Somatic Embryogenesis in Conifers: One Clade to Rule Them All?

Somatic embryogenesis (SE) in conifers is usually characterized as a multi-step process starting with the development of proembryogenic cell masses and followed by histodifferentiation, somatic embryo development, maturation, desiccation, and plant regeneration. Our current understanding of conifers' SE is mainly derived from studies using Pinaceae species as a model. However, the evolutionary relationships between conifers are not clear. Some hypotheses consider conifers as a paraphyletic group and Gnetales as a closely related clade. In this review, we used an integrated approach in order to cover the advances in knowledge on SE in conifers and Gnetales, discussing the state-of-the-art and shedding light on similarities and current bottlenecks. With this approach, we expect to be able to better understand the integration of these clades within current studies on SE. Finally, the points discussed raise an intriguing question: are non-Pinaceae conifers less prone to expressing embryogenic competence and generating somatic embryos as compared to Pinaceae species? The development of fundamental studies focused on this morphogenetic route in the coming years could be the key to finding a higher number of points in common between these species, allowing the success of the SE of one species to positively affect the success of another.

Enzymatic Antioxidant System Activation Assures the Viability of Guadua chacoensis (Bambusoideae, Poaceae) Embryogenic Cultures during Cryopreservation.

This study aimed to establish a cryopreservation protocol for G. chacoensis embryogenic cultures (ECs) and to investigate the role of antioxidant enzymes activities during cryopreservation. The growth dynamics of cell suspensions were also investigated, followed by a phytotoxicity test to assess the ECs' ability to tolerate the use of cryoprotective solutions for different incubation times (0, 30, 60, 120, and 240 min). We evaluated the EC redox state in three steps of cryopreservation: after incubation in cryoprotection solution, after thawing, and 60 days after regrowth. Our results showed that the ECs support the use of cryoprotective solution until 120 min, showing phytotoxic effects with 240 min of incubation. This study reports a 100% survival of the cultures and a 10% increase ratio in fresh material for both incubation times tested (60 and 120 min). Increased malonaldehyde content was identified after incubation in the cryoprotective solution. An increase in the activities of catalase and ascorbate peroxidase was also identified in the subsequent steps, suggesting that the activation of antioxidant enzymes is essential for maintaining cell homeostasis during cryopreservation.

Comparative proteomic analysis and antioxidant enzyme activity provide new insights into the embryogenic competence of Guadua chacoensis (Bambusoideae, Poaceae).

Somatic embryogenesis (SE) involves modifications of cellular, biochemical, genetic, and epigenetic patterns. Our work investigated proteins as markers of embryogenic response and characterized the redox state of embryogenic cultures (EC) of Guadua chacoensis. We identified a total of 855 proteins; 129 were up- and 136 down-accumulated in EC as compared with non-embryogenic culture (NEC). Additionally, 37 and 22 proteins were identified as unique in EC and NEC, respectively. Heat-shock proteins as unique proteins and increased activity in Superoxide Dismutase and Guaiacol Peroxidase in EC suggest that the embryogenic response requires activation of the stress response mechanism. Ribosomal, translational, and glycolytic proteins in EC seem to be associated with protein synthesis and energy sources for embryo development, respectively. Accumulation of cell wall-related proteins, such as Arabinogalactan and Polygalacturonase inhibitors, and signaling transduction proteins, including Chitinase, Phospholipase, and Guanine nucleotide-binding proteins in EC seems to be associated with embryogenic response. Enhancement of H2O2 content in EC compared to NEC suggests a possible role as a secondary messenger in SE. Altogether, the present study identified marker proteins of embryogenic response in G. chacoensis and revealed the activation of ROS scavenging enzymes to assure cell redox homeostasis and SE responses. SIGNIFICANCE: Somatic embryogenesis is a promising technique for the propagation and conservation of bamboo species; however, this route has been the least understood and studied until now. This study corresponds to the first work approaching proteomics complemented with biochemical analyses in the somatic embryogenesis of bamboo, bringing robust and precise information that can improve our understanding of this complex morphogenetic route.

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets.

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL-1 of L-glutamine, 1,050 mgL-1 of L-asparagine, 350 mgL-1 of L-arginine, and 35 mgL-1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.

