Research papers

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Regenerative potential, metabolic profile, and genetic stability of 'Brachypodium distachyon' embryogenic calli as affected by successive subcultures

T. C. Mamedes-Rodrigues; D.S. Batista; N. M. Vieira; E. M. Matos; D. Fernandes; A. Nunes-Nesi; C. D. Cruz; L. F. Viccini; Fabio Tebaldi Silveira Nogueira; W. C. Otoni

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L−1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.

Potential human health risk assessment of cylindrospermopsin accumulation and depuration in lettuce and arugula

Micheline Kézia Cordeiro-Araújo, Mathias Ahii Chiab, Maria do Carmo Bittencourt-Oliveira

The cyanobacterial toxin cylindrospermopsin (CYN) has become a globally important secondary metabolite due to the negative effect it has on human and animal health. As a means of evaluating the risk of human exposure to CYN, the bioaccumulation and depuration of the toxin in lettuce (Lactuca sativa L.) and arugula (Eruca sativa Mill.) were investigated, after irrigation with contaminated water. The vegetables were irrigated for 7 days with CYN (3, 5 and 10 mg/L) contaminated water (bioaccumulation phase), and subsequently, irrigated for 7 days with uncontaminated distilled water (depuration phase). In general, the bioaccumulation of CYN in both vegetables decreased with increasing exposure concentration. Bioconcentration factor (BCF) of CYN increased with the progression of the experiment at 3.0 mg/L CYN, while the reverse occurred at 5 and 10 mg/L CYN. In arugula, BCF increased at all CYN exposure concentrations throughout the study. The depuration of CYN decreased with increasing exposure concentration but was highest in the plants of both species with the highest bioaccumulation of CYN. Specifically, in plants previously irrigated with water contaminated with 3, 5 and 10 mg/L CYN, the depuration of the toxin was 60.68, 27.67 and 18.52% for lettuce, and 47, 46.21 and 27.67% for arugula, respectively. Human health risks assessment revealed that the consumption of approximately 10 to 40 g of vegetables per meal will expose children and adults to 1.00-6.00 ng CYN/kg body mass for lettuce and 2.22-7.70 ng CYN/kg body mass for arugula. The irrigation of lettuce and arugula with contaminated water containing low CYN concentrations constitutes a potential human exposure route.

Diversifying growth forms in tropical forest restoration: Enrichment with vascular epiphytes

Marina Melo Duarte, Sergius Gandolfi

Ecological restoration of forests is largely favored by tree planting, often leaving out other important growth forms. Despite their relevant ecological roles, in restoration plantations, epiphytic richness rarely reaches values found in reference ecosystems. At the same time, epiphytes are wasted when forests are cleared for infrastructure projects, instead of being properly relocated. The goal of this study is to improve the knowledge for epiphytic relocation and enrichment, in restoration forests. We seek to answer the following questions: (i) Over a one year period, can six species of epiphytes survive, attach to phorophytes and reproduce, after being transplanted to host trees? (ii) Is epiphyte development after transplantation affected by species of phorophytes, bark roughness, canopy cover and position of transplantation? (iii) Is performance of relocated epiphytes species specific? For this purpose, 360 adult individuals of vascular epiphytes (Bromeliaceae, Cactaceae and Orchidaceae) were transplanted onto host trees located in two semi-deciduous seasonal forests in the Atlantic Forest of Sao Paulo, Brazil. Epiphytes achieved high survival rates after one year (55.2–100% of individuals) and all species presented structures for either sexual or asexual reproduction. Their overall development was enhanced when we carried out transplantations at the beginning of wet season and using sisal string to attach epiphytes and palm fiber to cover phorophyte’s bark, which were relevant factors attributing to the success of transplantations. Species of phorophyte was not an attributing factor to the successful development of transplanted individuals, which only showed slight responses to conditions they provided. However, responses among epiphytes were speciesspecific, demonstrating the importance of studying their biology in order to successfully enrich restoration forests.

Manipulation of a senescence-associated gene improves fleshy fruit yield

Bruno S. Lira, Giovanna Gramegna, Bruna Trench, Frederico Rocha Rodrigues Alves, Éder Marques Silva, Geraldo Felipe Ferreira Silva, Venkatesh Periyakavanam Thirumalaikumar, Alessandra Cavalcanti Duarte Lupi, Diego Demarco, Eduardo Purgatto, Fabio Tebaldi Silveira Nogueira, Salma Balazadeh, Luciano Freschi, Magdalena Rossi

