The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the developmental landscape of a complex organ by monitoring the differentiation trajectory of every specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using this protocol, we generated viable cell suspensions suitable for an array of expression analyses, including scRNA-seq. The universal application of this protocol was further tested by successfully isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and gynophores) from representative species on the key branches of the angiosperm lineage. This work provides a robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species and opens a new avenue to study cell type specification, tissue function, and organ diversification in plants.

Actinomorphic flowers usually orient vertically (relative to the horizon) and possess symmetric nectar guides, while zygomorphic flowers often face horizontally and have asymmetric nectar guides, indicating that floral symmetry, floral orientation, and nectar guide patterning are correlated. The origin of floral zygomorphy is dependent on the dorsoventrally asymmetric expression of CYCLOIDEA (CYC)-like genes. However, how horizontal orientation and asymmetric nectar guides are achieved remains poorly understood. Here we selected Chirita pumila (Gesneriaceae) as a model plant to explore the molecular bases for these traits. By analyzing gene expression patterns, protein–DNA and protein–protein interactions, and encoded protein functions we identified multiple roles and functional divergence of two CYC-like genes, i.e., CpCYC1 and CpCYC2, in controlling floral symmetry, floral orientation, and nectar guide patterning. CpCYC1 positively regulates its own expression, whereas CpCYC2 does not regulate itself. In addition, CpCYC2 up-regulates CpCYC1, while CpCYC1 down-regulates CpCYC2. This asymmetric auto- and cross-regulation mechanism might explain the high expression levels of only one of these genes. We show that CpCYC1 and CpCYC2 determine asymmetric nectar guide formation, likely by directly repressing the flavonoid synthesis-related gene CpF3’5’H. We further suggest that CYC-like genes play multiple conserved roles in Gesneriaceae. These findings shed light on the repeated origins of zygomorphic flowers in angiosperms.

In animals and plants, organ shape is primarily determined during primordium development by carefully coordinated growth and cell division^1–3. Rare examples of post-primordial change in morphology (reshaping) exist that offer tractable systems for the study of mechanisms required for organ shape determination and diversification. One such example is morphogenesis in Capsella fruits whose heart-shaped appearance emerges by reshaping of the ovate spheroid gynoecium upon fertilization⁴. Here we use whole-organ live-cell imaging and single-cell RNA sequencing (scRNA-seq) analysis to show that Capsella fruit shape determination is based on dynamic changes in cell growth and cell division coupled with local maintenance of meristematic identity. At the molecular level, we reveal an auxin-induced mechanism that is required for morphological alteration and ultimately determined by a single cis-regulatory element. This element resides in the promoter of the Capsella rubella SHOOTMERISTEMLESS ⁵ (Cr STM) gene. The CrSTM meristem identity factor positively regulates its own expression through binding to this element, thereby providing a feed-forward loop at the position and time of protrusion emergence to form the heart. Independent evolution of the STM-binding element in STM promoters across Brassicaceae species correlates with those undergoing a gynoecium-to-fruit shape change. Accordingly, genetic and phenotypic studies show that the STM-binding element is required to facilitate the shape transition and suggest a conserved molecular mechanism for organ morphogenesis.

Plants bearing double flowers have long been cultivated as ornamental plants. Hose-in-hose flowers, bearing 2-whorled corolla tubes in whorls 1 and 2, are uncommon but recur in Sinningia (Gesnerioideae, Gesneriaceae). In this study, we selected 15 hose-in-hose cultivars as materials to explore the underlying molecular and genetic mechanisms of this floral architecture. We found that they originated from different hybridization events within the Dircaea clade. Three B-class MADS-box genes were globally expressed in all floral whorls, but only GLOBOSA1 (GLO1) has accumulated a dominant mutation, i.e., the insertion of a hAT-like miniature inverted-repeat transposable element (MITE) into its promoter, that co-segregated with the hose-in-hose phenotype. In addition, all 15 hose-in-hose cultivars contained the same dominant GLO1 allele. Transient gene expression assays confirmed the role of this MITE insertion in up-regulating the promoter activity of GLO1 by providing several cis-regulatory elements. Genetic transformation in heterologous Chirita pumila (Didymocarpoideae, Gesneriaceae) verified that this dominant GLO1 allele is sufficient to confer the hose-in-hose phenotype. We further demonstrated that both the GLO1 allele and the hAT-like MITE descended from wild S. cardinalis with single flowers. This study highlights the significance of wide hybridization in frequent gains of the dominant GLO1 allele and thereafter repeated occurrence of hose-in-hose flowers in Sinningia.

Gynoecium patterning is dependent on the dynamic distribution of auxin, the signalling of which is transduced through several distinct pathways. ETTIN (ETT)-mediated signalling occurs independently of the canonical auxin pathway, and ETT shares partial redundancy with Auxin Response Factor 4 (ARF4) in the gynoecium. ETT and ARF4 were previously hypothesized to translate auxin gradients into patterns of tissue polarity alongside other ARFs. As ARF repressors, ETT/ARF were assumed to antagonistically regulate targets shared with ARF activators of the canonical pathway. Here, comparative transcriptomics identified the distinct and overlapping targets of ETT/ARF4 in the Arabidopsis gynoecium. However, ETT/ARF4 targets with known roles in gynoecium development did not conform to models of A-B ARF antagonism, leaving the relationship with the canonical pathway unclear. Mutants in tir1 afb2 ett were therefore generated in Arabidopsis and Capsella to assess the relationship between the two pathways, and their conservation in species with distinct fruit shapes. The data presented indicate conserved synergism between the two pathways in gynoecium development and suggest a role for ARF4 in the integration of these pathways in Brassicaceae with distinct fruit shapes.