Yin-Zheng Wang

The tribe Trichosporeae is the most species‐rich, systemically complex, and morphologically diverse tribe in the Old World Gesneriaceae. It has long been a focal point and a challenge in the phylogeny of Trichosporeae, with frequent unclear relationships and delimitations among a lot of genera. Here, we conducted a molecular phylogenetic analysis by employing nine DNA fragments with a high sampling coverage for key clades in the tribe Trichosporeae. Meanwhile, we carried out a comprehensive morphological and anatomical investigation on vegetative and floral organs in related genera and species, and try to uncover morphological synapomorphies associated with molecular clades. Our results demonstrated a well‐supported phylogeny of major clades in the tribe, strongly corroborated by morphological data. We find that some genera, such as Raphiocarpus , Briggsia , and Boeica , are not monophyletic. Based on molecular phylogenetic and morphological analyses, we established five new genera and revived a genus in the tribe Trichosporeae, including Neoraphiocarpus , Macrophyllaea , Hispidopalata , Pseudobriggsia , and Kaiyua with revival of Boeicopsis . We further redefined the genera Raphiocarpus and Briggsiopsis . Our results would deepen our understanding about the phylogeny of the Old World Gesneriaceae.

Adaptive radiation is usually triggered by great in situ or ex situ environmental changes. How an adaptive radiation occurs on lands and how species richness relates to morphodisparity have been a major focus of evolutionary biology. Petrocodon, diversified in the southeastern Qinghai–Tibet Plateau (QTP), represents an ideal model to address these questions. To elucidate the dynamics of adaptive radiation of Petrocodon, we took an integrative approach, including phylogenetic, dating, disparity versus diversity, pollination, and gene expression analyses. Petrocodon with six clades has experienced radiation following the QTP uplift. Multiple modes of floral morphodisparity versus species diversity occur in the radiation process that are directly linked to their colonizing new environments and diversification with geographic expansion. Pollination and gene expression analyses suggest that accelerated emergence of de novo mutations might be relevant to the multiplex floral disparity and pollinator shifts in Petrocodon. For the first time in plants, we report that decreased genetic constraints on floral architecture triggered by QTP uplift might have generated abundant floral morphological variants, which were further targeted by selection for ecological divergence. The multiple modes of floral disparity versus species diversity may be attributed to accelerated mutations in colonizing new environments and repeated modifications of the already evolved traits in subsequent diversification and geographic expansion in Petrocodon. Our findings shed novel light on the interplay of ecological, developmental and evolutionary dynamics of disparity versus diversity in relation to trajectory changes of floral architecture in responses to environmental disturbance in a terrestrial plant group.

Although great progress has been made in transgenic technology, increasing the expression level and thus promising the expected phenotypes of exogenous genes in transgenic plants is still a crucial task for genetic transformation and crop engineering. Here, we conducted a comparative study of the enhancing efficiency of three putative translational enhancers, including Ω (natural leader from a plant virus), OsADH 5′ (natural leader from a plant gene), and ARC (active ribosomal RNA complementary), using the transient gene expression systems of Nicotiana benthamiana and Chirita pumila. We demonstrate that three tandem repeats of ARC (3 × ARC) are more efficient than other enhancers in expression. The enhancing efficiency of 6 × ARC is further increased, up to 130 times the expression level without the insertion of enhancers. We further evaluated the enhancing efficiency of 6 × ARC under agrobacterium-mediated transformation systems. In C. pumila, 6 × ARC significantly amplifies the phenotypic effect of CpCYC1 and CpCYC2 in repressing stamen development and yellow pigmentation. In Arabidopsis thaliana, 6 × ARC and the AtAP1 promoter work together to promote the accumulation of anthocyanin pigments in vegetative and reproductive organs. Most significantly, the fusion of 6 × ARC in a CpCYC1/2 transgenic system in C. pumila fully reveals that these genes have the complete function of repressing the yellow spots, displaying an advantage in manifesting the function of exogenous genes. This study highlights the application potential of the enhancer 6 × ARC in gene function research in plants.

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.

The genus Didymocarpus Wall. is a core group of the subfamily Didymocarpoideae in Gesneriaceae with a complex taxonomic history. It has long been controversial to delimit Didymocarpus and its allies. We conducted phylogenetic reconstructions with high‐density sampling of Didymocarpus and related genera by using four nuclear ribosomal DNAs (external transcribed spacer [ETS], internal transcribed spacer [ITS], 18S, 26S) and five chloroplast DNAs (atpB‐rbcL, rpl16, rps16, trnH‐psbA, and trnL‐F). The results indicated that neither Didymocarpus nor Allocheilos are monophyletic. Based on molecular phylogenetic and morphological analyses, we delimitated the range of the Didymocarpus sensu stricto (s. str.) with two‐section division, and established a new genus Palmatiboea for species originally placed in Didymocarpus. This genus is remarkably distinguished from Didymocarpus s. str. not only in molecular phylogeny and morphology but also in its specific geographic distribution, in that it is found only in Southeast China. In addition, we proposed a new monotypic genus Hequnia for Allocheilos guangxiensis that is completely isolated in phylogeny, consistent with a range of remarkably distinctive morphological characteristics. Our results clarify the systematics of Didymocarpus and the delimitation between it and related genera with establishment of two new genera.

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.

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.

Metapetrocosmea W. T. Wang and Deinostigma W. T. Wang & Z. Y. Li were originally monotypic with M. peltata (Merr. & Chun) W. T. Wang and D. poilanei (Pellegr.) W. T. Wang & Z. Y. Li, respectively. Recent molecular phylogenetic research expanded Deinostigma to include several species previously transferred from Chirita D. Don to Primulina Hance. However, the relationship between Metapetrocosmea and Deinostigma has not been well addressed. In the present study, the type species of Metapetrocosmea and Deinostigma were sampled together with related taxa, and the systematic relationships were reexamined using the nuclear ribosomal internal and external transcribed spacer regions (ITS and ETS) and four chloroplast regions (rpL32-trnL, rps16, trnH-psbA, trnL-F). Phylogenetic analyses demonstrate that M. peltata is embedded in the species of Deinostigma and sister to D. poilanei. All of these species form a highly supported branch, i.e., Metapetrocosmea–Deinostigma clade, which is sister to Oreocharis Benth. Morphological analyses show that species in the Metapetrocosmea–Deinostigma clade share a series of morphological traits, i.e., alternate leaves, arcuate filaments, densely lanate anthers with the hair surface covered with granular or vermiform appendages, bilobed stigma that vary from dorsal to ventral and almost equal in size to a dorsal-ventral oblique lamella with the dorsal one sterile, hooked hairs and T-shaped glandular hairs occurring on the whole plant, and riblike seed surfaces. Both molecular and morphological evidence strongly suggest that the Metapetrocosmea–Deinostigma clade is monophyletic and should be combined into a single genus, i.e., Metapetrocosmea W. T. Wang. Therefore, we present a taxonomic treatment for this group.

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.

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.