An evo-devo view of the gynoecium.

The appearance of the flower marks a key event in the evolutionary history of plants. Among the four types of floral organs, the gynoecium represents the major adaptive advantage of the flower. The gynoecium is an enclosing structure that protects and facilitates the fertilization of the ovules, which then mature as seeds. Upon fertilization, in many species, the gynoecium itself eventually becomes the fruit, which contributes to the dispersal of the seeds. However, despite its importance and the recent advances in our understanding of the genetic regulatory network guiding early gynoecium development, many questions remain to be resolved regarding the extent of the conservation of the molecular mechanisms for gynoecium development among different taxa, and how these mechanisms give origin and diversification to the gynoecium. In this review, we compile the existing knowledge about the evolution, development, and molecular mechanisms involved in the origin and evolution of the gynoecium.

Histone deacetylation regulates de novo shoot regeneration.

During de novo plant organ regeneration, auxin induction mediates the formation of a pluripotent cell mass called callus, which regenerates shoots upon cytokinin induction. However, molecular mechanisms underlying transdifferentiation remain unknown. Here, we showed that the loss of HDA19, a histone deacetylase (HDAC) family gene, suppresses shoot regeneration. Treatment with an HDAC inhibitor revealed that the activity of this gene is essential for shoot regeneration. Further, we identified target genes whose expression was regulated through HDA19-mediated histone deacetylation during shoot induction and found that ENHANCER OF SHOOT REGENERATION 1 and CUP-SHAPED COTYLEDON 2 play important roles in shoot apical meristem formation. Histones at the loci of these genes were hyperacetylated and markedly upregulated in hda19. Transient ESR1 or CUC2 overexpression impaired shoot regeneration, as observed in hda19. Therefore, HDA19 mediates direct histone deacetylation of CUC2 and ESR1 loci to prevent their overexpression at the early stages of shoot regeneration.

Non-cell-autonomous regulation of petal initiation in Arabidopsis thaliana.

In many flowering plants, petals initiate in alternate positions from first whorl sepals, suggesting possible signaling between sepal boundaries and petal initiation sites. PETAL LOSS (PTL) and RABBIT EARS (RBE) regulate petal initiation in Arabidopsis thaliana and their transcripts are expressed in sepal boundary and petal initiation sites, respectively, suggesting that PTL acts in a non-cell-autonomous manner. Here, we determined that cells expressing PTL and RBE fusion proteins did not overlap but were adjacent, confirming the non-cell-autonomous function of PTL. Genetic ablation of intersepal cells by expressing the diphtheria toxin-A chain gene driven by the PTL promoter resulted in flowers lacking petals, suggesting these cells are required for petal initiation. Transcriptome analysis combined with a PTL induction system revealed 42 genes that were upregulated under PTL activation, including UNUSUAL FLORAL ORGANS (UFO), which likely plays an important role in petal initiation. These findings suggest a molecular mechanism in which PTL indirectly regulates petal initiation and UFO mediates positional signaling between the sepal boundary and petal initiation sites.

Expression of the auxin biosynthetic genes YUCCA1 and YUCCA4 is dependent on the boundary regulators CUP-SHAPED COTYLEDON genes in the Arabidopsis thaliana embryo.

During embryogenesis of eudicots, the apical region of the embryo develops two cotyledon primordia and the shoot meristem. In Arabidopsis thaliana, this process is dependent on the functionally redundant activities of the CUP-SHAPED COTYLEDON (CUC) transcription factors, namely CUC1, CUC2, and CUC3, as well as the phytohormone auxin. However, the relationship between the CUC proteins and auxin has yet to be fully elucidated. In the present study, we examined whether the expression of auxin biosynthetic genes is dependent on CUC gene activities. Comprehensive quantitative RT-PCR analysis of the main auxin biosynthetic gene families of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATED and YUCCA (YUC) showed that YUC1 and YUC4 expression levels were lower in cuc double mutant embryos than the expression levels of these genes in wild type embryos. Reporter analysis also revealed that the expression of YUC1 and YUC4 in the cotyledon boundary region was reduced in cuc double mutant embryos. In contrast, the loss of function mutation in the SHOOT MERISTEMLESS gene, a shoot stem cell regulator that acts downstream of the CUC genes, did not markedly affect YUC1 expression levels. These results demonstrate that CUC genes play an important role in the regulation of auxin biosynthetic gene expression during embryogenesis; furthermore, they raise the possibility that the auxin produced by this regulation contributes to cotyledon boundary development.

