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Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon

Evelyn Jardim Oliveira, Andréa Dias Koehler, Diego Ismael Rocha, Lorena Melo Vieira, Marcos Vinícius Marques Pinheiro, Elyabe Monteiro de Matos, Ana Claudia Ferreira da Cruz, Thais Cristina Ribeiro da Silva, Francisco André Ossamu Tanaka, Fabio Tebaldi Silveira Nogueira, Wagner Campos Otoni
The wild grass species Brachypodium distachyon (L.) has been proposed as a new model for temperate grasses. Among the biotechnological tools already developed for the species, an efficient induction protocol of somatic embryogenesis (SE) using immature zygotic embryos has provided the basis for genetic transformation studies. However, a systematic work to better understanding the basic cellular and molecular mechanisms that underlie the SE process of this grass species is still missing. Here, we present new insights at the morpho-histological, histochemical, and molecular aspects of B. distachyon SE pathway. Somatic embryos arose from embryogenic callus formed by cells derived from the protodermal-dividing cells of the scutellum. These protodermal cells showed typical  meristematic features and high protein accumulation which were interpreted as the first observable steps towards the acquisition of a competent state. Starch content decreased along embryogenic callus differentiation supporting the idea that carbohydrate reserves are essential to morphogenetic processes. Interestingly, starch accumulation was also observed at late stages of SE process. Searches in databanks revealed three sequences available annotated as BdSERK, being two copies corresponding to SERK1 and one showing greater identity to SERK2. In silico analysis confirmed the presence of characteristic domains in a B. distachyon Somatic Embryogenesis Receptor Kinase genes candidates (BdSERKs), which suggests SERK functions are conserved in B. distachyon. In situ hybridization demonstrated the presence of transcripts of BdSERK1 in all development since globular until scutellar stages. The results reported in this study convey important information about the morphogenetic events in the embryogenic pathway which has been lacking in B. distachyon. This study also demonstrates that B. distachyon provides a useful model system for investigating the genetic regulation of SE in grass species. 

Genome editing as a tool to achieve the crop ideotype and de novo domestication of wild relatives: Case study in tomato

Agustin Zsögön; Tomas Cermak; Dan Voytas; Lázaro Eustáquio Pereira Peres
The ideotype is a theoretical model of an archetypal cultivated plant. Recent progress in genome editing is aiding the pursuit of this ideal in crop breeding. Breeding is relatively straightforward when the traits in question are monogenic in nature and show Mendelian inheritance. Conversely, traits with a diffuse, polygenic basis such as abiotic stress resistance are more difficult to harness. In recent years, many genes have been identified that are important for plant domestication and act by increasing yield, grain or fruit size or altering plant architecture. Here, we propose that (a) key monogenic traits whose physiology has been unveiled can be molecularly tailored to achieve the ideotype; and (b) wild relatives of crops harboring polygenic stress resistance genes or other traits of interest could be de novo domesticated by manipulating monogenic yield-related traits through state-of-the-art gene editing techniques. An overview of the genomic and physiological challenges in the world’s main staple crops is provided. We focus on tomato and its wild Solanum (section Lycopersicon) relatives as a suitable model for molecular design in the pursuit of the ideotype for elite cultivars and to test de novo domestication of wild relatives.

Are plant growth retardants a strategy to decrease lodging and increase yield of sunflower?

Marcia Eugenia Amaral Carvalho, Paulo Roberto de Camargo e Castro, Marcos Vinicius de Castro Ferraz Junior, Ana Carolina Cabrera Machado Mendes

One of the major disadvantages of sunflower cultivation is the increased plant height, making it prone to the lodging. The use of plant growth retardants can be an alternative strategy to reduce plant height; however, these compounds may affect productivity. The aim of this study was to evaluate the effects of plant growth retardants on sunflower development and yield. Four treatments were studied: 1- control; 2- gibberellic acid (GA) 10 mg L−1; 3- trinexapac-ethyl (TE) 5 mL L−1, and 4- maleic hidrazide (MH) 8 mL L−1. TE and MH decreased plant height (16.9 and 35.9%, respectively); however, only TE positively influenced capitulim diameter and dry mass (46.7 and 311%, when compared to control) at 60 days after planting (DAP). At 81 DAP, dry mass of capitulum did not differ among control and TE-treated plants. On the other hand, MH impaired diameter and dry mass of capitulum (92.9 and 74.7%, respectively). It can be concluded that the application of TE is a potential strategy to decrease lodging probability without affecting sunflower yield. Furthermore, although MH negatively affected sunflower development, its use on the crop cannot be excluded since other doses, frequencies and moment of application can be studied.


RNA interference as a gene silencing tool to control "Tuta absoluta" in tomato ("Solanum lycopersicum")

Roberto A. Camargo; Guilherme O. Barbosa; Isabella Presotto Possignolo; Lazaro Eustáquio Pereira Peres; Eric Lam; Joni E. Lima; Antonio Figueira; Henrique Marques-Souza
RNA interference (RNAi), a gene-silencing mechanism that involves providing doublestranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi.Weselected two target genes (Vacuolar ATPase-A and Arginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on "in planta-induced transient gene silencing" (PITGS), a well-established method for silencing plant genes, used here for the first time to deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes showed and 60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic `Micro-Tom' tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

Does anatoxin-a influence the physiology of 'Microcystis aeruginosa' and 'Acutodesmus acuminatus' under different light and nitrogen conditions?

