A new type of cell related to organ movement for selfing in plants.

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 a much-expanded rough endoplasmic reticulum (RER). Cryo-scanning electron microscopy and transmission electron microscopy analyses revealed that the RER is continuously distributed throughout the entirety of cells, confirmed by endoplasmic reticulum (ER)-specific fluorescent labeling, and is distinct from the common feature of plant ER. 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 microstructural changes in stigma movements convert an anti-selfing structure into a device promoting selfing-a unique cellular mechanism of reproductive adaptation for uncertain pollination environments.

CYCLOIDEA-like genes control floral symmetry, floral orientation, and nectar guide patterning.

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 2 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 upregulates CpCYC1, while CpCYC1 downregulates CpCYC2. This asymmetric auto-regulation and cross-regulation mechanism might explain the high expression levels of only 1 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.

An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing.

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.

An Optimized Transformation System and Functional Test of CYC-Like TCP Gene CpCYC in Chirita pumila (Gesneriaceae).

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.

Dosage imbalance of B- and C-class genes causes petaloid-stamen relating to F1 hybrid variation.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Evolution of Darwin's Peloric Gloxinia (Sinningia speciosa) Is Caused by a Null Mutation in a Pleiotropic TCP Gene.

Unlike most crops, which were domesticated through long periods of selection by ancient humans, horticultural plants were primarily domesticated through intentional selection over short time periods. The molecular mechanisms underlying the origin and spread of novel traits in the domestication process have remained largely unexplored in horticultural plants. Gloxinia (Sinningia speciosa), whose attractive peloric flowers influenced the thoughts of Darwin, have been cultivated since the early 19th century, but its origin and genetic basis are currently unknown. By employing multiple experimental approaches including genetic analysis, genotype-phenotype associations, gene expression analysis, and functional interrogations, we showed that a single gene encoding a TCP protein, SsCYC, controls both floral orientation and zygomorphy in gloxinia. We revealed that a causal mutation responsible for the development of peloric gloxinia lies in a 10-bp deletion in the coding sequence of SsCYC. By combining genetic inference and literature searches, we have traced the putative ancestor and reconstructed the domestication path of the peloric gloxinia, in which a 10-bp deletion in SsCYC under selection triggered its evolution from the wild progenitor. The results presented here suggest that a simple genetic change in a pleiotropic gene can promote the elaboration of floral organs under intensive selection pressure.

Distinct Regulatory Changes Underlying Differential Expression of TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR Genes Associated with Petal Variations in Zygomorphic Flowers of Petrocosmea spp. of the Family Gesneriaceae.

CYCLOIDEA (CYC)-like genes, belonging to the plant-specific TCP transcription factor family that is named after TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYC from Antirrhinum majus, and the PROLIFERATING CELL FACTORS (PCF) from rice (Oryza sativa), have conserved dorsal identity function in patterning floral zygomorphy mainly through specific expression in dorsal petals of a flower. Their expression changes are usually related to morphological diversity of zygomorphic flowers. However, it is still a challenge to elucidate the molecular mechanism underlying their expression differentiation. It is also unknown whether CINCINNATA (CIN)-like TCP genes, locally controlling cell growth and proliferation, are involved in the evolution of floral zygomorphy. To address these questions, we selected two closely related species, i.e. Petrocosmea glabristoma and Petrocosmea sinensis, with distinct petal morphology to conduct expression, hybridization, mutant, and allele-specific expression analyses. The results show that the size change of the dorsal petals between the two species is mainly mediated by the expression differentiation of CYC1C and CYC1D, while the shape variation of all petals is related to the expression change of CIN1. In reciprocal F1 hybrids, the expression of CYC1C, CYC1D, and CIN1 conforms to an additive inheritance mode, consistent with the petal phenotypes of hybrids. Through allele-specific expression analyses, we find that the expression differentiation of these TCP genes is underlain by distinctly different types of regulatory changes. We suggest that highly redundant paralogs with identical expression patterns and interspecific expression differentiation may be controlled by remarkably different regulatory pathways because natural selection may favor different regulatory modifications rather than coding sequence changes of key developmental genes in generating morphological diversity.