The boundary-expressed EPIDERMAL PATTERNING FACTOR-LIKE2 gene encoding a signaling peptide promotes cotyledon growth during Arabidopsis thaliana embryogenesis.

The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis suggests that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.

PUCHI Regulates Giant Cell Morphology During Root-Knot Nematode Infection in Arabidopsis thaliana.

Parasitic root-knot nematodes transform the host's vascular cells into permanent feeding giant cells (GCs) to withdraw nutrients from the host plants. GCs are multinucleated metabolically active cells with distinctive cell wall structures; however, the genetic regulation of GC formation is largely unknown. In this study, the functions of the Arabidopsis thaliana transcription factor PUCHI during GC development were investigated. PUCHI expression was shown to be induced in early developing galls, suggesting the importance of the PUCHI gene in gall formation. Despite the puchi mutant not differing significantly from the wild type in nematode invasion and reproduction rates, puchi GC cell walls appeared to be thicker and lobate when compared to the wild type, while the cell membrane sometimes formed invaginations. In three-dimensional (3D) reconstructions of puchi GCs, they appeared to be more irregularly shaped than those in the wild type, with noticeable cell-surface protrusions and folds. Interestingly, the loss-of-function mutant of 3-KETOACYL-COA SYNTHASE 1 showed GC morphology and cell wall defects similar to those of the puchi mutant, suggesting that PUCHI may regulate GC development via very long chain fatty acid synthesis.

Post-Embryonic Lateral Organ Development and Adaxial-Abaxial Polarity Are Regulated by the Combined Effect of ENHANCER OF SHOOT REGENERATION 1 and WUSCHEL in Arabidopsis Shoots.

The development of above-ground lateral organs is initiated at the peripheral zone of the shoot apical meristem (SAM). The coordination of cell fate determination and the maintenance of stem cells are achieved through a complex regulatory network comprised of transcription factors. Two AP2/ERF transcription factor family genes, ESR1/DRN and ESR2/DRNL/SOB/BOL, regulate cotyledon and flower formation and de novo organogenesis in tissue culture. However, their roles in post-embryonic lateral organ development remain elusive. In this study, we analyzed the genetic interactions among SAM-related genes, WUS and STM, two ESR genes, and one of the HD-ZIP III members, REV, whose protein product interacts with ESR1 in planta. We found that esr1 mutations substantially enhanced the wus and stm phenotypes, which bear a striking resemblance to those of the wus rev and stm rev double mutants, respectively. Aberrant adaxial-abaxial polarity is observed in wus esr1 at relatively low penetrance. On the contrary, the esr2 mutation partially suppressed stm phenotypes in the later vegetative phase. Such complex genetic interactions appear to be attributed to the distinct expression pattern of two ESR genes because the ESR1 promoter-driving ESR2 is capable of rescuing phenotypes caused by the esr1 mutation. Our results pose the unique genetic relevance of ESR1 and the SAM-related gene interactions in the development of rosette leaves.

Interpreting Cytokinin Action as Anterograde Signaling and Beyond.

Among the major phytohormones, the cytokinin exhibits unique features for its ability to positively affect the developmental status of plastids. Even early on in its research, cytokinins were known to promote plastid differentiation and to reduce the loss of chlorophyll in detached leaves. Since the discovery of the components of cytokinin perception and primary signaling, the genes involved in photosynthesis and plastid differentiation have been identified as those directly targeted by type-B response regulators. Furthermore, cytokinins are known to modulate versatile cellular processes such as promoting the division and differentiation of cells and, in concert with auxin, initiating the de novo formation of shoot apical meristem (SAM) in tissue cultures. Yet how cytokinins precisely participate in such diverse cellular phenomena, and how the associated cellular processes are coordinated as a whole, remains unclear. A plausible presumption that would account for the coordinated gene expression is the tight and reciprocal communication between the nucleus and plastid. The fact that cytokinins affect plastid developmental status via gene expression in both the nucleus and plastid is interpreted here to suggest that cytokinin functions as an initiator of anterograde (nucleus-to-plastid) signaling. Based on this viewpoint, we first summarize the physiological relevance of cytokinins to the coordination of plastid differentiation with de novo shoot organogenesis in tissue culture systems. Next, the role of endogenous cytokinins in influencing plastid differentiation within the SAM of intact plants is discussed. Finally, a presumed plastid-derived signal in response to cytokinins for coupled nuclear gene expression is proposed.

A ClearSee-Based Clearing Protocol for 3D Visualization of Arabidopsis thaliana Embryos.

