Development and Holocrine Secretion of Resin Ducts in Kielmeyera appariciana (Calophyllaceae).

The modes of formation and release of secretion are complex processes that occur in secretory ducts and their description has great divergence in some species. The use of modern techniques to detect hydrolytic enzymes, cytoskeleton arrangement and indicators of programmed cell death may help clarify the processes involved during the ontogeny of that gland. The goal of our study was to analyze subcellular changes during schizogenous formation and secretion production and release into the lumen in resin ducts of Kielmeyera appariciana. Our results demonstrate the participation of pectinase through the loosening of the central cells of the rosette, which subsequently split from each other through polarized growth mediated by a rearrangement of the microtubules. The resin is mainly synthesized in plastids and endoplasmic reticulum and is observed inside vesicles and small vacuoles. The secretion release is holocrine and occurs through programmed cell death related to the release of reactive oxygen species, causing cytoplasm darkening, chromatin condensation, vacuole rupture and plastid and mitochondria degeneration. Cellulase activity was identified prior to the rupture of the cell wall, causing the release of secretion into the lumen of the duct. The participation of the cytoskeleton was observed for the first time during schizogeny of ducts as well as programmed cell death as part of the process of the release of holocrine secretion. This type of secretion release may be a key innovation in Kielmeyera since it has not been observed in ducts of any other plant thus far.

Inaugural Description of Extrafloral Nectaries in Sapindaceae: Structure, Diversity and Nectar Composition.

Sapindales is a large order with a great diversity of nectaries; however, to date, there is no information about extrafloral nectaries (EFN) in Sapindaceae, except recent topological and morphological data, which indicate an unexpected structural novelty for the family. Therefore, the goal of this study was to describe the EFN in Sapindaceae for the first time and to investigate its structure and nectar composition. Shoots and young leaves of Urvillea ulmacea were fixed for structural analyses of the nectaries using light and scanning electron microscopy. For nectar composition investigation, GC-MS and HPLC were used, in addition to histochemical tests. Nectaries of Urvillea are circular and sunken, corresponding to ocelli. They are composed of a multiple-secretory epidermis located on a layer of transfer cells, vascularized by phloem and xylem. Nectar is composed of sucrose, fructose, xylitol and glucose, in addition to amino acids, lipids and phenolic compounds. Many ants were observed gathering nectar from young leaves. These EFNs have an unprecedented structure in the family and also differ from the floral nectaries of Sapindaceae, which are composed of secretory parenchyma and release nectar through stomata. The ants observed seem to protect the plant against herbivores, and in this way, the nectar increases the defence of vegetative organs synergistically with latex.

Corrigendum: Laticifer growth pattern is guided by cytoskeleton organization.

[This corrects the article DOI: 10.3389/fpls.2022.971235.].

Loss of S-nitrosoglutathione reductase disturbs phytohormone homeostasis and regulates shoot side branching and fruit growth in tomato.

S-Nitrosoglutathione plays a central role in nitric oxide (NO) homeostasis, and S-nitrosoglutathione reductase (GSNOR) regulates the cellular levels of S-nitrosoglutathione across kingdoms. Here, we investigated the role of endogenous NO in shaping shoot architecture and controlling fruit set and growth in tomato (Solanum lycopersicum). SlGSNOR silencing promoted shoot side branching and led to reduced fruit size, negatively impacting fruit yield. Greatly intensified in slgsnor knockout plants, these phenotypical changes were virtually unaffected by SlGSNOR overexpression. Silencing or knocking out of SlGSNOR intensified protein tyrosine nitration and S-nitrosation and led to aberrant auxin production and signaling in leaf primordia and fruit-setting ovaries, besides restricting the shoot basipetal polar auxin transport stream. SlGSNOR deficiency triggered extensive transcriptional reprogramming at early fruit development, reducing pericarp cell proliferation due to restrictions on auxin, gibberellin, and cytokinin production and signaling. Abnormal chloroplast development and carbon metabolism were also detected in early-developing NO-overaccumulating fruits, possibly limiting energy supply and building blocks for fruit growth. These findings provide new insights into the mechanisms by which endogenous NO fine-tunes the delicate hormonal network controlling shoot architecture, fruit set, and post-anthesis fruit development, emphasizing the relevance of NO-auxin interaction for plant development and productivity.