Proteomic Profile of Glyphosate-Resistant Soybean under Combined Herbicide and Drought Stress Conditions.

While some genetically modified (GM) plants have been targeted to confer tolerance to abiotic stressors, transgenes are impacted by abiotic stressors, causing adverse effects on plant physiology and yield. However, routine safety analyses do not assess the response of GM plants under different environmental stress conditions. In the context of climate change, the combination of abiotic stressors is a reality in agroecosystems. Therefore, the aim of this study was to analyze the metabolic cost by assessing the proteomic profiles of GM soybean varieties under glyphosate spraying and water deficit conditions compared to their non-transgenic conventional counterparts. We found evidence of cumulative adverse effects that resulted in the reduction of enzymes involved in carbohydrate metabolism, along with the expression of amino acids and nitrogen metabolic enzymes. Ribosomal metabolism was significantly enriched, particularly the protein families associated with ribosomal complexes L5 and L18. The interaction network map showed that the affected module representing the ribosome pathway interacts strongly with other important proteins, such as the chloro-plastic gamma ATP synthase subunit. Combined, these findings provide clear evidence for increasing the metabolic costs of GM soybean plants in response to the accumulation of stress factors. First, alterations in the ribosome pathway indicate that the GM plant itself carries a metabolic burden associated with the biosynthesis of proteins as effects of genetic transformation. GM plants also showed an imbalance in energy demand and production under controlled conditions, which was increased under drought conditions. Identifying the consequences of altered metabolism related to the interaction between plant transgene stress responses allows us to understand the possible effects on the ecology and evolution of plants in the medium and long term and the potential interactions with other organisms when these organisms are released in the environment.

Plastid genome evolution in Amazonian açaí palm (Euterpe oleracea Mart.) and Atlantic forest açaí palm (Euterpe edulis Mart.).

KEY MESSAGE: The plastomes of E. edulis and E. oleracea revealed several molecular markers useful for genetic studies in natural populations and indicate specific evolutionary features determined by vicariant speciation. Arecaceae is a large and diverse family occurring in tropical and subtropical ecosystems worldwide. E. oleracea is a hyperdominant species of the Amazon forest, while E. edulis is a keystone species of the Atlantic forest. It has reported that E. edulis arose from vicariant speciation after the emergence of the belt barrier of dry environment (Cerrado and Caatinga biomes) between Amazon and Atlantic forests, isolating the E. edulis in the Atlantic forest. We sequenced the complete plastomes of E. edulis and E. oleracea and compared them concerning plastome structure, SSRs, tandem repeats, SNPs, indels, hotspots of nucleotide polymorphism, codon Ka/Ks ratios and RNA editing sites aiming to investigate evolutionary traits possibly affected by distinct environments. Our analyses revealed 303 SNPs, 91 indels, and 82 polymorphic SSRs among both species. Curiously, the narrow correlation among localization of repetitive sequences and indels strongly suggests that replication slippage is involved in plastid DNA mutations in Euterpe. Moreover, most non-synonymous substitutions represent amino acid variants in E. edulis that evolved specifically or in a convergent manner across the palm phylogeny. Amino acid variants observed in several plastid proteins in E. edulis were also identified as positive signatures across palm phylogeny. The higher incidence of specific amino acid changes in plastid genes of E. edulis in comparison with E. oleracea probably configures adaptive genetic variations determined by vicariant speciation. Our data indicate that the environment generates a selective pressure on the plastome making it more adapted to specific conditions.

Untargeted Proteomics-Based Approach to Investigate Unintended Changes in Genetically Modified Maize for Environmental Risk Assessment Purpose.

