mostly observed in S1 vs. S2, followed by S0 vs. S1. Then, the contents of 12 endogenous hormones (e.g., irindole-3-acetic acid (IAA), salicylic acid (SA), abscisic acid (ABA), gibberellins, and cytokinins) were measured. The results showed that the ABA content was decreased from S0 to S1, while the gibberellic acid 1 (GA1) content exhibited an opposite trend, indicating the reduction in ABA levels combined with the increase in GA1 levels in S0 promoted the axillary bud dormancy breaking, preparing for the following spike initiation. The GA20 oxidase and ABA 8′-hydroxylase genes, which are involved in endogenous hormone metabolism and signaling pathways, displayed similar expression patterns, suggesting they were probably the key genes participating in the GA and ABA regulation. Taken together, the findings of this study indicate that GA and ABA may be the key endogenous hormones breaking the dormancy and promoting the germination of axillary buds in Phalaenopsis.