Histochemical Analysis of Plant Secretory Structures.

Histochemical analysis is essential for the study of plant secretory structures whose classification is based, at least partially, on the composition of their secretion. As each gland may produce one or more types of substances, a correct analysis of its secretion should be done using various histochemical tests to detect metabolites of different chemical classes. Here I describe some of the most used methods to detect carbohydrates, proteins, lipids, phenolic compounds, and alkaloids in the secretory structures.

Laticifer growth pattern is guided by cytoskeleton organization.

Laticifers are secretory structures that produce latex, forming a specialized defense system against herbivory. Studies using anatomical approaches to investigate laticifer growth patterns have described their origin; however, their mode of growth, i.e., whether growth is intrusive or diffuse, remains unclear. Studies investigating how cytoskeleton filaments may influence laticifer shape establishment and growth patterns are lacking. In this study, we combined microtubule immunostaining and developmental anatomy to investigate the growth patterns in different types of laticifers. Standard anatomical methods were used to study laticifer development. Microtubules were labelled through immunolocalization of α-tubulin in three types of laticifers from three different plant species: nonanastomosing (Urvillea ulmacea), anastomosing unbranched with partial degradation of terminal cell walls (Ipomoea nil), and anastomosing branched laticifers with early and complete degradation of terminal cell walls (Asclepias curassavica). In both nonanastomosing and anastomosing laticifers, as well as in differentiating meristematic cells, parenchyma cells and idioblasts, microtubules were perpendicularly aligned to the cell growth axis. The analyses of laticifer microtubule orientation revealed an arrangement that corresponds to those cells that grow diffusely within the plant body. Nonanastomosing and anastomosing laticifers, branched or not, have a pattern which indicates diffuse growth. This innovative study on secretory structures represents a major advance in the knowledge of laticifers and their growth mode.

Adnate Leaf-Base and the Origin of Ribs in Succulent Stems of Euphorbia L.

Stem succulence evolved independently in many plant lineages as an adaptation to arid environments. One of the most interesting cases is the convergence between Cactaceae and Euphorbia, which have anatomical adaptations mostly to increase photosynthetic capability and water storage. Our goal was to describe the shoot development in two succulent species of Euphorbia using light microscopy coupled with high-resolution X-ray-computed tomography. Collateral cortical bundles were observed associated with the stem ribs in both species. The analysis of vasculature demonstrated that these bundles are, in fact, leaf traces that run axially along a portion of the internode. That structural pattern is due to an ontogenetic alteration. During shoot development, the leaf-bases remain adnate to the stem near the SAM, forming an axial component. When the internode elongates, the leaf bundles stretch as cortical bundles. The meristematic activity associated with the bundles forms the stem ribs, as leaf veins near the node, and induce rib formation along the entire internode even in the portion where the leaf traces join the stele. In addition, heterochronic shifts are also involved in the evolution of the shoot system in these Euphorbia, being related to early deciduous reduced leaves and the transference of the main photosynthetic function to the stem. This study demonstrates for the first time the influence of leaf developmental shifts and stem rib formation in Euphorbia and sheds new light on the evolution of stem succulence.

Gynoecium structure in Sapindales and a case study of Trichilia pallens (Meliaceae).

