Antonio Chalfun-Junior

ASYMMETRIC LEAVES2-LIKE1gene, a member of the AS2/LOB family, controls proximal–distal patterning in Arabidopsis petals

The formation and the development of the floral organs require an intercalate expression of organ-specific genes. At the same time, meristem-specific genes are repressed to complete the differentiation of the organs in the floral whorls. In an Arabidopsis activation tagging population, a mutant affected in inflorescence architecture was identified. This gain-of-function mutant, designateddownwards siliques1 (dsl1-D), has shorter internodes and the lateral organs such as flowers are bending downwards, similar to the loss-of-function brevipedicellus (bp) mutant. The affected gene in dsl1-D appeared to be ASYMMETRIC LEAVES2-LIKE1 (ASL1)/LATERAL ORGAN BOUNDARIESdomain gene 36 (LBD36), which is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. Analysis of the loss-of-function mutant asl1/lbd36 did not show morphological aberration. Double mutant analysis of asl1/lbd36 together with as2, the ASL1/LBD36 closest homologue, demonstrates that these two members of the AS2/LOB family act partially redundant to control cell fate determination in Arabidopsis petals. Moreover, molecular analysis revealed that overexpression of ASL1/LBD36 leads to repression of the homeobox gene BP, which supports the model that an antagonistic relationship between ASL/LBD and homeobox members is required for the differentiation of lateral organs.

Low frequency of T-DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences

A powerful system to create gain‐of‐function mutants in plants is activation tagging using T‐DNA based vehicles to introduce transcriptional enhancer sequences. Large Arabidopsis populations of individual plants carrying a quadruple cauliflower mosaic virus (CaMV) 35S enhancer are frequently used for mutant screenings, however the frequency of morphological mutants remains very low. To clarify this low frequency we analyzed a subset of lines generated by this method. The correlation between the number of T‐DNA insertion sites, the methylation status of the 35S enhancer sequence and 35S enhancer activity was determined. All plants containing more than a single T‐DNA insertion showed methylation of the 35S enhancer and revealed a dramatic decrease in 35S enhancer activity. The results support the notion that in a large proportion of the T‐DNA based activation tagged lines the 35S transcriptional enhancer is silenced due to methylation, which is induced by multiple T‐DNA integrations.

In Silico and Quantitative Analyses of MADS-Box Genes in Coffea arabica

MADS-box genes comprise a family of transcription factors that act as key regulators in many cellular development processes of several organisms. Members of this family have highly conserved regions and play important roles as transcription factors, activating target genes. The present work aimed at analyzing the MADS-box gene family present in a database generated by the Brazilian Coffee Genome Project (CAFEST) as well as of observing their respective sites of expression within these data. Through the use of bioinformatics tools, it was possible to identify and classify 26 expressed sequence tag contigs, 13 of them were expressed exclusively in vegetative tissues, 11 in reproductive tissues, and two in both. Later, quantitative analysis by quantitative reverse transcriptase PCR was carried out for three of them belonging to the groups of genes APETALA3 (B genes), AGAMOUS (C genes), and SEPALLATAS (E genes), which could be compared with the expression profile of in silico analysis and of its putative orthologous genes. Therefore, this study aims at a better understanding of the development processes performed by this family of genes in Coffea arabica, mainly in reproductive organs, as well as to compare functions of MADS-box orthologous studied in other species.

Putative sugarcane FT/TFL1 genes delay flowering time and alter reproductive architecture in Arabidopsis

Agriculturally important grasses such as rice, maize, and sugarcane are evolutionarily distant from Arabidopsis, yet some components of the floral induction process are highly conserved. Flowering in sugarcane is an important factor that negatively affects cane yield and reduces sugar/ethanol production from this important perennial bioenergy crop. Comparative studies have facilitated the identification and characterization of putative orthologs of key flowering time genes in sugarcane, a complex polyploid plant whose genome has yet to be sequenced completely. Using this approach we identified phosphatidylethanolamine-binding protein (PEBP) gene family members in sugarcane that are similar to the archetypical FT and TFL1 genes of Arabidopsis that play an essential role in controlling the transition from vegetative to reproductive growth. Expression analysis of ScTFL1, which falls into the TFL1-clade of floral repressors, showed transcripts in developing leaves surrounding the shoot apex but not at the apex itself. ScFT1 was detected in immature leaves and apical regions of vegetatively growing plants and, after the floral transition, expression also occurred in mature leaves. Ectopic over-expression of ScTFL1 in Arabidopsis caused delayed flowering in Arabidopsis, as might be expected for a gene related to TFL1. In addition, lines with the latest flowering phenotype exhibited aerial rosette formation. Unexpectedly, over-expression of ScFT1, which has greatest similarity to the florigen-encoding FT, also caused a delay in flowering. This preliminary analysis of divergent sugarcane FT and TFL1 gene family members from Saccharum spp. suggests that their expression patterns and roles in the floral transition has diverged from the predicted role of similar PEBP family members.

New Insights on Coffea miRNAs: Features and Evolutionary Conservation

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5′ RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.

Expression of genes involved in lipid metabolism in the muscle of beef cattle fed soybean or rumen-protected fat, with or without monensin supplementation.