Profiling technologies, such as proteomics, allow the simultaneous measurement and comparison of thousands of plant components without prior knowledge of their identity. The combination of these non-targeted methods facilitates a more comprehensive approach than targeted methods and thus provides additional opportunities to identify genotypic changes resulting from genetic modification, including new allergens or toxins. The purpose of this study was to investigate unintended changes in GM Bt maize grown in South Africa. In the present study, we used bi-dimensional gel electrophoresis based on fluorescence staining, coupled with mass spectrometry in order to compare the proteome of the field-grown transgenic hybrid (MON810) and its near-isogenic counterpart. Proteomic data showed that energy metabolism and redox homeostasis were unequally modulated in GM Bt and non-GM maize variety samples. In addition, a potential allergenic protein-pathogenesis related protein -1 has been identified in our sample set. Our data shows that the GM variety is not substantially equivalent to its non-transgenic near-isogenic variety and further studies should be conducted in order to address the biological relevance and the potential risks of such changes. These finding highlight the suitability of unbiased profiling approaches to complement current GMO risk assessment practices worldwide.

Characterization of the complete plastid genome of Butia eriospatha (Arecaceae).

Butia eriospatha is an endemic palm species from the Atlantic Rainforest in Brazil, a biodiversity hotspot. This species is currently listed in the IUCN red list as vulnerable and lacks specific plastid markers for population genetics studies. In addition, the evolutionary relationship within the genus Butia is not yet well resolved. Here, we sequenced and characterized the complete plastid genome (plastome) sequence of B. eriospatha. The complete plastome sequence is 154,048 bp in length, with the typical quadripartite structure. This plastome length and genes content is consistent with other six species from tribe Cocoseae. However, the Inverted Repeat (IR) borders show some variation among the analyzed species from this tribe. Species from the Bactridinae (Astrocaryum and Acrocomia) and Elaeidinae (Elaeis) subtribes present the rps19 gene completely duplicated in the IR region. In contrast, all plastomes sequenced from the subtribe Attaleinae (Butia, Cocos, Syagrus) present one complete CDS of rps19 and one partial copy of rps19. The difference in the IR/LSC junctions between Attaleinae and the sister clades Bactridinae + Elaeidinae might be considered an evolutionary signal and the plastome sequence of B. eriopatha may be used in future studies of population genetics and phylogeny.

Molecular relationships of Campomanesia xanthocarpa within Myrtaceae based on the complete plastome sequence and on the plastid ycf2 gene.

Plastomes are very informative structures for comparative phylogenetic and evolutionary analyses. We sequenced and analyzed the complete plastome of Campomanesia xanthocarpa and compared its gene order, structure, and evolutionary characteristics within Myrtaceae. Analyzing 48 species of Myrtaceae, we identified six genes representing 'hotspots' of variability within the plastomes (ycf2, atpA, rpoC2, pcbE, ndhH and rps16), and performed phylogenetic analyses based on: (i) the ycf2 gene, (ii) all the six genes identified as 'hotspots' of variability, and (iii) the genes identified as 'hotspots' of variability, except the ycf2 gene. The structure, gene order, and gene content of the C. xanthocarpa plastome are similar to other Myrtaceae species. Phylogenetic analyses revealed the ycf2 gene as a promissing region for barcoding within this family, having also a robust phylogenetic signal. The synonymous and nonsynonymous substitution rates and the Ka/Ks ratio revealed low values for the ycf2 gene among C. xanthocarpa and the other 47 analyzed species of Myrtaceae, with moderate purifying selection acting on this gene. The average nucleotide identity (ANI) analysis of the whole plastomes produced phylogenetic trees supporting the monophyly of three Myrtaceae tribes. The findings of this study provide support for planning conservation, breeding, and biotechnological programs for this species.

Male sterility in Bambusa tuldoides Munro.

Despite their great economic importance, relatively little is known about bamboo sexual reproduction because they usually spread through rhizomes and have long intervals between flowering periods. Bambusa tuldoides is no exception; the intervals between flowering periods are about 23 years and often do not result in successful caryopsis production. The aim of the present work was to characterize Bambusa tuldoides sexual reproduction at three stages of flower development and investigate possible male sterility. Pollen was cultured onto several types of culture medium in order to encourage germination, but not a single of the thousands of observed pollen germinated under any condition. Anthers and microspores were analyzed by scanning electron microscopy, transmission electron microscopy, and optical microscopy techniques. Anther dehiscence appeared to be normal when compared to other species. In contrast, microspores began to develop abnormally starting as early as the first flower development stage: retraction of the cytoplasm and rupture of the nuclear and mitochondria membrane. As the interior machinery of the microspores degenerated, starch accumulated within numerous amyloplasts during stages two to four of flower development. The sporoderms of these microspores were similarly incomplete: though they possessed an exine, they lacked an intine. The results here obtained suggest that the non-viability of these abnormal pollen grains prevents the development of Bambusa tuldoides caryopses.