Tissue clearing methods combined with confocal microscopy have been widely used for studying developmental biology. In plants, ClearSee is a reliable clearing method that is applicable to a wide range of tissues and is suitable for gene expression analysis using fluorescent reporters, but its application to the Arabidopsis thaliana embryo, a model system to study morphogenesis and pattern formation, has not been described in the original literature. Here, we describe a ClearSee-based clearing protocol which is suitable for obtaining 3D images of Arabidopsis thaliana embryos. The method consists of embryo dissection, fixation, washing, clearing, and cell wall staining and enables high-quality 3D imaging of embryo morphology and expression of fluorescent reporters with the cellular resolution. Our protocol provides a reliable method that is applicable to the analysis of morphogenesis and gene expression patterns in Arabidopsis thaliana embryos.

Establishment of the Embryonic Shoot Meristem Involves Activation of Two Classes of Genes with Opposing Functions for Meristem Activities.

The shoot meristem, a stem-cell-containing tissue initiated during plant embryogenesis, is responsible for continuous shoot organ production in postembryonic development. Although key regulatory factors including KNOX genes are responsible for stem cell maintenance in the shoot meristem, how the onset of such factors is regulated during embryogenesis is elusive. Here, we present evidence that the two KNOX genes STM and KNAT6 together with the two other regulatory genes BLR and LAS are functionally important downstream genes of CUC1 and CUC2, which are a redundant pair of genes that specify the embryonic shoot organ boundary. Combined expression of STM with any of KNAT6, BLR, and LAS can efficiently rescue the defects of shoot meristem formation and/or separation of cotyledons in cuc1cuc2 double mutants. In addition, CUC1 and CUC2 are also required for the activation of KLU, a cytochrome P450-encoding gene known to restrict organ production, and KLU counteracts STM in the promotion of meristem activity, providing a possible balancing mechanism for shoot meristem maintenance. Together, these results establish the roles for CUC1 and CUC2 in coordinating the activation of two classes of genes with opposite effects on shoot meristem activity.

Primed histone demethylation regulates shoot regenerative competency.

Acquisition of pluripotency by somatic cells is a striking process that enables multicellular organisms to regenerate organs. This process includes silencing of genes to erase original tissue memory and priming of additional cell type specification genes, which are then poised for activation by external signal inputs. Here, through analysis of genome-wide histone modifications and gene expression profiles, we show that a gene priming mechanism involving LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3) specifically eliminates H3K4me2 during formation of the intermediate pluripotent cell mass known as callus derived from Arabidopsis root cells. While LDL3-mediated H3K4me2 removal does not immediately affect gene expression, it does facilitate the later activation of genes that act to form shoot progenitors when external cues lead to shoot induction. These results give insights into the role of H3K4 methylation in plants, and into the primed state that provides plant cells with high regenerative competency.

Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network.

The Arabidopsis homeodomain transcription factor SHOOT MERISTEMLESS (STM) is crucial for shoot apical meristem (SAM) function, yet the components and structure of the STM gene regulatory network (GRN) are largely unknown. Here, we show that transcriptional regulators are overrepresented among STM-regulated genes and, using these as GRN components in Bayesian network analysis, we infer STM GRN associations and reveal regulatory relationships between STM and factors involved in multiple aspects of SAM function. These include hormone regulation, TCP-mediated control of cell differentiation, AIL/PLT-mediated regulation of pluripotency and phyllotaxis, and specification of meristem-organ boundary zones via CUC1. We demonstrate a direct positive transcriptional feedback loop between STM and CUC1, despite their distinct expression patterns in the meristem and organ boundary, respectively. Our further finding that STM activates expression of the CUC1-targeting microRNA miR164c combined with mathematical modelling provides a potential solution for this apparent contradiction, demonstrating that these proposed regulatory interactions coupled with STM mobility could be sufficient to provide a mechanism for CUC1 localisation at the meristem-organ boundary. Our findings highlight the central role for the STM GRN in coordinating SAM functions.

Biosynthesis of volatile terpenes that accumulate in the secretory cavities of young leaves of Japanese pepper (Zanthoxylum piperitum): Isolation and functional characterization of monoterpene and sesquiterpene synthase genes.

