Hormones and Flower Development in Arabidopsis.

Sexual reproduction requires the participation of two gametes, female and male. In angiosperms, gametes develop in specialized organs, pollen (containing the male gametes) develops in the stamens, and the ovule (containing the female gamete) develops in the gynoecium. In Arabidopsis thaliana, the female and male sexual organs are found within the same structure called flower, surrounded by the perianth, which is composed of petals and sepals. During flower development, different organs emerge in an established order and throughout their development distinct tissues within each organ are differentiated. All this requires the coordination and synchronization of several biological processes. To achieve this, hormones and genes work together. These components can interact at different levels generating hormonal interplay and both positive and negative feedback loops, which in turn, gives robustness, stability, and flexibility to flower development. Here, we summarize the progress made on elucidating the role of different hormonal pathways during flower development in Arabidopsis thaliana.

Novel Roles of SPATULA in the Control of Stomata and Trichome Number, and Anthocyanin Biosynthesis.

The bHLH transcription factor SPATULA (SPT) has been identified as a regulator during different stages of Arabidopsis development, including the control of leaf size. However, the mechanism via which it performs this function has not been elucidated. To better understand the role of SPT during leaf development, we used a transcriptomic approach to identify putative target genes. We found putative SPT target genes related to leaf development, and to stomata and trichome formation. Furthermore, genes related to anthocyanin biosynthesis. In this work, we demonstrate that SPT is a negative regulator of stomata number and a positive regulator of trichome number. In addition, SPT is required for sucrose-mediated anthocyanin biosynthesis.

The protein-protein interaction landscape of transcription factors during gynoecium development in Arabidopsis.

Flowers are composed of organs whose identity is defined by the combinatorial activity of transcription factors (TFs). The interactions between MADS-box TFs and protein complex formation have been schematized in the floral quartet model of flower development. The gynoecium is the flower's female reproductive part, crucial for fruit and seed production and, hence, for reproductive success. After the establishment of carpel identity, many tissues arise to form a mature gynoecium. TFs have been described as regulators of gynoecium development, and some interactions and complexes have been identified. However, broad knowledge about the interactions among these TFs and their participation during development remains scarce. In this study, we used a systems biology approach to understand the formation of a complex reproductive unit-as the gynoecium-by mapping binary interactions between well-characterized TFs. We analyzed almost 4500 combinations and detected more than 250 protein-protein interactions (PPIs), resulting in a process-specific interaction map. Topological analyses suggest hidden functions and novel roles for many TFs. In addition, we observed a close relationship between TFs involved in auxin and cytokinin-signaling pathways and other TFs. Furthermore, we analyzed the network by combining PPI data, expression, and genetic data, which helped us to dissect it into several dynamic spatio-temporal subnetworks related to gynoecium development processes. Finally, we generated an extended PPI network that predicts new players in gynoecium development. Taken together, all these results serve as a valuable resource for the plant community.

Chili Pepper Jojutla Morelos (Capsicum annuum L.), CJ-2018: A Variety Resistant to Bactericera cockerelli.

Chili pepper is a vegetable of worldwide economic and gastronomic importance. The psyllid, Bactericera cockerelli, is an economically important pest in this crop, causing considerable losses in its production. Currently, the application of insecticides is the main way to control B. cockerelli. However, the use of varieties resistant to this insect is a viable alternative for its control and management. In this work, the oviposition rate, development, and survival of B. cockerelli in two native varieties of chili were evaluated. Choice and non-choice trials showed that the B. cockerelli oviposition was reduced on CJ-2018 by 92.17 and 80.18%, respectively, compared to the control. In CM-334, the insect showed a behavior similar to the control in the non-choice test, while in the choice test it laid more eggs on CM-334 compared to the control. The development and survival assay showed that only 1.33% of the eggs managed to reach the adult stage on CJ-2018. In contrast, on CM-334 the survival of B. cockerelli was similar to the control. These results suggest that CJ-2018 presented a resistance based on antixenosis and antibiosis against B. cockerelli.

GC/MS Analysis, Antioxidant Activity, and Antimicrobial Effect of Pelargonium peltatum (Geraniaceae).