Senescence is the process that marks the end of leaves lifespan. As it progresses, the massive macromolecular catabolism dismantles the chloroplasts and, consequently, decreases the photosynthetic capacity of these organs. Thus, senescence manipulation is a strategy to improve plant yield by extending the leaf photosynthetically active window of time. However, it remains to be addressed if this approach can improve fleshy fruit production and nutritional quality. One way to delay senescence initiation is by regulating key transcription factors (TFs) involved in triggering this process, such as the NAC TF ORESARA1 (ORE1). Here, three senescence-related NAC TFs from Solanum lycopersicum were identified, namely SlORE1S02, SlORE1S03 and SlORE1S06. All three genes showed to be responsive to senescence-inducing stimuli and post-transcriptionally regulated by the microRNA miR164. Moreover, the encoded proteins physically interacted with the chloroplast maintenance related TF SlGLKs. This characterization led to the selection of a putative tomato ORE1 as target gene for RNAi knockdown. Transgenic lines showed delayed senescence and enhanced carbon assimilation that, ultimately, increased the number of fruits and their total soluble solid content. Additionally, the fruit nutraceutical composition was enhanced. In conclusion, the data provide robust evidence that the manipulation of leaf senescence is an effective strategy for yield improvement in fleshy fruit-bearing species.

microRNA159-targeted SlGAMYB transcription factors are required for fruit set in tomato

Eder Marques da Silva, Geraldo Felipe Ferreira e Silva, Débora Brussolo Bidoia, Mariana da Silva Azevedo, Frederico Almeida de Jesus, Lilian Ellen Pino, Lázaro Eustáquio Pereira Peres, Esther Carrera, Isabel López-Díaz and Fabio Tebaldi Silveira Nogueira

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e., auxin and gibberelins) in controlling fruit set after pollination and fertilization, the role of microRNA-based regulation during ovary development and fruit set is still poorly understood. Here we show that microRNA159/GAMYB1 and -2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159-overexpressing tomato cv. Micro-Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159-overexpressing plants resulted in mis-regulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA-like genes and the miR167/SlARF8a module. Similarly to SlMIR159-overexpressing plants, SlGAMYB1 was down-regulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159-targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set

Effects of zooplankton and nutrients on phytoplankton: an experimental analysis in a eutrophic tropical reservoir

Juliana dos Santos Severiano, Viviane Lúcia dos Santos Almeida-Melo, Enaide Marinho de Melo-Magalhães, Maria do Carmo Bittencourt-Oliveira, Ariadne do Nascimento Moura

Experiments were conducted to evaluate the N : P ratio, as well as the effects of the interaction between this ratio and zooplankton, on phytoplankton in a tropical reservoir. Three experiments were performed in the presence (+Z) or absence (–Z) of zooplankton and the addition of N and P in different ratios (N : P molar ratio of 5, 16 and 60). In Experiment I, the total phytoplankton biomass and biomass by taxonomic class and species of the N : P 16–Z treatment did not differ significantly from that of the control, whereas for N : P 16+Z, there was a reduction in total phytoplankton. In Experiment II, there was a significant increase in Bacillariophyceae and the biomass of two species in the N : P 60–Z treatment. For the N : P 60+Z treatment, a significant reduction was observed in the total phytoplankton biomass and the biomass of three phytoplankton classes and three species. In Experiment III, there was an increase in the biomass of Dinophyceae with the N : P 5–Z treatment. In the N : P 5+Z treatment, there was a significant reduction in total phytoplankton biomass and the biomass of the phytoplankton class and five species. The findings of the present study reveal that zooplankton species native to a tropical reservoir can change the structure of the phytoplankton community and the response of these organisms to variations in nutrients.

Response of Microcystis aeruginosa BCCUSP 232 to barley ('Hordeum vulgare' L.) straw degradation extract and fractions

Gustavo Franciscatti Mecina, Anne Lígia Dokkedal, Luiz Leonardo Saldanha, Mathias Ahii Chia, Micheline Kézia Cordeiro-Araújo, Maria do Carmo Bittencourt-Oliveira, Regildo Márcio Gonçalves da Silva

The eutrophication of aquatic ecosystems is a serious environmental problemthat leads to increased frequency of cyanobacterial blooms and concentrations of cyanotoxins. These changes in aquatic chemistry can negatively affect animal and human health. Environment-friendly methods are needed to control bloom forming cyanobacteria. We investigated the effect of Hordeum vulgare L. (barley) straw degradation extract and its fractions on the growth, oxidative stress, antioxidant enzyme activities, and microcystins content of Microcystis aeruginosa (Kützing) Kützing BCCUSP232. Exposure to the extract significantly (p b 0.05) inhibited the growth of M. aeruginosa throughout the study, whereas only the highest concentration of fractions 1 and 2 significantly (p b 0.05) reduced the growth of the cyanobacterium on day 10 of the experiment. The production of reactive oxygen species (ROS), lipid peroxidation and antioxidant enzyme activities were significantly (p b 0.05) altered by the extract and fractions 1 and 2. Phytochemical profiling of the extract and its fractions revealed that the barley strawdegradation process yielded predominantly phenolic acids. These results demonstrate that barley straw extract and its fractions can efficiently interfere with the growth and development of M. aeruginosa under laboratory conditions.