Sapindales is a monophyletic order within the malvid clade of rosids. It represents an interesting group to address questions on floral structure and evolution due to a wide variation in reproductive traits. This review covers a detailed overview of gynoecium features, as well as a new structural study based on Trichilia pallens (Meliaceae), to provide characters to support systematic relationships and to recognize patterns of variations in gynoecium features in Sapindales. Several unique and shared characteristics are identified. Anacrostylous and basistylous carpels may have evolved multiple times in Sapindales, while ventrally bulging carpels are found in pseudomonomerous Anacardiaceae. Different from previous studies, similar gynoecium features, including degree of syncarpy, ontogenetic patterns, and PTTT structure, favors a closer phylogenetic proximity between Rutaceae and Simaroubaceae, or Rutaceae and Meliaceae. An apomorphic tendency for the order is that the floral apex is integrated in the syncarpous or apocarpous gynoecium, but with different length and shape among families. Nitrariaceae shares similar stigmatic features and PTTT structure with many Sapindaceae. As the current position of both families in Sapindales is uncertain, floral features should be investigated more extensively in future studies. Two different types of gynophore were identified in the order: either derived from intercalary growth below the gynoecium as a floral internode, or by extension of the base of the ovary locules as part of the gynoecium. Sapindales share a combination of gynoecial characters but variation is mostly caused by different degrees of development of the synascidiate part relative to the symplicate part of carpels, or the latter part is absent. Postgenital fusion of the upper part of the styles leads to a common stigma, while stylar lobes may be separate. Due to a wide variation in these features, a new terminology regarding fusion is proposed to describe the gynoecium of the order.

Secretory Patterns in Colleters of Apocynaceae.

Colleters of Apocynaceae are glands related to different types of protection of vegetative and floral meristems through the production of mucilage or a mixture of many different compounds. Although several anatomical papers have shown histological and histochemical aspects of colleters of the family, almost nothing is known about their secretory process. In this study, we analyzed two types of colleters in Apocynaceae: one produces mucilage and lipophilic compounds, while the other produces an exclusively mucilaginous secretion. The secretory epidermis of the colleters of Allamanda schottii and Blepharodon bicuspidatum has a dense cytoplasm with organelles responsible for the production of mucilage and lipids. This heterogeneous secretion is released through granulocrine and eccrine mechanisms and is temporarily stored in a subcuticular space before crossing the cuticle. Conversely, colleters of Mandevilla splendens and Peplonia axillaris produce only mucilage and have a very different secretory apparatus. The mechanism of secretion is granulocrine, and the exudate is firstly accumulated in a large periplasmic space and later in an intramural space before crossing the cuticle. Notably, the structure of the cuticle varies according to the secretion composition. Although the colleters of the family are histologically similar, this study demonstrates a metabolic and subcellular variability previously unknown for Apocynaceae.

Stinging Trichomes in Apocynaceae and Their Evolution in Angiosperms.

Stinging trichomes are rare in plants, occurring only in angiosperms, where they are reported for a few genera belonging to six families. Although there is no report of stinging trichomes in Apocynaceae, previous fieldwork collections of Fischeria and Matelea caused us a mild allergic reaction on the skin when we contacted the dense indumentum of the plants. This fact associated with the well-known presence of glandular trichomes with acute apex in both genera raised suspicions that stinging trichomes could be present in the family. Hence, this study aimed to investigate the likely occurrence of stinging trichomes in Fischeria and Matelea. We analyzed vegetative shoots and leaves of Fischeria stellata and Matelea denticulata through the usual procedures of light and scanning electron microscopy. We also performed several histochemical tests to investigate the chemical composition of trichome secretion. We detected that glandular trichomes occur throughout the surface of the leaf and stem. They are multicellular, uniseriate with an apical secretory cell, which has a dilated base and a needle-shaped apex. The secretion is compressed into the acuminate portion of the apical cell by a large vacuole, and crystals are deposited in the cell wall in a subapical position, providing a preferential site of rupture. The secretion, composed of amino acids and/or proteins, is released under mechanical action, causing skin irritation. Based on our detailed morphological and anatomical analyses, and in the functional aspects observed, we concluded that the glandular trichomes in Fischeria and Matelea can indeed be classified as stinging. Thus, Apocynaceae is the seventh family for which this type of trichome has been reported. We also compiled information on stinging trichomes in all families of angiosperms. Their phylogenetic distribution indicates that they have evolved at least 12 times during angiosperm evolution and may represent an evolutionary convergence of plant defense against herbivory.