Structural aspects of cypsela and seed development of Trichocline catharinensis (Cabrera): a Brazilian endemic species.

This is the first study to describe in a timescale morphohistological and ultrastructural characteristics of fruit (cypsela) and seed development in Trichocline catharinensis, which was completed 21 days after anthesis (DAA). At anthesis, we identified an ovary with three differentiated regions, including the inner epidermis, inner part, and outer epidermis. The mature ovule showed an integument with the outer epidermis, integumentary parenchyma, and endothelium. Cells around the endothelium form the periendothelial zone with thick cell walls that showed Periodic acid-Schiff (PAS)-positive reaction. The periendothelial zone and endothelium showed degradation of the cells during embryogenesis. The main stages of embryo development from fecundation through mature seed were identified. The ripe cypsela showed the pericarp (exocarp), seed coat (exotesta), and remaining endosperm surrounding the embryo. Mature embryos were straight with shoot apical meristem (SAM), and root apical meristem (RAM) was separated by the hypocotyl. Light microscopy (LM) and transmission electron microscopy (TEM) analyses indicate cells with characteristics of meristem cells, as well as proteins and lipid bodies and mitochondria with few cristae in cotyledon cells. Our findings provide insight into taxonomic and physiological studies by detailing cypsela and seed ontogenesis from an endemic and vulnerable Asteraceae from southern Brazil. This study is also a starting point for establishing the biological criteria for seed harvesting and future studies of seed physiology and conservation of plant genetic resource.

A new method for determination of the photosynthetic pathway in grasses.

An easy and inexpensive method of determining the photosynthetic pathway in grasses using a dye widely used in microscopy. To evaluate the efficiency of a new histochemical test for determination of the photosynthetic pathway in grasses (Poacea). Leaves of 58 grass species were sectioned transversally, and the sections treated with a 2% sodium hypochlorite solution to clarify the tissue. After discoloration, sections were washed with distilled water and double-stained with astra blue and safranin (1% each in 50% ethanol) for 1 min. Sections were then mounted between microscopy glass slides and coverslips using water. Grass species showing red staining of the bundle sheath cells were considered C4, and species with translucent bundle sheath were considered C3. The results of the histochemical test were then compared with results from carbon isotope composition analysis and the relevant scientific literature. Observations from the histochemical test were congruent with results from δ13C isotope composition analysis, and with data previously presented in the scientific literature. The proposed histochemical test proved efficient for characterization of the photosynthetic pathway in the tested grasses; however, the method should be further tested in a greater number of grass species, encompassing, preferably, all Poacea subfamilies. Future studies may elucidate if the proposed method can effectively be used in other botanical families. Furthermore, additional investigations may determine whether the phenolic compounds indicated by the histochemical test are exclusive to the bundle sheath of C4 grasses and if possible relations exist between these phenolic compounds and the C4 photosynthetic pathway in grasses.

Genetic, evolutionary and phylogenetic aspects of the plastome of annatto (Bixa orellana L.), the Amazonian commercial species of natural dyes.

MAIN CONCLUSION: The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.

The Crambe abyssinica plastome: Brassicaceae phylogenomic analysis, evolution of RNA editing sites, hotspot and microsatellite characterization of the tribe Brassiceae.