Volatile terpenes are ones of the characteristic aromas of Japanese pepper (Zanthoxylum piperitum). It has been hypothesized that the specialized epithelial cells surrounding the secretory cavities of Japanese pepper fruits and leaves are responsible for the synthesis of monoterpenes and sesquiterpenes, which are generally produced by terpene synthases (TPSs); however, direct evidence for the formation of terpenes in Japanese pepper remains elusive. Here we report that monoterpenes and sesquiterpenes accumulate inside the secretory cavities of Japanese pepper leaves, but not in other parts of leaf tissues that do not include secretory cavities. We have obtained cDNAs for ZpTPS1 and ZpTPS2, which are responsible for biosynthesis of the sesquiterpenes ß-caryophyllene and germacrene D, respectively, in Japanese pepper. In addition, we also identified a cDNA for the monoterpene synthase ZpTPS3. Expression of ZpTPS3 in Escherichia coli in addition to Agrobacterium-mediated transient ZpTPS3 expression in Nicotiana benthamiana demonstrated the catalytic activity of ZpTPS3 to form ß-phellandrene as the major product. In situ hybridization in Japanese pepper leaf tissue revealed that ZpTPS3 transcript specifically accumulated in the epithelial cells surrounding secretory cavities. Expression of ZpTPS3 in epithelial cells was only detectable during early stages of cavity development, whereas the formation of volatile terpenes occurred at a constant rate throughout the expansion of secretory cavities. Our studies have improved the understanding of the currently uncharacterized processes controlling volatile terpene biosynthesis in Japanese pepper leaves.

A Secreted Peptide and Its Receptors Shape the Auxin Response Pattern and Leaf Margin Morphogenesis.

Secreted peptides mediate intercellular communication [1, 2]. Several secreted peptides in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family regulate morphogenesis of tissues, such as stomata and inflorescences in plants [3-15]. The biological functions of other EPFL family members remain unknown. Here, we show that the EPFL2 gene is required for growth of leaf teeth. EPFL2 peptide physically interacts with ERECTA (ER) family receptor-kinases and, accordingly, the attenuation of ER family activities leads to formation of toothless leaves. During the tooth growth process, responses to the phytohormone auxin are maintained at tips of the teeth to promote their growth [16-19]. In the growing tooth tip of epfl2 and multiple er family mutants, the auxin response becomes broader. Conversely, overexpression of EPFL2 diminishes the auxin response, indicating that the EPFL2 signal restricts the auxin response to the tooth tip. Interestingly, the tip-specific auxin response in turn organizes characteristic expression patterns of ER family and EPFL2 by enhancing ER family expression at the tip while eliminating the EPFL2 expression from the tip. Our findings identify the novel ligand-receptor pairs promoting the tooth growth, and further reveal a feedback circuit between the peptide-receptor system and auxin response as a mechanism for maintaining proper auxin maxima during leaf margin morphogenesis.

Three-Dimensional Imaging of Plant Organs Using a Simple and Rapid Transparency Technique.

Clearing techniques eliminate factors that interfere with microscopic observation, including light scattering and absorption by pigments and cytoplasmic components. The techniques allow fluorescence-based detailed analyses of materials and characterization of the three-dimensional structure of organs. We describe a simple and rapid clearing and imaging method, termed 'TOMEI' (Transparent plant Organ MEthod for Imaging), which enables microscopic observation of intact plant organs. This method involves a clearing reagent containing 2,2'-thiodiethanol. Conveniently, transparent plant organs were prepared within only 3-6 h. We detected fluorescent stains at a depth of approximately 200 µm using confocal laser scanning microscopy and analyzed fluorescent proteins in internal tissues of transparent organs cleared using TOMEI. We adapted TOMEI for various plant organs of Arabidopsis thaliana and Oryza sativa, including leaves, flower buds, flower stalks, root and nematode-infected root-knots. We visualized whole leaves of A. thaliana from the adaxial epidermis to the abaxial epidermis as well as protoxylem and metaxylem vessels of vascular bundles embedded in spongy mesophyll cells. Inner floral organs were observed in flower buds cleared using TOMEI without the need to prepare sections or remove sepals. Multicolor imaging of fluorescent proteins and dyes, and analyses of the three-dimensional structure of plant organs based on optical sections are possible using TOMEI. We analyzed root-knots cleared using TOMEI and revealed that nematodes induce giant cell expansion in a DNA content-dependent manner. The TOMEI method is applicable to analysis of fluorescent proteins and dyes quantitatively with cell morphological characteristics in whole plant organs.

Mechanical stress contributes to the expression of the STM homeobox gene in Arabidopsis shoot meristems.