Degree of unsaturation of fatty acids, which is influenced by lipid source and level of metabolism in the rumen, is a major determinant in how dietary lipids affect genes that regulate beef marbling. A total of 28 Red Norte bulls with an initial live weight of 361±32 kg (P>0.05) were used in a completely randomized experimental design to analyze the expression of genes that are involved in lipid metabolism in the longissimus dorsi (LD) when diets contained soybean grain or rumen-protected fat, with or without monensin. Treatments were arranged as a 2×2 factorial, with 4 treatments and 7 replicates per treatment. Half of the animals that received soybean or rumen-protected fat were supplemented with 230 mg head(-1) d(-1) of monensin. Gene expression was analyzed by reverse-transcription quantitative PCR (RT-qPCR). Expression of sterol regulatory element-binding protein-1c (SREBP-1c) in the LD muscle was not affected by lipid source or monensin (P>0.05). There was an interaction effect (P<0.05) between lipid source and monensin for peroxisome proliferator-activated receptor α (PPAR-α) and stearoyl-CoA desaturase (SCD) expression, where greater gene expression was found in animals fed soybean plus monensin and the lower gene expression was found in animals fed rumen-protected fat plus monensin. Expression of lipoprotein lipase (LPL) and fatty acid-binding protein 4 (FABP4) were greater (P<0.05) in the LD muscle of animals fed soybean. Monensin had no effect on LPL and FABP4 expression when soybean without monensin was fed, but when rumen-protected fat was fed, monensin increased LPL expression and decreased FABP4 expression (P<0.05). Linoleic and arachidonic acids had negative correlations (P<0.05) with the expression of PPAR-α, SCD, FABP4, and LPL genes. PPAR-α gene expression was not correlated with SREBP-1c but was positively correlated with SCD, FABP4, LPL, and glutathione peroxidase (GPX1) gene expression (P<0.001). Lipid sources and monensin interact and alter the expression of PPAR-α, SCD, acetyl CoA carboxylase α (ACACA), LPL, FABP4, and GPX1. These changes in gene expression were most associated with arachidonic and α-linolenic acids and the ability of lipid sources and monensin to increase these fatty acids in tissues.

Strategies to increase zinc deficiency tolerance and homeostasis in plants

Zinc deficiency is a global problem of considerable importance for agriculture and human health. Under zinc deficiency conditions, many essential zinc-dependent physiological functions are unable to operate normally, and the cellular homeostasis is adversely affected. This paper described the potential damages that low-zinc bioavailability in soil can have for plants, humans, and animals. In addition, current knowledge on physiological and molecular aspects of zinc homeostasis in plants and strategies used to increase zinc deficiency tolerance were discussed.

In Silico and Quantitative Analyses of the Putative FLC-like Homologue in Coffee ( Coffea arabica L.)

The sequential pattern of coffee flowering is a major constraint that directly affects productivity, increases harvest costs, and generates a final product of lower quality for mixing dry fruits with ripe and unripe ones. The objective of this work was to identify and analyze one of the main genes involved in flowering regulation, FLOWERING LOCUS C (FLC) in coffee (Coffea arabica L.). The identification of this gene was conducted in silico using a coffee EST database (CAFEST) and bioinformatics tools. Quantitative PCR results suggest that the identified CaFLC-like homologue is directly involved in flowering regulation in coffee. This expands our knowledge on evolutionary conservation of flowering pathways in dicot species. The functional studies of CaFLC-like with mutants of a more tractable species will lead to a better understanding of the molecular regulation as well as the specific functions of each gene flowering during floral induction in coffee.

Effects of 60 Hz sinusoidal magnetic field on in vitro establishment, multiplication, and acclimatization phases of Coffea arabica seedlings

The influence of extremely low frequency electromagnetic fields on net photosynthesis, transpiration, photosynthetic pigment concentration, and gene expression of ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS1), during in vitro establishment, in vitro multiplication and acclimatization phases of coffee seedlings were investigated. Untreated coffee plants were considered as control, whereas treated plants were exposed to a 60 Hz sinusoidal magnetic field of 2 mT of magnetic induction during 3 min. This magnetic field was generated by an electromagnet, connected to a wave generator. The results revealed that magnetically treated plants showed a significant increase in net photosynthesis (85.4% and 117.9%, in multiplication and acclimatization phases, respectively), and in photosynthetic pigment concentration (66.6% for establishment phase, 79.9% for multiplication phase, and 43.8% for acclimatization phase). They also showed a differential RBCS1 gene expression (approximately twofold) and a decrease of transpiration rates in regard to their control plants. In conclusion, the findings suggest that the application of 60 Hz magnetic field to in vitro coffee plants may improve the seedlings quality by modifying some photosynthetic physiological and molecular processes, increasing their vigor, and ensuring better plant development in later stages.

Validation of reference genes for qPCR analysis of Coffea arabica L. somatic embryogenesis-related tissues

Quantitative real-time PCR (qPCR) is a sensitive method used to investigate relevant changes in gene expression during somatic embryogenesis. Understanding its regulatory network might be helpful to the process of induction of embryos and facilitate the development of efficient plant regeneration procedures. In this study, a set of 12 genes was selected and their stability was assessed in different tissues of somatic embryogenesis-related cultures of Coffea arabica. Analyses of gene expression stability were performed using the RefFinder tool that integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms. Among the all candidate reference genes studied, APRT/EF1a, UBQ/ACT, ACT/24S, RPL39/24S, PP2A/RPL39, PP2A/AP47, emerged as the most stable for normalization of qPCR analyses of embryogenic cell suspensions, non-embryogenic calli, embryogenic calli, combined embryogenic and non-embryogenic calli, somatic embryos and plantlet, respectively. A combination of two genes, 24S and PP2A, was identified as most suitable reference genes across all samples for the C. arabica tissues studied. The commonly employed reference gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found to be inappropriate as a reference gene for embryogenic tissues of C. arabica. In addition, Baby boom (BBM) gene expression was investigated to confirm the validity of the selected reference genes, the transcript levels of gene were overestimated when unsuitable reference genes were used for normalization. The results shown herein will permit a more precise and reliable normalization of qPCR in experiments involving somatic embryogenesis of C. arabica.