Adriana P. Martinelli

Brassinosteroids interact negatively with jasmonates in the formation of anti-herbivory traits in tomato

Given the susceptibility of tomato plants to pests, the aim of the present study was to understand how hormones are involved in the formation of tomato natural defences against insect herbivory. Tomato hormone mutants, previously introgressed into the same genetic background of reference, were screened for alterations in trichome densities and allelochemical content. Ethylene, gibberellin, and auxin mutants indirectly showed alteration in trichome density, through effects on epidermal cell area. However, brassinosteroids (BRs) and jasmonates (JAs) directly affected trichome density and allelochemical content, and in an opposite fashion. The BR-deficient mutant dpy showed enhanced pubescence, zingiberene biosynthesis, and proteinase inhibitor expression; the opposite was observed for the JA-insensitive jai1-1 mutant. The dpy x jai1-1 double mutant showed that jai1-1 is epistatic to dpy, indicating that BR acts upstream of the JA signalling pathway. Herbivory tests with the poliphagous insect Spodoptera frugiperda and the tomato pest Tuta absoluta clearly confirmed the importance of the JA-BR interaction in defence against herbivory. The study underscores the importance of hormonal interactions on relevant agricultural traits and raises a novel biological mechanism in tomato that may differ from the BR and JA interaction already suggested for Arabidopsis.

Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition

During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins.

Biochemical and histological characterization of tomato mutants

Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT), we observed that the malondialdehyde (MDA) content was enhanced in the diageotropica (dgt) and lutescent (l) mutants, whilst the highest levels of hydrogen peroxide (H(2)O(2)) were observed in high pigment 1 (hp1) and aurea (au) mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT) activity when compared to MT. Guaiacol peroxidase (GPOX) was enhanced in both sitiens (sit) and notabilis (not) mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX). Based on PAGE analysis, the activities of glutathione reductase (GR) isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD) isoform III was reduced in leaves of sit, epi, Never ripe (Nr) and green flesh (gf) mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.

Direct determination of plant-growth related metabolites by capillary electrophoresis with spectrophotometric UV detection

The detection of plant hormones and growth regulators is of great interest for many biological studies especially in the determination of metabolites related to plan growth and differentiation. In this work, we propose a simple method based on capillary electrophoresis (CE) for the separation of different classes of plant growth regulators such as auxins, cytokinins, gibberelic acid and abscisic acid. CE with UV detection was used and the analytical conditions were as follows: phosphate buffer 25 mmol L-1, for all the measurements and the separation conditions pH 12 or 2.5, by hydrodynamic injection 5 s at 10 cm and separation voltage of 22 kV. The absorbance detection was fixed at either 220 nm or 270 nm depending on a given phytohormone class. Under these conditions, phytohormones (Indole-3-acetic acid (IAA), Gibberellic acid (GA3), Abscisic acid (ABA), picloram, zeatin and 6-Benzylaminopurine (BAP) were separated in approximately 3 to 5 min. The plant material used to verify the possibility of detection of hormone/plant growth regulators was citro (Citrus sinensis L. Osbeck) callus in the multiplication stage. In the plant tissue sample, zeatin was successfully detected. The results confirmed the potential use of CE as an efficient alternative and simple method to the classical procedures used for phytohormone detection in plant tissues.

Plant regeneration from embryogenic callus and cell suspensions of Brachiaria brizantha

In monocots, in vitro plant regeneration can be obtained through somatic embryogenesis (Vasil and Vasil 1980, 1982; Vasil 2005). Evidence of more than one morphogenic response in the same explant, under the control of different auxin/cytokinin ratios and concentrations, was observed in species of the Poaceae family such as sorghum (Sorghum bicolor, (L.) Moench.) , minor mil le t (Paspalum scrobiculatum L.), sugar cane (Saccharum officinarum L.), and baby bamboo (Pogonatherum paniceum Lam. Hack.) (reviewed by Wang et al. 2008). Brachiaria callus has been induced using seeds as explants from Brachiaria brizantha (Hochst. ex A. Rich.) Stapf, Brachiaria decumbens Stapf, Brachiaria ruziziensis Germain et Evrard and Brachiaria dictyoneura [Brachiaria humidicola (Rendle) Schweick vr. Lanero, ex B. dictyoneura] (Tohme et al. 1996). Subsequently, using the same methodology, calli were induced from 76% of isolated embryos from seeds of B. brizantha (Lenis-Manzano 1998). The formation of somatic embryos and multiple shoots from seedling apical meristems was demonstrated for B. ruziziensis (Ishigaki et al. 2009). In this system, mature seeds were used as source of explants and cultured on a medium containing 4 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Ishigaki et al. (2012) reported that embryogenic calli were more suitable than shoot apex explants for genetic transformation of ruzigrass. B. brizantha cv. Marandu is the most important and cultivated forage grass in Brazil. It is tolerant to spittlebugs (Homoptera: Cercopidae complex, mainly genera Deois and Zulia), an economically important pest for Brachiaria (Felismino et al. 2012). This cultivar is considered as the key forage in beef cattle production in Brazil. Breeding is hindered by its apomictic reproductive mode (the production of clones of the mother plant), which reduces the possibility of being used for hybridizations (Valle and Savidan 1996). Alternatives to conventional breeding of B. brizantha would include genetic modification via transformation. However, methods of transformation and tissue culture of these plants are not yet established. The development of embryogenic calli has been reported from 73% of the B. brizantha cv. Marandu isolated seed embryos in culture and 67% of calli regenerated plants (Silveira et al. 2003). More recently, the histology of embryos isolated from the in vitro cultivated seed was shown (Lenis-Manzano et al. 2010). The formation of multiple shoots was reported in cultured basal segments from micropropagated plantlets of this cultivar (Pinheiro et al. 2000), a system that was efficient for in vitro chromosome duplication in Brachiaria. Somatic embryogenesis and organogenesis in B. brizantha is influenced by several factors such as genotype, explant type, and culture conditions (Cabral et al. 2011). A high rate of cell division in explants used for genetic transformation ofmonocots is a prerequisite for the integration of exogenous DNA (Hiei et al. 1997; Vasil 2005). Rapid cell proliferation is a characteristic of embryogenic tissue. Cell suspension cultures (CS) are well known for their cell division capacity and the possibility of developmental-stage Electronic supplementary material The online version of this article (doi:10.1007/s11627-015-9690-0) contains supplementary material, which is available to authorized users. G. B. Cabral (*) :V. T. C. Carneiro : J. P. da Silva :D. M. A. Dusi Embrapa Genetic Resources and Biotechnology, Brasília, Brazil e-mail: glaucia.cabral@embrapa.br

