Somatic Embryogenesis of Brachiaria brizantha (Syn. Urochloa brizantha) Analyzed by In Situ Hybridization.

In situ hybridization with mRNA probes enables the detection and localization of gene expression in plant somatic embryogenesis samples. BbrizSERK is a gene that is expressed in embryogenic cells and tissues of Brachiaria. Here we describe methods used for in situ hybridization to localize BbrizSERK transcripts during somatic embryogenesis of Brachiaria brizantha according to the plant material and observations intended, using paraffin or butyl methyl methacrylate resin-embedded samples, as well as a method for whole-mount preparation applicable for the analysis of other genes involved in embryogenic processes, along with other in vitro processes.

Cell type-specific dynamics underlie cellular growth variability in plants.

Coordination of growth, patterning and differentiation is required for shaping organs in multicellular organisms. In plants, cell growth is controlled by positional information, yet the behavior of individual cells is often highly heterogeneous. The origin of this variability is still unclear. Using time-lapse imaging, we determined the source and relevance of cellular growth variability in developing organs of Arabidopsis thaliana. We show that growth is more heterogeneous in the leaf blade than in the midrib and petiole, correlating with higher local differences in growth rates between neighboring cells in the blade. This local growth variability coincides with developing stomata. Stomatal lineages follow a specific, time-dependent growth program that is different from that of their surroundings. Quantification of cellular dynamics in the leaves of a mutant lacking stomata, as well as analysis of floral organs, supports the idea that growth variability is mainly driven by stomata differentiation. Thus, the cell-autonomous behavior of specialized cells is the main source of local growth variability in otherwise homogeneously growing tissue. Those growth differences are buffered by the immediate neighbors of stomata and trichomes to achieve robust organ shapes.

Using positional information to provide context for biological image analysis with MorphoGraphX 2.0.

Positional information is a central concept in developmental biology. In developing organs, positional information can be idealized as a local coordinate system that arises from morphogen gradients controlled by organizers at key locations. This offers a plausible mechanism for the integration of the molecular networks operating in individual cells into the spatially coordinated multicellular responses necessary for the organization of emergent forms. Understanding how positional cues guide morphogenesis requires the quantification of gene expression and growth dynamics in the context of their underlying coordinate systems. Here, we present recent advances in the MorphoGraphX software (Barbier de Reuille et al., 2015⁠) that implement a generalized framework to annotate developing organs with local coordinate systems. These coordinate systems introduce an organ-centric spatial context to microscopy data, allowing gene expression and growth to be quantified and compared in the context of the positional information thought to control them.

The TCP transcription factor HvTB2 heterodimerizes with VRS5 and controls spike architecture in barley.

KEY MESSAGE: Understanding the molecular network, including protein-protein interactions, of VRS5 provide new routes towards the identification of other key regulators of plant architecture in barley. The TCP transcriptional regulator TEOSINTE BRANCHED 1 (TB1) is a key regulator of plant architecture. In barley, an important cereal crop, HvTB1 (also referred to as VULGARE SIX-ROWED spike (VRS) 5), inhibits the outgrowth of side shoots, or tillers, and grains. Despite its key role in barley development, there is limited knowledge on the molecular network that is utilized by VRS5. In this work, we performed protein-protein interaction studies of VRS5. Our analysis shows that VRS5 potentially interacts with a diverse set of proteins, including other class II TCP's, NF-Y TF, but also chromatin remodelers. Zooming in on the interaction capacity of VRS5 with other TCP TFs shows that VRS5 preferably interacts with other class II TCP TFs in the TB1 clade. Induced mutagenesis through CRISPR-Cas of one of the putative VRS5 interactors, HvTB2 (also referred to as COMPOSITUM 1 and BRANCHED AND INDETERMINATE SPIKELET 1), resulted in plants that have lost their characteristic unbranched spike architecture. More specifically, hvtb2 mutants exhibited branches arising at the main spike, suggesting that HvTB2 acts as inhibitor of branching. Our protein-protein interaction studies of VRS5 resulted in the identification of HvTB2 as putative interactor of VRS5, another key regulator of spike architecture in barley. The study presented here provides a first step to underpin the protein-protein interactome of VRS5 and to identify other, yet unknown, key regulators of barley plant architecture.