Crambe abyssinica is an important oilseed crop that accumulates high levels of erucic acid, which is being recognized as a potential oil platform for several industrial purposes. It belongs to the family Brassicaceae, assigned within the tribe Brassiceae. Both family and tribe have been the subject of several phylogenetic studies, but the relationship between some lineages and genera remains unclear. Here, we report the complete sequencing and characterization of the C. abyssinica plastome. Plastome structure, gene order, and gene content of C. abyssinica are similar to other species of the family Brassicaceae. The only exception is the rps16 gene, which is absent in many genera within the family Brassicaceae, but seems to be functional in the tribe Brassiceae, including C. abyssinica. However, the analysis of gene divergence shows that the rps16 is the most divergent gene in C. abyssinica and within the tribe Brassiceae. In addition, species of the tribe Brassiceae also show similar SSR loci distribution, with some regions containing a high number of SSRs, which are located mainly at the single copy regions. Six hotspots of nucleotide divergence among Brassiceae species were located in the single copy regions by sliding window analysis. Brassicaceae phylogenomic analysis, based on the complete plastomes of 72 taxa, resulted in a well-supported and well-resolved tree. The genus Crambe is positioned within the Brassiceae clade together with the genera Brassica, Raphanus, Sinapis, Cakile, Orychophragmus and Sinalliaria. Moreover, we report several losses and gains of RNA editing sites that occurred in plastomes of Brassiceae species during evolution.

Determination of endogenous IAA and carbohydrates during the induction and development of protocorm-like bodies of Cattleya tigrina A. Richard / Determinação de AIA e carboidratos endógenos durante a indução e desenvolvimento deestruturas semelhantes a protocormos de Cattleya tigrina A. Richard

Tissue culture techniques have been employed for orchid mass propagation by means of themorphogenetic route of protocorm like-bodies (PLBs). This study aimed to analyze and compare Indoleaceticacid (IAA) and sugar endogenous levels in protocorm-like bodies (PLBs) induction and developmentin Cattleya tigrina, in order to better understand this process and to optimize micropropagation procedureprotocols. Leaves grown on MS (Murashige and Skoog) culture medium, suplemented with 9 μM TDZfor PLBs induction and development were collected after 0, 2, 7, 14, 30, 60 and 100 days of cultivation, forfurther analysis. Increase of IAA and reduction of sugar levels are strongly related to morphogeneticresponse, that is, PLBs formation over the preexisting ones and leaf primordia formation. Sucrose, fructoseand glucose presence in this study is related to cell signaling. Thus, hormonal signals and carbohydratesalter metabolism, triggering PLBs initiation and development in C. tigrina.

Técnicas de cultura de tecidos têm sido empregadas para a propagação em massa de orquídeaspor meio da via morfogenética de estruturas semelhantes a protocormos (ESPs). O estudo teve comoobjetivo analisar e comparar os níveis endógenos de ácido indol-3-acético (AIA) e de açúcares na indução eno desenvolvimento de estruturas semelhantes a protocormos (ESPs) em Cattleya tigrina, visando à melhorcompreensão deste processo e à otimização de protocolos de micropropagação. Folhas cultivadas em meiode cultura MS (Murashige & Skoog), suplementadas com 9 μM de TDZ para indução e desenvolvimentode ESPs foram coletadas após 0, 2, 7, 14, 30, 60 e 100 dias de cultivo, para posterior análise. O aumento dosníveis de AIA e a redução dos níveis de açúcar estão fortemente relacionados à resposta morfogenética, ouseja, à formação de ESPs sobre os pré-existentes e à formação de primórdios foliares. A presença desacarose, frutose e glicose neste estudo está relacionada à sinalização celular. Assim, sinais hormonais ecarboidratos alteram o metabolismo, desencadeando a iniciação e o desenvolvimento de ESPs em C. tigrina.

Effects of glutathione supplementation and carbon source duringsomatic embryogenesis of Acca sellowiana (O. Berg) Burret(Myrtaceae) / Efeitos da suplementação de glutationa e fontes de carbono durante a embriogênesesomática de Acca sellowiana (O. Berg) Burret (Myrtaceae)

The present study intended to investigate the effects of different glutathione (GSH) levels (0, 0.1, 0.5 and 1 mM) on the somatic embryogenesis (SE) induction of Acca sellowiana. Besides, we evaluated the effect of different carbon sources (sucrose and maltose) on the somatic embryos conversion. GSH-supplemented treatments resulted in improved SE induction rates (~70%) as compared to the control GSH-free (~35%) after 50 days of culture. The total number of somatic embryos obtained did not differ between treatments, but significant differences were observed for the embryonic stages after 80 days of culture. After 80 days of culture, 0.5 and 1 mM GSH-supplemented treatments showed the largest amount of torpedo-staged somatic embryos. In contrast, treatments supplemented with 0 and 0.1 mM GSH showed equal amounts of somatic embryos at all embryonic stages. These results indicate that GSH accelerates the SE induction process and increases the synchrony of the somatic embryo formation of A. sellowiana. The use of maltose for the somatic embryos conversion, as compared to sucrose, did not influence the conversion rate of normal chlorophyllous somatic embryos, but increased the formation of normal achlorophyllous somatic plantlets. This finding can be attributed to the rapid hydrolysis of sucrose, contributing to an enhanced chlorophyll synthesis.