Two Origins, Two Functions: The Discovery of Distinct Secretory Ducts Formed during the Primary and Secondary Growth in Kielmeyera.

Secretory ducts have been reported for more than 50 families of vascular plants among primary and secondary tissues. A priori, all ducts of a plant are of the same type, and only slight variations in the concentration of their compounds have been reported for few species. However, two types of secretion were observed in primary and secondary tissues of Kielmeyera appariciana, leading us to investigate the possible influence of duct origins on the structure and metabolism of this gland. Kielmeyera appariciana has primary ducts in the cortex and pith and secondary ducts in the phloem. Both ducts are composed of uniseriate epithelium surrounded by a sheath and a lumen formed by a schizogenous process. Despite their similar structure and formation, the primary ducts produce resin, while the secondary ducts produce gum. This is the first report of two types of ducts in the same plant. The distinct origin of the ducts might be related to the metabolic alteration, which likely led to suppression of the biosynthetic pathway of terpenoids and phenolics in the secondary ducts. The functional and evolutionary implications of this innovation are discussed in our study and may be related to the diversification of Kielmeyera and Calophyllaceae in tropical environments.

Corrigendum: Laticifers in Sapindaceae: Structure, Evolution and Phylogenetic Importance.

[This corrects the article DOI: 10.3389/fpls.2020.612985.].

Crystal structure and specific location of a germin-like protein with proteolytic activity from Thevetia peruviana.

Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 Å (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical ß-barrel fold and two glycosylation sites (Asn55 and Asn144). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory.

Phytochrome-Dependent Temperature Perception Modulates Isoprenoid Metabolism.

Changes in environmental temperature influence many aspects of plant metabolism; however, the underlying regulatory mechanisms remain poorly understood. In addition to their role in light perception, phytochromes (PHYs) have been recently recognized as temperature sensors affecting plant growth. In particular, in Arabidopsis (Arabidopsis thaliana), high temperature reversibly inactivates PHYB, reducing photomorphogenesis-dependent responses. Here, we show the role of phytochrome-dependent temperature perception in modulating the accumulation of isoprenoid-derived compounds in tomato (Solanum lycopersicum) leaves and fruits. The growth of tomato plants under contrasting temperature regimes revealed that high temperatures resulted in coordinated up-regulation of chlorophyll catabolic genes, impairment of chloroplast biogenesis, and reduction of carotenoid synthesis in leaves in a PHYB1B2-dependent manner. Furthermore, by assessing a triple phyAB1B2 mutant and fruit-specific PHYA- or PHYB2-silenced plants, we demonstrated that biosynthesis of the major tomato fruit carotenoid, lycopene, is sensitive to fruit-localized PHY-dependent temperature perception. The collected data provide compelling evidence concerning the impact of PHY-mediated temperature perception on plastid metabolism in both leaves and fruit, specifically on the accumulation of isoprenoid-derived compounds.

Beyond the limits of photoperception: constitutively active PHYTOCHROME B2 overexpression as a means of improving fruit nutritional quality in tomato.

Photoreceptor engineering has recently emerged as a means for improving agronomically beneficial traits in crop species. Despite the central role played by the red/far-red photoreceptor phytochromes (PHYs) in controlling fruit physiology, the applicability of PHY engineering for increasing fleshy fruit nutritional content remains poorly exploited. In this study, we demonstrated that the fruit-specific overexpression of a constitutively active GAF domain Tyr252 -to-His PHYB2 mutant version (PHYB2Y252H ) significantly enhances the accumulation of multiple health-promoting antioxidants in tomato fruits, without negative collateral consequences on vegetative development. Compared with the native PHYB2 overexpression, PHYB2Y252H -overexpressing lines exhibited more extensive increments in transcript abundance of genes associated with fruit plastid development, chlorophyll biosynthesis and metabolic pathways responsible for the accumulation of antioxidant compounds. Accordingly, PHYB2Y252H -overexpressing fruits developed more chloroplasts containing voluminous grana at the green stage and overaccumulated carotenoids, tocopherols, flavonoids and ascorbate in ripe fruits compared with both wild-type and PHYB2-overexpressing lines. The impacts of PHYB2 or PHYB2Y252H overexpression on fruit primary metabolism were limited to a slight promotion in lipid biosynthesis and reduction in sugar accumulation. Altogether, these findings indicate that mutation-based adjustments in PHY properties represent a valuable photobiotechnological tool for tomato biofortification, highlighting the potential of photoreceptor engineering for improving quality traits in fleshy fruits.