Phylogenetic Relationships and Evolution of the Androecia in Ruteae (Rutaceae).

Ruta, which belongs to tribe Ruteae, is the type genus of the subfamily Rutoideae and the family Rutaceae. Molecular systematic studies have shown that the genera in Ruteae are closer related to Aurantioideae than to most other genera of Rutoideae, some of the genera traditionally placed in Ruteae have been shown to be nested within the Aurantioideae clade, but the diagnostic characters for determining new patterns in the relationship are poor. In this study, we investigated the floral development of Boenninghausenia in Ruteae (sensu stricto), Haplophyllum in the basal position of Aurantioideae and Murraya in traditional Aurantioideae using scanning electron microscopy. The androecium of Boenninghausenia is obdiplostemony. As androecia in other genera within Ruteae (s.s.) are also obdiplostemonous, reconstruction of the ancestral state indicates that obdiplostemony is an ancestral character in this clade. Because the androecia of Haplophyllum and Murraya are also obdiplostemonous, obdiplostemony is also an ancestral character in Aurantioideae clade. The ancestral state reconstruction indicates this character can serve as a synapomorphy of the Ruteae-Aurantioideae clade. The results of our work also shed light on the evolution of the androecium in Rutaceae, as the obdiplostemony of this group is clearly derived from haplostemony in the ancestral genera in Rutaceae and has develop into polyandry by increasing antepetalous stamens.

Seed shattering: from models to crops.

Seed shattering (or pod dehiscence, or fruit shedding) is essential for the propagation of their offspring in wild plants but is a major cause of yield loss in crops. In the dicot model species, Arabidopsis thaliana, pod dehiscence necessitates a development of the abscission zones along the pod valve margins. In monocots, such as cereals, an abscission layer in the pedicle is required for the seed shattering process. In the past decade, great advances have been made in characterizing the genetic contributors that are involved in the complex regulatory network in the establishment of abscission cell identity. We summarize the recent burgeoning progress in the field of genetic regulation of pod dehiscence and fruit shedding, focusing mainly on the model species A. thaliana with its close relatives and the fleshy fruit species tomato, as well as the genetic basis responsible for the parallel loss of seed shattering in domesticated crops. This review shows how these individual genes are co-opted in the developmental process of the tissues that guarantee seed shattering. Research into the genetic mechanism underlying seed shattering provides a premier prerequisite for the future breeding program for harvest in crops.

Origin and evolution of Petrocosmea (Gesneriaceae) inferred from both DNA sequence and novel findings in morphology with a test of morphology-based hypotheses.

BACKGROUND: Petrocosmea Oliver (Gesneriaceae) currently comprises 38 species with four non-nominate varieties, nearly all of which have been described solely from herbarium specimens. However, the dried specimens have obscured the full range of extremely diverse morphological variation that exists in the genus and has resulted in a poor subgeneric classification system that does not reflect the evolutionary history of this group. It is important to develop innovative methods to find new morphological traits and reexamine and reevaluate the traditionally used morphological data based on new hypothesis. In addition, Petrocosmea is a mid-sized genus but exhibits extreme diverse floral variants. This makes the genus of particular interest in addressing the question whether there are any key factors that is specifically associated with their evolution and diversification. RESULTS: Here we present the first phylogenetic analyses of the genus based on dense taxonomic sampling and multiple genes combined with a comprehensive morphological investigation. Maximum-parsimony, maximum likelihood and Bayesian analyses of molecular data from two nuclear DNA and six cpDNA regions support the monophyly of Petrocosmea and recover five major clades within the genus, which is strongly corroborated by the reconstruction of ancestral states for twelve new morphological characters directly observed from living material. Ancestral area reconstruction shows that its most common ancestor was likely located east and southeast of the Himalaya-Tibetan plateau. The origin of Petrocosmea from a potentially Raphiocarpus-like ancestor might have involved a series of morphological modifications from caulescent to acaulescent habit as well as from a tetrandrous flower with a long corolla-tube to a diandrous flower with a short corolla-tube, also evident in the vestigial caulescent habit and transitional floral form in clade A that is sister to the remainder of the genus. Among the five clades in Petrocosmea, the patterns of floral morphological differentiation are consistent with discontinuous lineage-associated morphotypes as a repeated adaptive response to alternative environments. CONCLUSION: Our results suggest that the lineage-specific morphological differentiations reflected in the upper lip, a functional organ for insect pollination, are likely adaptive responses to pollinator shifts. We further recognize that the floral morphological diversification in Petrocosmea involves several evolutionary phenomena, i.e. evolutionary successive specialization, reversals, parallel evolution, and convergent evolution, which are probably associated with adaptation to pollination against the background of heterogeneous abiotic and biotic environments in the eastern wing regions of Himalaya-Tibetan plateau.