O presente estudo teve como objetivo investigar o efeito de diferentes níveis de glutationa (GSH) (0, 0,1, 0,5 e 1 mM) na indução da embriogênese somática (ES) de Acca sellowiana. Além disso, avaliamos o efeito de diferentes fontes de carbono (sacarose e maltose) na conversão de embriões somáticos em plântulas. Os tratamentos suplementados com GSH resultaram em melhores taxas de indução de ES (~70%) em comparação com o controle isento de GSH (~35%) após 50 dias de cultivo. Após 80 dias as taxas de indução foram iguais. O número total de embriões somáticos obtidos não diferiu entre os tratamentos, mas diferenças expressivas foram observadas nos estágios embrionários. No dia 80 em cultura, os tratamentos suplementados com 0,5 e 1 mM de GSH mostraram a maior porção de embriões somáticos no estádio torpedo. Diferentemente, tratamentos suplementados com 0 e 0,1 mM de GSH mostraram quantidades iguais de embriões somáticos em todos os estágios embrionários. Estes resultados indicam que o GSH acelera o processo de indução do ES e aumenta a sincronia na formação de embriões somáticos de A. sellowiana. O uso de maltose no meio de cultura de conversão de embriões somáticos, em comparação com a sacarose, não influenciou a taxa de conversão de embriões somáticos clorofílados normais, mas aumentou a formação de plântulas aclorofiladas normais. Esse resultado pode ser atribuído à rápida hidrólise da sacarose, apresentando translocação de plantas mais eficiente e aumento da osmolaridade do meio de cultura, contribuindo para uma síntese melhorada de clorofila.

Effect of Thermal Stress on Tissue Ultrastructure and Metabolite Profiles During Initiation of Radiata Pine Somatic Embryogenesis.

Climate change will inevitably lead to environmental variations, thus plant drought tolerance will be a determinant factor in the success of plantations and natural forestry recovery. Some metabolites, such as soluble carbohydrates and amino acids, have been described as being the key to both embryogenesis efficiency and abiotic stress response, contributing to phenotypic plasticity and the adaptive capacity of plants. For this reason, our main objectives were to evaluate if the temperature during embryonal mass initiation in radiata pine was critical to the success of somatic embryogenesis, to alter the morphological and ultrastructural organization of embryonal masses at cellular level and to modify the carbohydrate, protein, or amino acid contents. The first SE initiation experiments were carried out at moderate and high temperatures for periods of different durations prior to transfer to the control temperature of 23°C. Cultures initiated at moderate temperatures (30°C, 4 weeks and 40°C, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50°C, 5 min). No significant differences were observed either for the percentage of embryogenic cell lines that produced somatic embryos, or for the number of somatic embryos per gram of embryonal mass. Based on the results from the first experiments, initiation was carried out at 40°C 4 h; 50°C, 30 min; and a pulse treatment of 60°C, 5 min. No significant differences were found for the initiation or number of established lines or for the maturation of somatic embryos. However, large morphological differences were observed in the mature somatic embryos. At the same time, changes observed at cellular level suggested that strong heat shock treatments may trigger the programmed cell death of embryogenic cells, leading to an early loss of embryogenic potential, and the formation of supernumerary suspensor cells. Finally, among all the differences observed in the metabolic profile, it is worth highlighting the accumulation of tyrosine and isoleucine, both amino acids involved in the synthesis of abiotic stress response-related secondary metabolites.

The Linum usitatissimum L. plastome reveals atypical structural evolution, new editing sites, and the phylogenetic position of Linaceae within Malpighiales.