In recent years, the increase in antibiotic resistance demands searching for new compounds with antimicrobial activity. Phytochemicals found in plants offer an alternative to this problem. The genus Pelargonium contains several species; some have commercial use in traditional medicine such as P. sinoides, and others such as P. peltatum are little studied but have promising potential for various applications such as phytopharmaceuticals. In this work, we characterized the freeze-dried extracts (FDEs) of five tissues (root, stem, leaf, and two types of flowers) and the ethyl acetate fractions from leaf (Lf-EtOAc) and flower (Fwr-EtOAc) of P. peltatum through the analysis by thin-layer chromatography (T.L.C.), gas chromatography coupled to mass spectrometry (GC-MS), phytochemicals quantification, antioxidant capacity, and antimicrobial activity. After the first round of analysis, it was observed that the FDE-Leaf and FDE-Flower showed higher antioxidant and antimicrobial activities compared to the other FDEs, for which FDE-Leaf and FDE-Flower were fractionated and analyzed in a second round. The antioxidant activity determined by ABTS showed that Lf-EtOAc and Fwr-EtOAc had the lowest IC50 values with 27.15 ± 1.04 and 28.11 ± 1.3 µg/mL, respectively. The content of total polyphenols was 264.57 ± 7.73 for Lf-EtOAc and 105.39 ± 4.04 mg G.A./g FDE for Fwr-EtOAc. Regarding the content of flavonoid, Lf-EtOAc and Fw-EtOAc had the highest concentration with 34.4 ± 1.06 and 29.45 ± 1.09 mg Q.E./g FDE. In addition, the minimum inhibitory concentration (M.I.C.) of antimicrobial activity was evaluated: Lf-EtOAc and Fwr-EtOAc were effective at 31.2 µg/mL for Staphylococcus aureus and 62.5 µg/mL for Salmonella enterica, while for the Enterococcus feacalis strain, Fwr-EtOAc presented 31.2 µg/mL of M.I.C. According to the GC-MS analysis, the main compounds were 1,2,3-Benzenetriol (Pyrogallol), with 77.38% of relative abundance in the Lf-EtOAc and 71.24% in the Fwr-EtOAc, followed by ethyl gallate (13.10%) in the Fwr-EtOAc and (Z)-9-Octadecenamide (13.63% and 6.75%) in both Lf-EtOAc and Fwr-EtOAc, respectively.

Green Synthesis of Gold and Silver Nanoparticles Using Leaf Extract of Capsicum chinense Plant.

So far, several studies have focused on the synthesis of metallic nanoparticles making use of extracts from the fruit of the plants from the genus Capsicum. However, as the fruit is the edible, and highly commercial, part of the plant, in this work we focused on the leaves, a part of the plant that is considered agro-industrial waste. The biological synthesis of gold (AuNPs) and silver (AgNPs) nanoparticles using aqueous extracts of root, stem and leaf of Capsicum chinense was evaluated, obtaining the best results with the leaf extract. Gold and silver nanoparticles synthesized using leaf extract (AuNPs-leaf and AgNPs-leaf, respectively) were characterized by UV-visible spectrophotometry (UV-Vis), Fourier Transform Infrared Spectroscopy with Attenuated Total Reflection (FTIR-ATR), X-ray Photoelectron Spectroscopy (XPS), Ultra Hight Resolution Scanning Electron Microscopy coupled to Energy-Dispersive X-ray spectroscopy (UHR-SEM-EDX) and Transmission Electron Microscopy (TEM), and tested for their antioxidant and antimicrobial activities. In addition, different metabolites involved in the synthesis of nanoparticles were analyzed. We found that by the use of extracts derived from the leaf, we could generate stable and easy to synthesize AuNPs and AgNPs. The AuNPs-leaf were synthesized using microwave radiation, while the AgNPs-leaf were synthesized using UV light radiation. The antioxidant activity of the extract, determined by ABTS, showed a decrease of 44.7% and 60.7% after the synthesis of the AuNPs-leaf and AgNPs-leaf, respectively. After the AgNPs-leaf synthesis, the concentration of polyphenols, reducing sugars and amino acids decreased by 15.4%, 38.7% and 46.8% in the leaf extract, respectively, while after the AuNPs-leaf synthesis only reducing sugars decreased by 67.7%. These results suggest that these groups of molecules are implicated in the reduction/stabilization of the nanoparticles. Although the contribution of these compounds in the synthesis of the AuNPs-leaf and the AgNPs-leaf was different. Finally, the AgNPs-leaf inhibited the growth of S. aureus, E. coli, S. marcescens and E. faecalis. All of them are bacterial strains of clinical importance due to their fast antibiotic resistance development.