Plant growth regulators on sweet sorghum: physiological and nutritional value analysis

Willian Rodrigues Macedo, Diego Kitahara Araújo, Valdinei Moreira Santos, Paulo Roberto de Camargo e Castro, Gisele Machado Fernandes

Sweet sorghum has gained attention in tropical and subtropical regions because of its potential as a bioenergy crop. The present research was carried out to evaluate the physiological, biochemical and nutritional characteristics of sweet sorghum submitted to six plant growth regulators (thiamethoxam, biostimulant mixture, gibberellic acid, chlormequat chloride, ethephon, and trinexapac-ethyl). The compounds were applied via foliar spraying in order to increase the productive potential of plants and reduce the carbohydrates sink strength by inflorescences. The experiment was conducted in pots and the following variables were evaluated: plant height, inflorescence dry matter, soluble solids content, shoot dry matter, crude protein, ashes, neutral detergent fiber and in vitro digestibility. It were observed the action of trinexapac-ethyl and ethephon to reduce the inflorescence dry matter, chlormequat chloride to increase the shoot dry matter, and trinexapac-ethyl to improve ashes content and reduce the neutral detergent fiber contents. These results indicate that chlormequat chloride and trinexapac-ethyl are effective in restricting the plant growth and increasing sorghum nutritional quality.

Loss of type-IV glandular trichomes is a heterochronic trait in tomatoand can be reverted by promoting juvenility

Eloisa Vendemiatti, Agustin Zsögön, Geraldo Felipe Ferreira e Silva,Frederico Almeida de Jesus, Lucas Cutri, Cassia Regina Fernandes Figueiredo,Francisco André Ossamu Tanaka, Fábio Tebaldi Silveira Nogueiraa,Lázaro Eustáquio Pereira Peres

Glandular trichomes are structures with widespread distribution and deep ecological significance. In the Solanum genus, type-IV glandular trichomes provide resistance to insect pests. The occurrence of thesestructures is, however, poorly described and controversial in cultivated tomato (Solanum lycopersicum).Optical and scanning electron microscopy were used to screen a series of well-known commercial tomatocultivars, revealing the presence of type-IV trichomes on embryonic (cotyledons) and juvenile leaves. Atomato line overexpressing the microRNA miR156, known to promote heterochronic development, andmutants affecting KNOX and CLAVATA3 genes possessed type-IV trichomes in adult leaves. A re-analysis ofthe Woolly (Wo) mutant, previously described as enhancing glandular trichome density, showed that thiseffect only occurs at the juvenile phase of vegetative development. Our results suggest the existence ofat least two levels of regulation of multicellular trichome formation in tomato: one enhancing differenttypes of trichomes, such as that controlled by the WOOLLY gene, and another dependent on develop-mental stage, which is fundamental for type-IV trichome formation. Their combined manipulation couldrepresent an avenue for biotechnological engineering of trichome development in plants.

Genetic and physiological characterization of three natural allelic variations affecting the organogenic capacity in tomato ('Solanum lycopersicum' cv. Micro-Tom)

Maísa de Siqueira Pinto, Chanaka Roshan Abeyratne, Vagner Augusto Benedito, Lázaro Eustáquio Pereira Peres

The study of allelic variations affecting organogenic capacity is not only relevant for manipulating plant traits but also to understand the fundamental mechanisms involved in plant development. Here, we report the characterization of three tomato (Solanum lycopersicum) loci (RG3C, RG7H and RG8F) whose alleles from its wild relative Solanum pennellii enhance in vitro shoot and root regeneration. S. pennellii alleles were introgressed into tomato cv. Micro-Tom (MT), creating near-isogenic lines. We evaluated the time taken for shoot induction and acquisition of competence by quantifying organogenesis after transferring explants, respectively, from the shoot-inducing medium (SIM) to the basal medium (BM) and from root-inducing medium (RIM) to the SIM. Concomitantly, we monitored the expression of key developmental genes. MT-Rg3C and MT-Rg7H started shoot induction, respectively, at 48 and 24 h earlier than MT and MT-Rg8F, while MT-Rg3C and MT-Rg8F acquired competence 24 h before MT. The impact of MT-Rg3C and MT-Rg8F in the acquisition of competence to assume different fates is consistent with their effect enhancing both shoot and root regeneration. MT-Rg7H seems to affect shoot induction specifically, which is in agreement with the enhanced expression of the shoot-related genes WUSCHEL and SHOOT MERISTEMLESS. Phenotypic characterization of greenhouse-grown plants showed that Rg3C has increased branching when compared to MT. Conversely, the normal branching observed in MT-Rg7H and MT-Rg8F indicates that adventitious in vitro shoot formation and ex vitro axillary bud formation/outgrowth are induced by different genetic pathways. These natural variations are thus useful for breeding highly regenerating varieties without undesirable effects on plant architecture.