KEY MESSAGE: The plastome of Linum usitatissimum was completely sequenced allowing analyses of evolution of genome structure, RNA editing sites, molecular markers, and indicating the position of Linaceae within Malpighiales. Flax (Linum usitatissimum L.) is an economically important crop used as food, feed, and industrial feedstock. It belongs to the Linaceae family, which is noted by high morphological and ecological diversity. Here, we reported the complete sequence of flax plastome, the first species within Linaceae family to have the plastome sequenced, assembled and characterized in detail. The plastome of flax is a circular DNA molecule of 156,721 bp with a typical quadripartite structure including two IRs of 31,990 bp separating the LSC of 81,767 bp and the SSC of 10,974 bp. It shows two expansion events from IRB to LSC and from IRB to SSC, and a contraction event in the IRA-LSC junction, which changed significantly the size and the gene content of LSC, SSC and IRs. We identified 109 unique genes and 2 pseudogenes (rpl23 and ndhF). The plastome lost the conserved introns of clpP gene and the complete sequence of rps16 gene. The clpP, ycf1, and ycf2 genes show high nucleotide and aminoacid divergence, but they still possibly retain the functionality. Moreover, we also identified 176 SSRs, 20 tandem repeats, and 39 dispersed repeats. We predicted in 18 genes a total of 53 RNA editing sites of which 32 were not found before in other species. The phylogenetic inference based on 63 plastid protein-coding genes of 38 taxa supports three major clades within Malpighiales order. One of these clades has flax (Linaceae) sister to Chrysobalanaceae family, differing from earlier studies that included Linaceae into the euphorbioid clade.

Dynamics in global DNA methylation and endogenous polyamine levels during protocorm-like bodies induction of Cattleya tigrina A. Richard / Dinâmica da metilação do DNA global e níveis endógenos de poliaminas durante a indução de estruturas semelhantes a protocormos de Cattleya tigrina A. Richard

Cattleya tigrina is endemic to the Atlantic forest biome and classified as vulnerable in the Red Book of Brazilian Flora. In vitro techniques comprise valuable tools for the conservation of endangered plant species. The aim of the present study was to evaluate the morphological features, global DNA methylation levels and free polyamines during protocorm- like bodies (PLBs) induction of C. tigrina. Along with that, an efficient protocol for in vitro propagation of this species is proposed. The first evidences of PLBs induction in C. tigrina occurred at seven days in culture, starting from the basal portion of the leaf abaxial surface. A hypomethylation marked the beginning of cell differentiation, followed by an increased global DNA methylation at 35 days in culture, coinciding with a subtle change in the structures morphogenetic development. During PLBs induction, putrescine exhibited higher levels as compared to spermidine and spermine, and apparently presents a major role during the PLBs induction in C. tigrina. Due to the apparent secondary PLBs formation, this protocol can represent a highly efficient method for in vitro propagation of this species.

Cattleya tigrina é uma espécie endêmica do bioma Mata Atlântica e classificada como vulnerável no Livro Vermelho da Flora Brasileira. As técnicas in vitro compreendem ferramentas valiosas a serem empregadas na conservação de espécies de plantas ameaçadas. O objetivo do presente estudo foi avaliar as características morfológicas, os níveis globais de metilação do DNA e as poliaminas livres durante a indução de estruturas semelhantes a protocormos (ESPs). Paralelamente, um protocolo eficiente para a propagação in vitro desta espécie é apresentado. As primeiras evidências de indução de ESPs em C. tigrina foram observadas aos sete dias de cultivo, a partir da porção basal da superfície abaxial da folha. Uma hipometilação foi observada concomitante ao início da diferenciação celular, e um aumento da metilação global do DNA foi encontrada aos 35 dias de cultivo, coincidindo com uma sutil mudança no desenvolvimento morfogenético das estruturas. Durante a indução de ESPs, a putrescina exibiu níveis aumentados em comparação a espermidina e espermina e, aparentemente, apresenta um papel importante durante a indução dessas estruturas em C. tigrina. Devido à aparente formação secundária de ESPs, este protocolo pode representar um método altamente eficiente para a propagação in vitro desta espécie.