Perspectives for a Framework to Understand Aril Initiation and Development

A differentiated structure called “aril” has been described in seeds of several plant species during the course of evolution and might be considered as a supernumerary integument. Besides its ecological function in seed dispersal, the structure also represents a relevant character for systematic classification and exhibits important properties that impart agronomic value in certain species. Little is known about the molecular pathways underlying this morphological innovation because it is absent in currently used model species. A remarkable feature of the seeds of Passiflora species is the presence of a conspicuous aril. This genus is known for the ornamental, medicinal, and food values of its species. In view of the molecular resources and tools available for some Passiflora species, we highlight the potential of these species as models for developmental studies of the aril.

Pollen morphology and viability in Bromeliaceae

Pollen morphology characterization is important in taxonomy, conservation and plant breeding, and pollen viability studies can support breeding programs. This study investigated pollen morphology and male fertility in 18 species of Bromeliaceae with ornamental potential. For morphological characterization, pollen grains were acetolyzed and characterization of exine was done using scanning and transmission electron microscopy. Pollen viability was investigated by in vitro germination and histochemical tests. Species belonging to Aechmea and Ananas genera presented medium size pollen, except for Ae. fasciata, with large pollen. Al. nahoumii, P. sagenarius and the Vriesea species analyzed showed large pollen, except for V. carinata, with very large pollen. Pollen of Aechmea, Ananas and P. sagenarius presented bilateral symmetry, diporate, exine varying from tectate to semitectate. Al. nahoumii and Vriesea species presented pollen with bilateral symmetry, monocolpate; exine was semitectate, reticulate and heterobrochate. Germination percentage and tube growth were greater in SM and BKM media. Histochemical tests showed pollen viability above 70% for all species, except for Ananas sp. (40%). Pollen morphology is important for the identification of species, especially in this family, which contains a large number of species. High rates of viability favor fertilization and seed production, essential for efficient hybrid production and conservation.

Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis

Summary The flowers of most dicotyledons have petals that, together with the sepals, initially protect the reproductive organs. Later during development petals are required to open the flower and to attract pollinators. This diverse set of functions demands tight temporal and spatial regulation of petal development. We studied the functioning of the Arabidopsis thaliana TCP5‐like transcription factors (TFs) in petals. Overexpression of TCP5 in petal epidermal cells results in smaller petals, whereas tcp5 tcp13 tcp17 triple knockout lines have wider petals with an increased surface area. Comprehensive expression studies revealed effects of TCP5‐like TFs on the expression of genes related to the cell cycle, growth regulation and organ growth. Additionally, the ethylene biosynthesis genes 1‐amino‐cyclopropane‐1‐carboxylate (ACC) synthase 2 (ACS2) and ACC oxidase 2 (ACO2) and several ETHYLENE RESPONSE FACTORS (ERFs) are found to be differentially expressed in TCP5 mutant and overexpression lines. Chromatin immunoprecipitation–quantitative PCR showed direct binding of TCP5 to the ACS2 locus in vivo. Ethylene is known to influence cell elongation, and the petal phenotype of the tcp5 tcp13 tcp17 mutant could be complemented by treatment of the plants with an ethylene pathway inhibitor. Taken together, this reveals a novel role for TCP5‐like TFs in the regulation of ethylene‐mediated petal development and growth.

Correction to: Somatic embryogenesis of a seedless sweet orange (Citrus sinensis (L.) Osbeck)

In the original article Fig. 2 was not processed correctly.

Post-seminal development and morphoanatomy of vegetative and reproductive organs in Stevia rebaudiana (Bert.) Bertoni (Asteraceae)

Stevia rebaudiana belongs to the Asteraceae family with high economic and medicinal potential. This article describes and illustrates morphological and histological aspects of leaves and reproductive organs, and the germination process, to provide detailed information on this species and to contribute to taxonomic, phylogenetic and pharmacobotanical projects. The fruit is a cypsela, small, simple, dry, indehiscent, monospermic, light or dark colored, with aristate pappus, and the seed presents a spatulate axile embryo. Germination is phaneroepigeal with a pivotal root system and many absorbing root hairs. The leaves are simple, elliptical to obovate, with two types of trichomes (glandular and tector), with a short petiole, exhibiting an opposite decussate phyllotaxy. Our results showed 37.5% germination after 12 days, only in the dark cypsela, the light colored being considered unviable. The inflorescence is paniculate and the florets are grouped in capitula with isomorphic ends, monoclinous (bisexual), dichlamydeous, heterochlamydeous, pentamerous calyx and corolla, gamossepalous and gamopetalous. The androecium is gamostemone comprised of five stamens with free filaments, isodynamous and epipetalous stamens, synandrous and rimose anthers. The flower presents an inferior ovary, bicarpelar, unilocular and ovules with a basal placentation. The pollen grains are small, isopolar, radial symmetry, tricolporate, with echinate ornamentation.