Pod shattering resistance associated with domestication is mediated by a NAC gene in soybean.

Loss of seed dispersal is a key agronomical trait targeted by ancient human selection and has been regarded as a milestone of crop domestication. In this study, in the legume crop soybean Glycine max (L.) Merr. which provides vegetable oils and proteins for humans, we show that the key cellular feature of the shattering-resistant trait lies in the excessively lignified fibre cap cells (FCC) with the abscission layer unchanged in the pod ventral suture. We demonstrate that a NAC (NAM, ATAF1/2 and CUC2) gene shattering1-5 (SHAT1-5) functionally activates secondary wall biosynthesis and promotes the significant thickening of FCC secondary walls by expression at 15-fold the level of the wild allele, which is attributed to functional disruption of the upstream repressor. We show that strong artificial selection of SHAT1-5 has caused a severe selective sweep across ~ 116 kb on chromosome 16. This locus and regulation mechanism could be applicable to legume crop improvement.

Evolution of double positive autoregulatory feedback loops in CYCLOIDEA2 clade genes is associated with the origin of floral zygomorphy.

Members of the CYCLOIDEA2 (CYC2) clade of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF transcription factor genes are widely involved in controlling floral zygomorphy, a key innovation in angiosperm evolution, depending on their persistently asymmetric expression in the corresponding floral domains. However, it is unclear how this asymmetric expression is maintained throughout floral development. Selecting Primulina heterotricha as a model, we examined the expression and function of two CYC2 genes, CYC1C and CYC1D. We analyzed the role of their promoters in protein-DNA interactions and transcription activation using electrophoresis mobility shift assays, chromatin immunoprecipitation, and transient gene expression assays. We find that CYC1C and CYC1D positively autoregulate themselves and cross-regulate each other. Our results reveal a double positive autoregulatory feedback loop, evolved for a pair of CYC2 genes to maintain their expression in developing flowers. Further comparative genome analyses, together with the available expression and function data of CYC2 genes in the core eudicots, suggest that this mechanism might have led to the independent origins of floral zygomorphy, which are associated with plant-insect coevolution and the adaptive radiation of angiosperms.

Multiple mechanisms underlie rapid expansion of an invasive alien plant.

• With growing concerns over serious ecological problems, a particular challenge is to reveal the complex mechanisms underlying rapid expansion of invasive species. Ageratina adenophora is of particular interest in addressing this question. • We used geographic information systems and logistic regression to identify the geographic and environmental factors contributing to the presence of A. adenophora. Join-count spatial statistics with reproduction mode examination were employed to elucidate the spatiotemporal dispersal mechanisms. • Multiple factors have significantly contributed to the rapid expansion of A. adenophora. Its biological traits, favoring dispersal by water and wind coupled with local spatiotemporally heterogeneous geography and ecology, promote invasion downstream and upstream along river valleys, while other factors associated with human activities facilitate its invasion over high mountains and across river valleys, providing new scope for progressive invasions. We further identified an unusual invasion event of A. adenophora subsequent to a great flood that amplified its dispersal ability from vegetative propagules and seeds. • These findings suggest that dynamic interactions of multiple factors in heterogeneous ecogeographical environments - a 'combinatorial' invasion mechanism - would generate an unexpected invasion rate of an alien species or a seemingly stochastic invasion event.

