Successful plant reproduction and fruit formation depend on adequate pollen and pistil developmen... more Successful plant reproduction and fruit formation depend on adequate pollen and pistil development, and pollen–pistil interactions. In Nicotiana tabacum, pollen tubes grow through the intercellular spaces of pistil-specialized tissues, stigmatic secretory zone, and stylar transmitting tissue (STT). These intercellular spaces are supposed to be formed by the modulation of cell wall pectin esterification. Previously we have identified a gene preferentially expressed in pistils encoding a putative pectin acetylesterase (PAE), named NtPAE1. Here, we characterized the NtPAE1 gene and performed genome-wide and phylogenetic analyses of PAEs. We identified 30 PAE sequences in the N. tabacum genome, distributed in four clades. The expression of NtPAE1 was assessed by RT-qPCR and in situ hybridization. We confirmed NtPAE1 preferential expression in stigmas/styles and ovaries and demonstrated its high expression in the STT. Structural predictions and comparisons between NtPAE1 and functional e...
Additional file 2: Figure S2. Typical dissociation curves for better concentration of pair primer... more Additional file 2: Figure S2. Typical dissociation curves for better concentration of pair primer in shoot roots samples. Pictures were taken using the qPCR instrument's software.
Additional file 1: Figure S1. Typical dissociation curves for better concentration of pair primer... more Additional file 1: Figure S1. Typical dissociation curves for better concentration of pair primer in leaves samples. Pictures were taken using the qPCR instrument's software.
Additional file 3: Table S2. Primer pairs sequences, amplicon size (A) in basepairs (bp), melting... more Additional file 3: Table S2. Primer pairs sequences, amplicon size (A) in basepairs (bp), melting temperature (Tm), coefficient of variation (CV), PCR reaction efficiency (E) and coefficient of determination (R2) of genes selected for stability assessment under SCMV infection. Table S3. Individual Ct values of each gene in sugarcane leaf samples. Table S4. RT-qPCR Cycles, Fluorescence (Rn) and change in Fluorescence (ΔRn) of each candidate reference gene in sugarcane leaf samples used as input in LinRegPCR for reaction efficiency assessment.
Additional file 2: Figure S1. Evaluation of Ct values of seven candidate reference genes across a... more Additional file 2: Figure S1. Evaluation of Ct values of seven candidate reference genes across all leaf samples. The box indicates 25-75% while the line across the box represents the median and whiskers represent the range from minimum to maximum.
Additional file 5: Figure S3. qRT-PCR amplicon size verification in agarose gel 1% of three newly... more Additional file 5: Figure S3. qRT-PCR amplicon size verification in agarose gel 1% of three newly designed primer pairs in cDNA bulks and genomic DNA from IACSP95-5000 and IAC91-1099 sugarcane cultivars.
Additional file 4: Figure S2. Dissociation curve of seven candidate reference genes, with picture... more Additional file 4: Figure S2. Dissociation curve of seven candidate reference genes, with pictures taken using the qPCR instrument's software. The dissociation curves for no template controls (NTCs) are indicated by an arrow.
Sugarcane (Saccharum spp.) is an important economic crop for both sugar and biomass, the yields o... more Sugarcane (Saccharum spp.) is an important economic crop for both sugar and biomass, the yields of which are negatively affected by flowering. The molecular mechanisms controlling flowering in sugarcane are nevertheless poorly understood. RNA-seq data analysis and database searches have enabled a comprehensive description of the PEBP gene family in sugarcane. It is shown to consist of at least 13 FLOWERING LOCUS T (FT)-like genes, two MOTHER OF FT AND TFL (MFT)-like genes, and four TERMINAL FLOWER (TFL)-like genes. As expected, these genes all show very high homology to their corresponding genes in Sorghum, and also to FT-like, MFT-like, and TFL-like genes in maize, rice, and Arabidopsis. Functional analysis in Arabidopsis showed that the sugarcane ScFT3 gene can rescue the late flowering phenotype of the Arabidopsis ft-10 mutant, whereas ScFT5 cannot. High expression levels of ScFT3 in leaves of short day-induced sugarcane plants coincided with initial stages of floral induction in...
The dirigent proteins (DP) families and resembling the DP (DP-like) are exclusive in land plants ... more The dirigent proteins (DP) families and resembling the DP (DP-like) are exclusive in land plants and related to plants defense and forming phenylpropanoids com-pounds optically active, mainly pinoresinol [1]. Pinoresi-nol is converted into a variety of lignans. The dirigent protein involvement is not confirmed in lignin forma-tion, a biopolymer which is a negative factor for the ethanol cellulosic production. However, recently it was demonstrated Arabidopsis DP-like, AtDIR10, localization in the lignin polymerization site and a determinant role in the formation of a lignin specific root structure, named as Casparian strip [2]. Despite of the controversy of the DP forming-lignin its substrate diversity is consis-tent. Since DPs and DPs-like are represented by numer-ous members of gene families with high diversified
Although reference genes have previously been used in the expression analysis of genes involved i... more Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use ...