Arabidopsis cysteine-rich receptor-like protein kinase CRK33 affects stomatal density and drought tolerance.

Cysteine-rich receptor-like protein kinases (CRKs) are transmembrane proteins containing two domains of unknown function 26 (DUF26) RLKs in their ectodomain. Despite that CRKs control important aspects of plant development, only few proteins have functionally been characterized. In this work, we analyzed the function of CRK33 by characterizing two insertional lines. The stomatal density and stomatal index were decreased in crk33-2 and crk33-3 plants in comparison to wild-type plants, correlating with a decreased transpiration in transgenic plants and a higher drought tolerance. Furthermore, photosynthesis and stomatal conductance changed. Finally, all four stomata cell fate genes were upregulated, especially the expression of TMM and SPCH in the mutant background, suggesting a role for CRK33 in stomatal spacing.

Redundant and Non-redundant Functions of the AHK Cytokinin Receptors During Gynoecium Development.

The phytohormone cytokinin is crucial for plant growth and development. The site of action of cytokinin in the plant is dependent on the expression of the cytokinin receptors. In Arabidopsis, there are three cytokinin receptors that present some overlap in expression pattern. Functional studies demonstrated that the receptors play highly redundant roles but also have specialized functions. Here, we focus on gynoecium development, which is the female reproductive part of the plant. Cytokinin signaling has been demonstrated to be important for reproductive development, positively affecting seed yield and fruit production. Most of these developmental processes are regulated by cytokinin during early gynoecium development. While some information is available, there is a gap in knowledge on cytokinin function and especially on the cytokinin receptors during early gynoecium development. Therefore, we studied the expression patterns and the role of the cytokinin receptors during gynoecium development. We found that the three receptors are expressed in the gynoecium and that they have redundant and specialized functions.

Gynoecium development: networks in Arabidopsis and beyond.

Life has always found a way to preserve itself. One strategy that has been developed for this purpose is sexual reproduction. In land plants, the gynoecium is considered to be at the top of evolutionary innovation, since it has been a key factor in the success of the angiosperms. The gynoecium is composed of carpels with different tissues that need to develop and differentiate in the correct way. In order to control and guide gynoecium development, plants have adapted elements of pre-existing gene regulatory networks (GRNs) but new ones have also evolved. The GRNs can interact with internal factors (e.g. hormones and other metabolites) and external factors (e.g. mechanical signals and temperature) at different levels, giving robustness and flexibility to gynoecium development. Here, we review recent findings regarding the role of cytokinin-auxin crosstalk and the genes that connect these hormonal pathways during early gynoecium development. We also discuss some examples of internal and external factors that can modify GRNs. Finally, we make a journey through the flowering plant lineage to determine how conserved are these GRNs that regulate gynoecium and fruit development.

Conserved and novel responses to cytokinin treatments during flower and fruit development in Brassica napus and Arabidopsis thaliana.

Hormones are an important component in the regulatory networks guiding plant development. Cytokinins are involved in different physiological and developmental processes in plants. In the model plant Arabidopsis thaliana, cytokinin application during gynoecium development produces conspicuous phenotypes. On the other hand, Brassica napus, also known as canola, is a crop plant belonging to the Brassicaceae family, as A. thaliana. This makes B. napus a good candidate to study whether the cytokinin responses observed in A. thaliana are conserved in the same plant family. Here, we observed that cytokinin treatment in B. napus affects different traits of flower and fruit development. It increases ovule and seed number, affects stamen filament elongation and anther maturation, and causes a conspicuous overgrowth of tissue in petals and gynoecia. Furthermore, cytokinin recovers replum development in both wild type B. napus and in the A. thaliana rpl ntt double mutant, in which no replum is visible. These results indicate both conserved and novel responses to cytokinin in B. napus. Moreover, in this species, some cytokinin-induced phenotypes are inherited to the next, untreated generation, suggesting that cytokinins may trigger epigenetic modifications.