Expressions of ECE-CYC2 clade genes relating to abortion of both dorsal and ventral stamens in Opithandra (Gesneriaceae).

BACKGROUND: ECE-CYC2 clade genes known in patterning floral dorsoventral asymmetry (zygomorphy) in Antirrhinum majus are conserved in the dorsal identity function including arresting the dorsal stamen. However, it remains uncertain whether the same mechanism underlies abortion of the ventral stamens, an important morphological trait related to evolution and diversification of zygomorphy in Lamiales sensu lato, a major clade of predominantly zygomorphically flowered angiosperms. Opithandra (Gesneriaceae) is of particular interests in addressing this question as it is in the base of Lamiales s.l., an early representative of this type zygomorphy. RESULTS: We investigated the expression patterns of four ECE-CYC2 clade genes and two putative target cyclinD3 genes in Opithandra using RNA in situ hybridization and RT-PCR. OpdCYC gene expressions were correlated with abortion of both dorsal and ventral stamens in Opithandra, strengthened by the negatively correlated expression of their putative target OpdcyclinD3 genes. The complement of OpdcyclinD3 to OpdCYC expressions further indicated that OpdCYC expressions were related to the dorsal and ventral stamen abortion through negative effects on OpdcyclinD3 genes. CONCLUSION: These results suggest that ECE-CYC2 clade TCP genes are not only functionally conserved in the dorsal stamen repression, but also involved in arresting ventral stamens, a genetic mechanism underlying the establishment of zygomorphy with abortion of both the dorsal and ventral stamens evolved in angiosperms, especially within Lamiales s.l.

Familial placement and relations of Rehmannia and Triaenophora (Scrophulariaceae s.l.) inferred from five gene regions.

Accurate classification systems based on evolution are imperative for biological investigations. The recent explosion of molecular phylogenetics has resulted in a much improved classification of angiosperms. More than five phylogenetic lineages have been recognized from Scrophulariaceae sensu lato since the family was determined to be polyphyletic; however, questions remain about the genera that have not been assigned to one of the segregate families of Scrophulariaceae s.l. Rehmannia Liboschitz and Triaenophora Solereder are such genera with uncertain familial placement. There also is debate whether Triaenophora should be segregated from Rehmannia. To evaluate the phylogenetic relations between Rehmannia and Triaenophora, to find their closest relatives, and to verify their familial placement, we conducted phylogenetic analyses of the sequences of one nuclear DNA (ITS) region and four chloroplast DNA gene regions (trnL-F, rps16, rbcL, and rps2) individually and combined. The analyses showed that Rehmannia and Triaenophora are each strongly supported as monophyletic and together are sister to Orobanchaceae. This relation was corroborated by phytochemical and morphological data. Based on these data, we suggest that Rehmannia and Triaenophora represent the second nonparasitic branch sister to the remainder of Orobanchaceae (including Lindenbergia).

A preliminary phylogeny of the 'didymocarpoid Gesneriaceae' based on three molecular data sets: Incongruence with available tribal classifications.