Live-imaging provides an atlas of cellular growth dynamics in the stamen.

Development of multicellular organisms is a complex process involving precise coordination of growth among individual cells. Understanding organogenesis requires measurements of cellular behaviors over space and time. In plants, such a quantitative approach has been successfully used to dissect organ development in both leaves and external floral organs, such as sepals. However, the observation of floral reproductive organs is hampered as they develop inside tightly closed floral buds, and are therefore difficult to access for imaging. We developed a confocal time-lapse imaging method, applied here to Arabidopsis (Arabidopsis thaliana), which allows full quantitative characterization of the development of stamens, the male reproductive organs. Our lineage tracing reveals the early specification of the filament and the anther. Formation of the anther lobes is associated with a temporal increase of growth at the lobe surface that correlates with intensive growth of the developing locule. Filament development is very dynamic and passes through three distinct phases: (1) initial intense, anisotropic growth, and high cell proliferation; (2) restriction of growth and proliferation to the filament proximal region; and (3) resumption of intense and anisotropic growth, displaced to the distal portion of the filament, without cell proliferation. This quantitative atlas of cellular growth dynamics provides a solid framework for future studies into stamen development.

Replacement of Soybean Meal With Soybean Cake Reduces Methane Emissions in Dairy Cows and an Assessment of a Face-Mask Technique for Methane Measurement.

The objective of this study was to (a) evaluate the effect of replacing soybean meal (SBM) with soybean cake (SBC) on feeding behavior, rumen fermentation, milk production, nutrient digestibility and CH4 emissions and (b) investigate whether a face-mask technique could be used to predict daily methane (CH4) emissions in dairy cattle. The experiment was conducted as a completely randomized design, with 32 crossbred Holstein × Gyr cows (days in milk (DIM): 112 ± 25.1) randomly assigned to the following treatments (n = 8/group) for 75 days: (1) 0% SBC, (2) 6% SBC, (3) 14% SBC, and (4) 23% SBC, in place of SBM on a dry matter (DM) basis. Across the final 4 weeks of the study, CH4 production was estimated using the proposed face-mask technique subsequent to a respiration chamber measurement for an evaluation of treatment efficacy and face-mask accuracy. There was no effect of SBM replacement by SBC on intake, feeding or drinking behavior (P > 0.21). Total VFA concentration, the individual proportions of VFA and blood metabolites were not altered (P > 0.17) by SBC, however there was a tendency for decreased (P = 0.08) lactate and plasma urea nitrogen (P = 0.07) concentration associated with SBC addition. Fat-corrected milk yield (FCM4%) and composition was not affected (P > 0.27) by SBC; however, there was a tendency for decreased total milk solids (P = 0.07) and milk fat (P = 0.08) associated with 23% SBC treatment. There was no treatment × technique interaction (P > 0.05) effect on gas measurements. A maximum reduction (P = 0.01) in CH4 yield (g/kg DM) and intensity (g/kg milk) of 11 and 20%, respectively, was observed for the 14% SBC inclusion. Compared to the week of mask measurements, chambers decreased (P = 0.01) intake (kg/d, %BW) and increased (P = 0.05) FCM4%. The face-mask method over estimated O2 consumption by 5%. The face-mask method accurately predicted daily CH4 emissions when compared to the chamber at the same time-point. However, there was a linear bias of CH4 outputs so further evaluation of the calculation of total CH4 from a spot measurement is required.

Comprehensive phenotyping reveals interactions and functions of Arabidopsis thaliana TCP genes in yield determination.

Members of the Arabidopsis thaliana TCP transcription factor (TF) family affect plant growth and development. We systematically quantified the effect of mutagenizing single or multiple TCP TFs and how altered vegetative growth or branching influences final seed yield. We monitored rosette growth over time and branching patterns and seed yield characteristics at the end of the lifecycle. Subsequently, an approach was developed to disentangle vegetative growth and to determine possible effects on seed yield. Analysis of growth parameters showed all investigated tcp mutants to be affected in certain growth aspects compared with wild-type plants, highlighting the importance of TCP TFs in plant development. Furthermore, we found evidence that all class II TCPs are involved in axillary branch outgrowth, either as inhibitors (BRANCHED-like genes) or enhancers (JAW- and TCP5-like genes). Comprehensive phenotyping of plants mutant for single or multiple TCP TFs reveals that the proposed opposite functions of class I and class II TCPs in plant growth needs revision and shows complex interactions between closely related TCP genes instead of full genetic redundancy. In various instances, the alterations in vegetative growth or in branching patterns result into negative trade-off effects on seed yield that were missed in previous studies, showing the importance of comprehensive and quantitative phenotyping.