C4 and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation.

Although biochemically related, C4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown. Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C4 . The putative CAM-specific genes were predominantly involved in night-time primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-abundance regulation and photosynthetic physiology indicated that C4 and CAM coexist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C4 to C4 -CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs. These findings provide insights into C4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C4 crop species.

Laticifers in Sapindaceae: Structure, Evolution and Phylogenetic Importance.

Laticifer occurrence and structure are poorly known in Sapindaceae. Occurrence is likely underestimated owing to the low production of latex in most species. We investigated 67 species from 23 genera of Sapindaceae to verify laticifer occurrence and their structural, developmental and chemical features, as well as their evolutionary history in the family. Shoots were collected from herbarium and fresh specimens for histological analyses. Three characters derived from laticifer features were coded and their ancestral states reconstructed through Bayesian stochastic mapping and maximum likelihood estimation. Only articulated non-anastomosing laticifers were found in Sapindaceae. Laticifers differentiate early during shoot development and are found in the cortex, phloem, and pith. Latex is mostly composed of lipids. Callose and suberin were detected in laticifer cell walls in some genera. Reconstruction of laticifer ancestral states showed that laticifers are present in most clades of Sapindaceae with some reversals. Callose in the laticifer cell wall was found exclusively in Serjania and Paullinia (tribe Paullinieae), a character regarded as independently derived. Occurrence of laticifers in Sapindaceae is broader than previously reported. Articulated non-anastomosing laticifers had five independent origins in Sapindaceae with some secondary losses, occurring in five out of six genera of Paullinieae and 10 other genera outside Paullinieae. Particularly, callose in the laticifer cell wall evolved independently twice in the family, and its occurrence may be interpreted as a key-innovation that promoted the diversification of Paullinia and Serjania. Our study suggests that laticifer characters may be useful in understanding the generic relationships within the family.

Solanum lycopersicum GOLDEN 2-LIKE 2 transcription factor affects fruit quality in a light- and auxin-dependent manner.

Plastids are organelles responsible for essential aspects of plant development, including carbon fixation and synthesis of several secondary metabolites. Chloroplast differentiation and activity are highly regulated by light, and several proteins involved in these processes have been characterised. Such is the case of the GOLDEN 2-LIKE (GLK) transcription factors, which induces the expression of genes related to chloroplast differentiation and photosynthesis. The tomato (Solanum lycopersicum) genome harbours two copies of this gene, SlGLK1 and SlGLK2, each with distinct expression patterns. While the former predominates in leaves, the latter is mainly expressed in fruits, precisely at the pedicel region. During tomato domestication, the selection of fruits with uniform ripening fixed the mutation Slglk2, nowadays present in most cultivated varieties, what penalised fruit metabolic composition. In this study, we investigated how SlGLK2 is regulated by light, auxin and cytokinin and determined the effect of SlGLK2 on tocopherol (vitamin E) and sugar metabolism, which are components of the fruit nutritional and industrial quality. To achieve this, transcriptional profiling and biochemical analysis were performed throughout fruit development and ripening from SlGLK2, Slglk2, SlGLK2-overexpressing genotypes, as well as from phytochrome and hormonal deficient mutants. The results revealed that SlGLK2 expression is regulated by phytochrome-mediated light perception, yet this gene can induce chloroplast differentiation even in a phytochrome-independent manner. Moreover, auxin was found to be a negative regulator of SlGLK2 expression, while SlGLK2 enhances cytokinin responsiveness. Additionally, SlGLK2 enhanced chlorophyll content in immature green fruits, leading to an increment in tocopherol level in ripe fruits. Finally, SlGLK2 overexpression resulted in higher total soluble solid content, possibly by the regulation of sugar metabolism enzyme-encoding genes. The results obtained here shed light on the regulatory network that interconnects SlGLK2, phytohormones and light signal, promoting the plastidial activity and consequently, influencing the quality of tomato fruit.

