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.

Great advances have been achieved in our understanding of flower development and evolution since the establishment of the ABC model. However, it remains a challenge to define the exact context of organ identity in the component interactions of the ABC model. Through hybridization, we detected a homeotic mutant in Petrocosmea (Gesneriaceae) uniquely displayed by the ‘petaloid-stamen’ in the third whorl with petal identity. Comparative Real-time PCR analyses demonstrate that both two B-class genes DEF2 and GLO are excessively expressed while the transcripts of the C-class gene PLE are reduced in the third floral whorl in the mutant compared to that in the wild-type F1 hybrids. Further allele-specific expression (ASE) analyses indicate that an allele-specific change in PgPLE might be responsible for up-regulation of both B-class genes and down-regulation of the C-class gene in the petaloid-stamen mutants. Our findings suggest that the petaloid-stamen is consequent upon an evident dosage imbalance between B- and C-class products that is probably triggered by a cis-regulatory change. In addition, the genetic pathway for the floral organ identity might be in parallel with that for the floral symmetry. The extreme variation in hybrids further suggests that interspecific hybridization may represent a major factor for evolutionary innovation and diversification in plants.

The development of an ideal model plant located at a key phylogenetic node is critically important to advance functional and regulatory studies of key regulatory genes in the evolutionary developmental (evo-devo) biology field. In this study, we selected Chirita pumila in the family Gesneriaceae, a basal group in Lamiales, as a model plant to optimize its genetic transformation system established previously by us through investigating a series of factors and further conduct functional test of the CYC-like floral symmetry gene CpCYC. By transforming a RNAi:CpCYC vector, we successfully achieved the desired phenotypes of upright actinomorphic flowers, which suggest that CpCYC actually determines the establishment of floral zygomorphy and the horizontal orientation of flowers in C. pumila. We also confirmed the activities of CpCYC promoter in dorsal petals, dorsal/lateral staminodes, as well as the pedicel by transferring a CpCYC promoter:GUS vector into C. pumila. Furthermore, we testified the availability of a transient gene expression system using C. pumila mesophyll protoplasts. The improved transformation system together with the inherent biological features would make C. pumila an attractive new model in functional and regulatory studies for a broad range of evo-devo issues.

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.

Many plants employ osmotic and hydrostatic pressure to generate movement for survival; but little is known about the cellular mechanisms involved. Here, we report a new cell type in angiosperms termed ‘contractile cells’ in the stigmas of the flowering plant Chirita pumila with much expanded rough endoplasmic reticulum (RER). Cryo-SEM and TEM analyses revealed that the RER is continuously distributed throughout the entirety of cells, confirmed by ER-specific fluorescent labeling, and is distinct from the common feature of plant endoplasmic reticulum. The RER is water sensitive and extremely elongated with water absorption. We show that the contractile cells drive circadian stigma closing-bending movements in response to day-to-night moisture changes. RNA-seq analyses demonstrated that contractile cells have distinct molecular components. Furthermore, multiple micro structural changes in stigma movements convert an anti-selfing structure to a device promoting selfing, a unique cellular mechanism of reproductive adaptation for uncertain pollination environments.