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

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.

Tithonia diversifolia as a Supplementary Feed for Dairy Cows.

The objective of this study was to examine the effects of Tithonia diversifolia as a supplementary forage on dairy cow performance and methane production. Nine lactating Holstein × Zebu dairy cows (519 ± 53.3 kg of body weight and 66 ± 13.3 d in milk) were paired by milk yield (21.3 ± 2.34 kg/d) and body weight and randomly assigned to three dietary treatments in a Latin square design with 21-d experimental periods (14 d for diet adaptation and 7 d for measurements and sample collection). The dietary treatments included the control diet consisting of fresh sugar cane plus concentrate (44:56, % of diet DM), and two treatment diets containing different levels of fresh T. diversifolia (6.5 and 15.4%, DM basis) which partially replaced both sugarcane and concentrates. Methane production was measured using the sulphur hexafluoride (SF6) technique from d 16 to d 21 of each experimental period. Analysis of the gas samples was performed by gas chromatography. The inclusion of T. diversifolia at 15.4% DM had no effects on DM intake, milk production, nitrogen balance or methane production. There was no effect on the concentrations of total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) in milk fat (P ≥ 0.28), though individual milk fatty acids were affected. Serum concentrations of glucose, urea nitrogen (BUN), triglycerides, ß-hydroxybutyrate (BHBA), and cholesterol were unaffected by the dietary treatments (P ≥ 0.13). There was a time (2 and 6 h post-feeding) and dietary treatment effect (P < 0.01) on the acetate to propionate ratio in the rumen. A denaturing gradient gel electrophoresis analysis of the archaeal community showed distinct clustering of the archaea populations for control and treatment diets. Taken together, our results indicate the potential of T. diversifolia as a supplementary forage for dairy cattle in the tropics.

Productivity and nutritional quality of Flechinha grass ( Echinolaena inflexa ), native grass of Brazilian Cerrado / Produção e qualidade nutricional do capim-flechinha (Echinolaena inflexa), gramínea nativa do Cerrado brasileiro

ABSTRACT: Due to scarce nutritional data, this study assessed the productivity and nutritional value of Echinolaena inflexa (EI) grass, native to the Cerrado biome. It was compared to B. brizantha (BB), one of the most cultivated grasses in Brazil, during a whole year (rainy; RS and dry season; DS). Sampling was held in accordance with pasture management (entry / exit height; 50 / 5cm and 80 / 25cm for EI and BB, respectively). Dry matter production (DMP), crude protein (CP), neutral and acid detergent fiber (NDF; ADF), hemicellulose (HCEL), PB insoluble neutral and acid detergent (PIDN; PIDA), total and non-fibrous carbohydrates (TC; NFC), ether extract (EE), and mineral matter (MM), and in vitro fermentation kinetics and DM degradability (DMD) were evaluated. A completely randomized design (season as a fixed term) and average treatment compared by Tukey post test were applied. EI produced 38.5% of the DMP of BB. A higher CP (75.3; 73.5 in the RS and DS), PIDA (12.5; 8.7), PIDN (47.1; 40.1), NDF (714.4; 749.5) and ADF (396.0; 419.0) were obtained by EI in relation to BB (CP (60.3; 33.5), PIDA (6.0; 3.5), PIDN (21.4; 10.8), NDF (673.0; 675.1) and ADF (335.5; 351.4) during the RS and DS, respectively (g kg-1 DM). In vitro data were directly associated with chemical composition, resulting in lower DMD of EI compared to BB. EI showed productive similarity (DMP) during RS and DS (939.3; 809.8kg DM respectively). Although EI showed greater nutritional stability (CP) between seasons, 17% of CP was linked to ADF and therefore, not available for rumen microorganisms.