The role of mechanical signals in cell identity determination remains poorly explored in tissues. Furthermore, because mechanical stress is widespread, mechanical signals are difficult to uncouple from biochemical-based transduction pathways. Here we focus on the homeobox gene SHOOT MERISTEMLESS (STM), a master regulator and marker of meristematic identity in Arabidopsis. We found that STM expression is quantitatively correlated to curvature in the saddle-shaped boundary domain of the shoot apical meristem. As tissue folding reflects the presence of mechanical stress, we test and demonstrate that STM expression is induced after micromechanical perturbations. We also show that STM expression in the boundary domain is required for organ separation. While STM expression correlates with auxin depletion in this domain, auxin distribution and STM expression can also be uncoupled. STM expression and boundary identity are thus strengthened through a synergy between auxin depletion and an auxin-independent mechanotransduction pathway at the shoot apical meristem.

A conserved role for CUP-SHAPED COTYLEDON genes during ovule development.

The evolution of plant reproductive strategies has led to a remarkable diversity of structures, especially within the flower, a structure characteristic of the angiosperms. In flowering plants, sexual reproduction depends notably on the development of the gynoecium that produces and protects the ovules. In Arabidopsis thaliana, ovule initiation is promoted by the concerted action of auxin with CUC1 (CUP-SHAPED COTYLEDON1) and CUC2, two genes that encode transcription factors of the NAC family (NAM/ATAF1,2/CUC). Here we highlight an additional role for CUC2 and CUC3 in Arabidopsis thaliana ovule separation. While CUC1 and CUC2 are broadly expressed in the medial tissue of the gynoecium, CUC2 and CUC3 are expressed in the placental tissue between developing ovules. Consistent with the partial overlap between CUC1, CUC2 and CUC3 expression patterns, we show that CUC proteins can physically interact, both in yeast cells and in planta. We found that the cuc2;cuc3 double mutant specifically harbours defects in ovule separation, producing fused seeds that share the seed coat, and suggesting that CUC2 and CUC3 promote ovule separation in a partially redundant manner. Functional analyses show that CUC transcription factors are also involved in ovule development in Cardamine hirsuta. Additionally we show a conserved expression pattern of CUC orthologues between ovule primordia in other phylogenetically distant species with different gynoecium architectures. Taken together these results suggest an ancient role for CUC transcription factors in ovule separation, and shed light on the conservation of mechanisms involved in the development of innovative structures.

The CUP-SHAPED COTYLEDON2 and 3 genes have a post-meristematic effect on Arabidopsis thaliana phyllotaxis.

BACKGROUND AND AIMS: The arrangement of flowers in inflorescence shoots of Arabidopsis thaliana represents a regular spiral Fibonacci phyllotaxis. However, in the cuc2 cuc3 double mutant, flower pedicels are fused to the inflorescence stem, and phyllotaxis is aberrant in the mature shoot regions. This study examined the causes of this altered development, and in particular whether the mutant phenotype is a consequence of defects at the shoot apex, or whether post-meristematic events are involved. METHODS: The distribution of flower pedicels and vascular traces was examined in cross-sections of mature shoots; sequential replicas were used to investigate the phyllotaxis and geometry of shoot apices, and growth of the young stem surface. The expression pattern of CUC3 was analysed by examining its promoter activity. KEY RESULTS: Phyllotaxis irregularity in the cuc2 cuc3 double mutant arises during the post-meristematic phase of shoot development. In particular, growth and cell divisions in nodes of the elongating stem are not restricted in the mutant, resulting in pedicel-stem fusion. On the other hand, phyllotaxis in the mutant shoot apex is nearly as regular as that of the wild type. Vascular phyllotaxis, generated almost simultaneously with the phyllotaxis at the apex, is also much more regular than pedicel phyllotaxis. The most apparent phenotype of the mutant apices is a higher number of contact parastichies. This phenotype is associated with increased meristem size, decreased angular width of primordia and a shorter plastochron. In addition, the appearance of a sharp and deep crease, a characteristic shape of the adaxial primordium boundary, is slightly delayed and reduced in the mutant shoot apices. CONCLUSIONS: The cuc2 cuc3 double mutant displays irregular phyllotaxis in the mature shoot but not in the shoot apex, thus showing a post-meristematic effect of the mutations on phyllotaxis. The main cause of this effect is the formation of pedicel-stem fusions, leading to an alteration of the axial positioning of flowers. Phyllotaxis based on the position of vascular flower traces suggests an additional mechanism of post-meristematic phyllotaxis alteration. Higher density of flower primordia may be involved in the post-meristematic effect on phyllotaxis, whereas delayed crease formation may be involved in the fusion phenotype. Promoter activity of CUC3 is consistent with its post-meristematic role in phyllotaxis.

Heterotrimeric G proteins control stem cell proliferation through CLAVATA signaling in Arabidopsis.

Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gß mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.