Synergistic relationship between auxin and cytokinin in the ovary and the participation of the transcription factor SPATULA.

The phytohormones auxin and cytokinin are key regulators of plant development, and both regulate almost all aspects of plant growth and development. Communication between auxin-cytokinin signaling pathways has been the subject of intense research. However, few studies have focused specifically on the development of the early gynoecium. We have recently discovered that cytokinin signaling plays a role in the regulation of auxin biosynthesis and transport in the ovary region of the gynoecium, and that the transcription factor SPATULA (SPT) is necessary. Here, we provide evidence that indicates that cytokinin and auxin have a synergistic relationship at the medial domain during gynoecium development, and that SPT is important for this interaction.

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium.

Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

The NTT transcription factor promotes replum development in Arabidopsis fruits.

Fruits are complex plant structures that nurture seeds and facilitate their dispersal. The Arabidopsis fruit is termed silique. It develops from the gynoecium, which has a stigma, a style, an ovary containing the ovules, and a gynophore. Externally, the ovary consists of two valves, and their margins lay adjacent to the replum, which is connected to the septum that internally divides the ovary. In this work we describe the role for the zinc-finger transcription factor NO TRANSMITTING TRACT (NTT) in replum development. NTT loss of function leads to reduced replum width and cell number, whereas increased expression promotes replum enlargement. NTT activates the homeobox gene BP, which, together with RPL, is important for replum development. In addition, the NTT protein is able to bind the BP promoter in yeast, and when this binding region is not present, NTT fails to activate BP in the replum. Furthermore, NTT interacts with itself and different proteins involved in fruit development: RPL, STM, FUL, SHP1 and SHP2 in yeast and in planta. Moreover, its genetic interactions provide further evidence about its biological relevance in replum development.

Cytokinin treatments affect the apical-basal patterning of the Arabidopsis gynoecium and resemble the effects of polar auxin transport inhibition.

The apical-basal axis of the Arabidopsis gynoecium is established early during development and is divided into four elements from the bottom to the top: the gynophore, the ovary, the style, and the stigma. Currently, it is proposed that the hormone auxin plays a critical role in the correct apical-basal patterning through a concentration gradient from the apical to the basal part of the gynoecium, as chemical inhibition of polar auxin transport through 1-N-naphtylphtalamic acid (NPA) application, severely affects the apical-basal patterning of the gynoecium. In this work, we show that the apical-basal patterning of gynoecia is also sensitive to exogenous cytokinin (benzyl amino purine, BAP) application in a similar way as to NPA. BAP and NPA treatments were performed in different mutant backgrounds where either cytokinin perception or auxin transport and perception were affected. We observed that cytokinin and auxin signaling mutants are hypersensitive to NPA treatment, and auxin transport and signaling mutants are hypersensitive to BAP treatment. BAP effects in apical-basal gynoecium patterning are very similar to the effects of NPA, therefore, it is possible that BAP affects auxin transport in the gynoecium. Indeed, not only the cytokinin-response TCS::GFP marker, but also the auxin efflux carrier PIN1 (PIN1::PIN1:GFP) were both affected in BAP-induced valveless gynoecia, suggesting that the BAP treatment producing the morphological changes has an impact on both in the response pattern to cytokinin and on auxin transport. In summary, we show that cytokinin affects proper apical-basal gynoecium patterning in Arabidopsis in a similar way to the inhibition of polar auxin transport, and that auxin and cytokinin mutants and markers suggest a relation between both hormones in this process.

Inside the gynoecium: at the carpel margin.

The gynoecium, which is produced at the center of most flowers, is the female reproductive organ and consists of one or more carpels. The Arabidopsis gynoecium consists of two fused carpels. Its inner tissues possess meristematic characteristics and are called the carpel margin meristem (CMM), because they are located at the margins of the carpels and generate the 'marginal' tissues of the gynoecium (placenta, ovules, septum, transmitting tract, style, and stigma). A key question is which factors are guiding the correct development of all these tissues, many of which are essential for reproduction. Besides regulatory genes, hormones play an important part in the development of the marginal tissues, and recent reports have highlighted the role of cytokinins, as discussed in this review.