The 'didymocarpoid Gesneriaceae' (traditional subfam. Cyrtandroideae excluding Epithemateae) are the largest group of Old World Gesneriaceae, comprising 85 genera and 1800 species. We attempt to resolve their hitherto poorly understood generic relationships using three molecular markers on 145 species, of which 128 belong to didymocarpoid Gesneriaceae. Our analyses demonstrate that consistent topological relationships can be retrieved from data sets with missing data using subsamples and different combinations of gene sequences. We show that all available classifications in Old World Gesneriaceae are artificial and do not reflect natural relationships. At the base of the didymocarpoids are grades of clades comprising isolated genera and small groups from Asia and Europe. These are followed by a clade comprising the African and Madagascan genera. The remaining clades represent the advanced Asiatic and Malesian genera. They include a major group with mostly twisted capsules. The much larger group of remaining genera comprises exclusively genera with straight capsules and the huge genus Cyrtandra with indehiscent fruits. Several genera such as Briggsia, Henckelia, and Chirita are not monophyletic; Chirita is even distributed throughout five clades. This degree of incongruence between molecular phylogenies, traditional classifications, and generic delimitations indicates the problems with classifications based on, sometimes a single, morphological characters.

Expression differentiation of CYC-like floral symmetry genes correlated with their protein sequence divergence in Chirita heterotricha (Gesneriaceae).

CYCLOIDIEA (CYC) and its homologues have been studied intensively in the model organism Antirrhinum majus and related species regarding their function in controlling floral dorsoventral (adaxial-abaxial) asymmetry, including aborting the adaxial and lateral stamens. This raises the question whether the same mechanism underlies the great morphological diversity of zygomorphy in angiosperms, especially in Lamiales sensu lato, a major clade predominantly with zygomorphic flowers. To address this, we selected a representative in Gesneriaceae, the sister to the remainder of Lamiales s.l., to isolate CYC homologues and further investigate their expression patterns using locus-specific semiquantitative reverse transcriptase polymerase chain reaction. Our results showed that four CYC homologues in Chirita heterotricha differentiated spatially and temporally in expression, in which ChCYC1D was only expressed in the adaxial regions, and transcripts of ChCYC1C were distributed in both the adaxial and lateral regions, while ChCYC2A and ChCYC2B transcripts were only detected in the young inflorescences. ChCYC1C expression in the lateral regions correlated with abortion of the lateral stamens in C. heterotricha hinted at its gain of function, i.e., expanding from the adaxial to the lateral regions in expression. Correlatively, the protein sequences of ChCYC genes exhibited remarkable divergences, in which some lineage-specific amino acids between GCYC1 and GCYC2 in conserved functional domains and two sublineage-specific motifs between GCYC1C and GCYC1D in GCYC1 genes had further been identified. Our results indicated that ChCYC genes had probably undergone an expressional differentiation and specialization in establishing the floral dorsoventral asymmetry in C. heterotricha responding to different selective pressure after gene duplication.

Altered expression patterns of TCP and MYB genes relating to the floral developmental transition from initial zygomorphy to actinomorphy in Bournea (Gesneriaceae).

The shift from zygomorphy to actinomorphy has been intensively studied in molecular genetic model organisms. However, it is still a key challenge to explain the great morphological diversity of derived actinomorphy in angiosperms, since different underlying mechanisms may be responsible for similar external morphologies. Bournea (Gesneriaceae) is of particular interest in addressing this question, as it is a representative of primarily derived actinomorphy characteristic of a unique developmental transition from zygomorphy to actinomorphic flowers at anthesis. Using RNA in situ hybridization, the expression patterns were investigated of three different Bournea orthologues of TCP and MYB genes that have been shown to control floral symmetry in model species. Here, it is shown that the initial zygomorphic pattern in Bournea is likely a residual zygomorphy resulting from conserved expression of the adaxial (dorsal) identity gene BlCYC1. As a key novel event, the late downregulation of BlCYC1 and BlRAD and the correlative changes in the late specific expression of the abaxial (ventral) identity gene BlDIV should be responsible for the origin of the derived actinomorphy in Bournea. These results further indicate that there might be diverse pathways in the origin and evolution of derived actinomorphy through modifications of pre-existing zygomorphic developmental programs under dynamics of regulatory networks.