RESUMO: Existem escassas informações a respeito da qualidade nutricional e potencial produtivo de Echinolaena Inflexa, planta nativa do Cerrado Brasileiro e que possui potencial valor forrageiro. Dessa forma, este trabalho objetivou avaliar o seu valor nutritivo e produtividade, comparando-a com a B. brizantha (BB), uma das mais cultivadas gramíneas no Brasil, durante um no agrícola (Estação das Chuvas; CH e seca; SE). A coleta do material experimental foi realizada de acordo com as recomendações de manejo (altura de entrada / altura de saída 50/5cm e 80/25cm para EI e BB, respectivamente). Foram avaliadas a produção de matéria seca (PMS), proteína bruta (PB), fibra detergente neutro e ácido (FDN e FDA), hemicelulose (HCEL), PB insolúvel em detergente neutro e ácido (PIDN e PIDA), carboidratos totais e não fibrosos (CT e CNF), extrato etéreo (EE) e matéria mineral (MM), e a cinética de fermentação ruminal e degradabilidade in vitro da MS (DMS). Utilizou-se o delineamento inteiramente casualizado e as médias de tratamento comparadas pelo post test de Tukey. A EI apresentou 38,5% da PMS da BB. Foram obtidos maiores teores de PB (75,3 e 73,5), PIDA (12,5 e 8,7), PIDN (47,1 e 40,1), FDN (714,4 e 749,5) e FDA (396,0 e 419,0) para a EI em relação a BB (PB; 60,3 e 33,5), PIDA (6,0 e 3.5), PIDN (21,4 e 10,8), FDN (673,0 e 675,1) e FDA (335,5 e 351,4) na estação chuvosa e seca, respectivamente (g kg-1 MS). Os dados in vitro da EI foram diretamente associados à composição química, resultando em menor DMS comparativamente BB. A EI apresentou estabilidade produtiva (PMS) durante a estação chuvosa e seca (939,3 e 809,8kg MS), respectivamente. Apesar da similaridade nutricional (PB) nas estações, cerca de 17% da PB está ligada a FDA e, portanto, não disponível para os microrganismos ruminais.

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.

Transcriptome analysis of Gossypium hirsutum flower buds infested by cotton boll weevil (Anthonomus grandis) larvae.

BACKGROUND: Cotton is a major fibre crop grown worldwide that suffers extensive damage from chewing insects, including the cotton boll weevil larvae (Anthonomus grandis). Transcriptome analysis was performed to understand the molecular interactions between Gossypium hirsutum L. and cotton boll weevil larvae. The Illumina HiSeq 2000 platform was used to sequence the transcriptome of cotton flower buds infested with boll weevil larvae. RESULTS: The analysis generated a total of 327,489,418 sequence reads that were aligned to the G. hirsutum reference transcriptome. The total number of expressed genes was over 21,697 per sample with an average length of 1,063 bp. The DEGseq analysis identified 443 differentially expressed genes (DEG) in cotton flower buds infected with boll weevil larvae. Among them, 402 (90.7%) were up-regulated, 41 (9.3%) were down-regulated and 432 (97.5%) were identified as orthologues of A. thaliana genes using Blastx. Mapman analysis of DEG indicated that many genes were involved in the biotic stress response spanning a range of functions, from a gene encoding a receptor-like kinase to genes involved in triggering defensive responses such as MAPK, transcription factors (WRKY and ERF) and signalling by ethylene (ET) and jasmonic acid (JA) hormones. Furthermore, the spatial expression pattern of 32 of the genes responsive to boll weevil larvae feeding was determined by "in situ" qPCR analysis from RNA isolated from two flower structures, the stamen and the carpel, by laser microdissection (LMD). CONCLUSION: A large number of cotton transcripts were significantly altered upon infestation by larvae. Among the changes in gene expression, we highlighted the transcription of receptors/sensors that recognise chitin or insect oral secretions; the altered regulation of transcripts encoding enzymes related to kinase cascades, transcription factors, Ca2+ influxes, and reactive oxygen species; and the modulation of transcripts encoding enzymes from phytohormone signalling pathways. These data will aid in the selection of target genes to genetically engineer cotton to control the cotton boll weevil.