Osmophores and floral fragrance in Anacardium humile and Mangifera indica (Anacardiaceae): an overlooked secretory structure in Sapindales.

Flowers of Anacardiaceae and other Sapindales typically produce nectar, but scent, often associated with a reward for pollinators, has surprisingly been mentioned only rarely for members of the family and order. However, flowers of Anacardium humile and Mangifera indica produce a strong sweet scent. The origin and composition of these floral scents is the subject of this study. Screening of potential osmophores on the petals and investigations of their anatomy were carried out by light, scanning and transmission electron microscopy. The composition of the floral fragrance was characterized by gas chromatography-mass spectrometry. In both species, the base of the adaxial side of each petal revealed specialized secretory epidermal cells which are essentially similar in structure and distinct from all other neighbouring cells. These cells also showed evidence of granulocrine secretory mechanisms and slight specific variations in their subcellular apparatus coinciding with the respective composition of the floral fragrance, predominantly composed of sesquiterpenes in A. humile and monoterpenes in M. indica. This study reports the presence of osmophores for the first time in flowers of Anacardiaceae and confirms the link between the ultrastructural features of their secretory cells and the volatiles produced by the flowers. The flowers of most Sapindales, including Anacardiaceae, are nectariferous. However, the presence of osmophores has only been described for very few genera of Rutaceae and Sapindaceae. Both the occurrence of osmophores and fragrance may have largely been overlooked in Anacardiaceae and Sapindales until now. Further studies are needed to better understand the nature and diversity of the interactions of their nectariferous flowers with their pollinators.

Structure and development of flowers and inflorescences in Peraceae and Euphorbiaceae and the evolution of pseudanthia in Malpighiales.

Pseudanthia are reduced and compact inflorescences which apparently had independent evolution in Euphorbiaceae and Peraceae within Malpighiales. In order to analyze the hypothesis that the different pseudanthia found in Malpighiales have non-homologous developmental steps, we studied the inflorescence and flower development in the three Malpighiales genera that present this type of inflorescence-Dalechampia (Acalyphoideae/Euphorbiaceae), Euphorbia (Euphorbioideae/Euphorbiaceae), and Pera (Peraceae)-and compared them to that of Joannesia (Crotonoideae/Euphorbiaceae), which does not present a pseudanthium. Inflorescences and flowers were analyzed using light microscopy and scanning electron microscopy. Dalechampia and Euphorbia have protogynic bisexual pseudanthia, with unisexual perianthed flowers in Dalechampia, and achlamydeous flowers in Euphorbia. Pera has unisexual pseudanthia and the male flowers have a vestigial calyx and the female flowers are achlamydeous. Joannesia flowers are very distinct when compared to the pseudanthia flowers, as they are composed of all the whorls and there is no reduction. In the early stages of development, the first structures to be formed in the pseudanthia are the different series of bracts, including outer, involucral and involucel bracts. The floral primordia are initiated almost simultaneously with the involucre. Although the different morphology, the early inflorescence followed the same branching pattern in all studied genera, and the number and elongation of the branches were affected by the early female flower development in the terminal position. We suggest that the different pseudanthia evolved via process of floral whorl reduction and reorganization of flowers in the inflorescence axes, especially the position of female and male flowers and elongation or shortening of the branches. The sex of the terminal flower is a developmental key, i.e., the protogynic development deeply affects the pseudanthia growth, reducing the ramification and elongation of the axes.