Mathias Ahii Chia; Micheline Kézia Cordeiro-Araújo; Adriana Sturion Lorenzi; Maria do Carmo Bittencourt-Oliveira

Due to changing global climatic conditions, a lot of attention has been given to cyanobacteria and their bioactive secondary metabolites. These conditions are expected to increase the frequency of cyanobacterial blooms, and consequently, the concentrations of cyanotoxins in aquatic ecosystems. Unfortunately, there are very few studies that address the effects of cyanotoxins on the physiology of phytoplankton species under different environmental conditions. In the present study, we investigated the effect of the cyanotoxin anatoxin-a (ATX-A) on Microcystis aeruginosa (cyanobacteria) and Acutodesmus acuminatus (chlorophyta) under varying light and nitrogen conditions. Low light (LL) and nitrogen limitation (LN) resulted in significant cell density reduction of the two species, while the effect of ATX-A on M. aeruginosa was not significant. However, under normal (NN) and high nitrogen (HN) concentrations, exposure to ATX-A resulted in significantly (p < 0.05) lower cell density of A. acuminatus. Pigment content of M. aeruginosa significantly (p < 0.05) declined in the presence of ATX-A, regardless of the light condition. Under each light condition, exposure to ATX-A caused a reduction in total microcystin (MC) content of M. aeruginosa. The detected MC levels varied as a function of nitrogen and ATX-A concentrations. The production of reactive oxygen species (H2O2) and antioxidant enzyme activities of both species were significantly altered by ATX-A under different light and nitrogen conditions. Our results revealed that under different light and nitrogen conditions, the response of M. aeruginosa and A. acuminatus to ATX-A was variable, which demonstrated the need for different endpoints of environmental factors during ecotoxicological investigations.


Root growth restraint can be an acclimatory response to low pH and is associated with reduced cell mortality: a possible role of class III peroxidases and NADPH oxidases

J. P. Gracas; R. Ruiz-Romero; L. D. Figueiredo; L. Mattiello; Lázaro Eustáquio Pereira Peres; Victor Augusto Vitorello

Low pH (<5.0) can significantly decrease root growth but whether this is a direct effect of H+ or an active plant response is examined here. Tomato (Solanum lycopersicum cv Micro-Tom) roots were exposed directly or gradually to low pH through step-wise changes in pH over periods ranging from 4 to 24 h. Roots exposed gradually to pH 4.5 grew even less than those exposed directly, indicating a plant-coordinated response. Direct exposure to pH 4.0 suppressed root growth and caused high cell mortality, in contrast to roots exposed gradually, in which growth remained inhibited but cell viability was maintained. Total class III peroxidase activity increased significantly in all low pH treatments, but was not correlated with the observed differential responses. Use of the enzyme inhibitors salicylhydroxamic acid (SHAM) or diphenyleneiodonium chloride (DPI) suggest that peroxidase and, to a lesser extent, NADPH oxidase were required to prevent or reduce injury in all low pH treatments. However, a role for other enzymes, such as the alternative oxidase is also possible. The results with SHAM, but not DPI, were confirmed in tobacco BY-2 cells. Our results indicate that root growth inhibition from low pH can be part of an active plant response, and suggest that peroxidases may have a critical early role in reducing loss of cell viability and in the observed root growth constraint.


Allelopathic interactions between microcystin-producing and non-microcystin-producing cyanobacteria and green microalgae: implications for microcystins production

Maria do Carmo Bittencourt-Oliveira; Mathias Ahii Chia; Helton Soriano Bezerra de Oliveira; Micheline Kézia Cordeiro Araújo; Renato José Reis Molica; Carlos Tadeu Santos Dias
Most mixed culture studies on the allelopathic interactions between toxic and nontoxic cyanobacteria with phytoplankton species rarely investigate the role of microcystins (MC) production and regulation in the course of the studies. This study investigated the interactions between intact cells of toxic (Microcystis aeruginosa (Kützing) Kützing) and nontoxic (Microcystis panniformis Komárek et al.) cyanobacteria with those of green algae (Monoraphidium convolutum (Corda) Komárková-Legnerová and Scenedesmus acuminatus (Largerheim) Chodat) as well as the effects of their respective crude extracts (5 and 10 μg.L−1) on their growth under controlled conditions. M. aeruginosa and M. panniformis were able to significantly (p<0.05) inhibit the growth of the green algae with M. convolutum being the most affected. The green alga S. acuminatus in return was able to inhibit the growth of the both cyanobacteria. In response to the presence of a competing species in the growth medium, M. aeruginosa significantly increased itsMCproduction per cell with the progression of the experiment, having the highest concentration at the end of the experiment. On the other hand, the extracts of the cyanobacteria had no significant inhibitory effect on the green algal strains investigated, while those of the green algae also had significant inhibitory effect on the growth of M. aeruginosa. In conclusion, both cyanobacterial and green algal strains investigated were negatively affected by the presence of competing species. M. aeruginosa responded to the presence of green algae by increasing its MC production.
The green algal strains significantly inhibited the growth of M. aeruginosa.

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