Hormones talking: does hormonal cross-talk shape the Arabidopsis gynoecium?

The proper development of fruits is important for the sexual reproduction and propagation of many plant species. The fruit of Arabidopsis derives from the fertilized gynoecium, which initiates at the center of the flower and obtains its final shape, size, and functional tissues through progressive stages of development. Hormones, specially auxins, play important roles in gynoecium and fruit patterning. Cytokinins, which act as counterparts to auxins in other plant tissues, have been studied more in the context of ovule formation and parthenocarpy. We recently studied the role of cytokinins in gynoecium and fruit patterning and found that they have more than one role during gynoecium and fruit patterning. We also compared the cytokinin response localization to the auxin response localization in these organs, and studied the effects of spraying cytokinins in young flowers of an auxin response line. In this addendum, we discuss further the implications of the observed results in the knowledge about the relationship between cytokinins and auxins at the gynoecium.

The class II HD-ZIP JAIBA gene is involved in meristematic activity and important for gynoecium and fruit development in Arabidopsis.

Development and patterning of the gynoecium - and later the fruit - must be finely regulated to ensure the survival of the species that produces them. The process that leads to successful fruit formation starts at early stages of floral meristem development and follows a series of chronologically successive events. In a recent work we reported the functional characterization of the class II HD-ZIP JAIBA (JAB) gene. Mutant jab plants showed sporophytic defects in male and female reproductive development, and combined with the mutant crabs claw (crc) caused defects in the floral meristem (FM) determination process and gynoecium medial tissue development. Furthermore, the JAB protein interacted with transcription factors known to regulate meristematic activity, fruit development and FM determinacy. Preliminary results presented here suggest a genetic interaction between JAB and the gene SHOOT MERISTEMLESS (STM).

The role of cytokinin during Arabidopsis gynoecia and fruit morphogenesis and patterning.

Cytokinins have many essential roles in embryonic and post-embryonic growth and development, but their role in fruit morphogenesis is currently not really known. Moreover, information about the spatio-temporal localization pattern of cytokinin signaling in gynoecia and fruits is lacking. Therefore, the synthetic reporter line TCS::GFP was used to visualize cytokinin signaling during gynoecium and fruit development. Fluorescence was detected at medial regions of developing gynoecia, and, unexpectedly, at the valve margin in developing fruits, and was severely altered in mutants that lack or ectopically acquire valve margin identity. Comparison to developing gynoecia and fruits in a DR5rev::GFP line showed that the transcriptional responses to cytokinin and auxin are frequently present in complementary patterns. Moreover, cytokinin treatments in early gynoecia produced conspicuous changes, and treatment of valve margin mutant fruits restored this tissue. The results suggest that the phytohormone cytokinin is important in gynoecium and fruit patterning and morphogenesis, playing at least two roles: an early proliferation-inducing role at the medial tissues of the developing gynoecia, and a late role in fruit patterning and morphogenesis at the valve margin of developing fruits.

JAIBA, a class-II HD-ZIP transcription factor involved in the regulation of meristematic activity, and important for correct gynoecium and fruit development in Arabidopsis.

The gynoecium is one of the most complex organs of a plant. After fertilization, it becomes a fruit, which has two important functions: to protect the seeds while they develop and to disperse them at maturity. The development and patterning of the gynoecium and later fruit must be finely regulated to ensure the survival of the species that produces them. The process that leads to successful fruit formation starts at early stages of floral meristem development and follows a series of chronologically successive events. In this work we report the functional characterization of the class-II homeodomain leucine zipper (HD-ZIP) JAIBA (JAB) gene. Mutant jab plants show sporophytic defects in male and female reproductive development, and combined with crabs claw cause defects in the floral meristem (FM) determination process and gynoecium medial tissue development. This suggests that proper FM determination is required for gynoecium medial tissue development, and indicates that JAB and CRC are necessary for both processes. Furthermore, the JAB protein interacts with transcription factors known to regulate meristematic activity, fruit development, and floral meristem determinacy. The sporophytic effect on pollen and embryo sac development might be an independent and later function of JAB. In summary, we present data that indicates that the JAB gene regulates meristematic activity in different tissues, and that it is necessary for the correct formation of the gynoecium at different stages, contributing to a crucial process in plant life: proper fruit development.