Sugarcane mosaic virus (SCMV) is the causal agent of sugarcane mosaic disease (SMD) in Brazil; it... more Sugarcane mosaic virus (SCMV) is the causal agent of sugarcane mosaic disease (SMD) in Brazil; it is mainly controlled by using resistant cultivars. Studies on the changes in sugarcane transcriptome provided the first insights about the molecular basis underlying the genetic resistance to SMD; nonetheless, epigenetic modifications such as cytosine methylation is also informative, considering its roles in gene expression regulation. In our previous study, differentially transcribed fragments (DTFs) were obtained using cDNA-amplified fragment length polymorphism by comparing mock- and SCMV-inoculated plants from two sugarcane cultivars with contrasting responses to SMD. In this study, the identification of unexplored DTFs was continued while the same leaf samples were used to evaluate SCMV-mediated changes in the cytosine methylation pattern by using methylation-sensitive amplification polymorphism. This analysis revealed minor changes in cytosine methylation in response to SCMV infec...
Successful plant reproduction and fruit formation depend on adequate pollen and pistil developmen... more Successful plant reproduction and fruit formation depend on adequate pollen and pistil development, and pollen–pistil interactions. In Nicotiana tabacum, pollen tubes grow through the intercellular spaces of pistil-specialized tissues, stigmatic secretory zone, and stylar transmitting tissue (STT). These intercellular spaces are supposed to be formed by the modulation of cell wall pectin esterification. Previously we have identified a gene preferentially expressed in pistils encoding a putative pectin acetylesterase (PAE), named NtPAE1. Here, we characterized the NtPAE1 gene and performed genome-wide and phylogenetic analyses of PAEs. We identified 30 PAE sequences in the N. tabacum genome, distributed in four clades. The expression of NtPAE1 was assessed by RT-qPCR and in situ hybridization. We confirmed NtPAE1 preferential expression in stigmas/styles and ovaries and demonstrated its high expression in the STT. Structural predictions and comparisons between NtPAE1 and functional e...
Additional file 2: Figure S2. Typical dissociation curves for better concentration of pair primer... more Additional file 2: Figure S2. Typical dissociation curves for better concentration of pair primer in shoot roots samples. Pictures were taken using the qPCR instrument's software.
Additional file 1: Figure S1. Typical dissociation curves for better concentration of pair primer... more Additional file 1: Figure S1. Typical dissociation curves for better concentration of pair primer in leaves samples. Pictures were taken using the qPCR instrument's software.
Additional file 3: Table S2. Primer pairs sequences, amplicon size (A) in basepairs (bp), melting... more Additional file 3: Table S2. Primer pairs sequences, amplicon size (A) in basepairs (bp), melting temperature (Tm), coefficient of variation (CV), PCR reaction efficiency (E) and coefficient of determination (R2) of genes selected for stability assessment under SCMV infection. Table S3. Individual Ct values of each gene in sugarcane leaf samples. Table S4. RT-qPCR Cycles, Fluorescence (Rn) and change in Fluorescence (ΔRn) of each candidate reference gene in sugarcane leaf samples used as input in LinRegPCR for reaction efficiency assessment.
Additional file 2: Figure S1. Evaluation of Ct values of seven candidate reference genes across a... more Additional file 2: Figure S1. Evaluation of Ct values of seven candidate reference genes across all leaf samples. The box indicates 25-75% while the line across the box represents the median and whiskers represent the range from minimum to maximum.
Additional file 5: Figure S3. qRT-PCR amplicon size verification in agarose gel 1% of three newly... more Additional file 5: Figure S3. qRT-PCR amplicon size verification in agarose gel 1% of three newly designed primer pairs in cDNA bulks and genomic DNA from IACSP95-5000 and IAC91-1099 sugarcane cultivars.
Additional file 4: Figure S2. Dissociation curve of seven candidate reference genes, with picture... more Additional file 4: Figure S2. Dissociation curve of seven candidate reference genes, with pictures taken using the qPCR instrument's software. The dissociation curves for no template controls (NTCs) are indicated by an arrow.
Sugarcane (Saccharum spp.) is an important economic crop for both sugar and biomass, the yields o... more Sugarcane (Saccharum spp.) is an important economic crop for both sugar and biomass, the yields of which are negatively affected by flowering. The molecular mechanisms controlling flowering in sugarcane are nevertheless poorly understood. RNA-seq data analysis and database searches have enabled a comprehensive description of the PEBP gene family in sugarcane. It is shown to consist of at least 13 FLOWERING LOCUS T (FT)-like genes, two MOTHER OF FT AND TFL (MFT)-like genes, and four TERMINAL FLOWER (TFL)-like genes. As expected, these genes all show very high homology to their corresponding genes in Sorghum, and also to FT-like, MFT-like, and TFL-like genes in maize, rice, and Arabidopsis. Functional analysis in Arabidopsis showed that the sugarcane ScFT3 gene can rescue the late flowering phenotype of the Arabidopsis ft-10 mutant, whereas ScFT5 cannot. High expression levels of ScFT3 in leaves of short day-induced sugarcane plants coincided with initial stages of floral induction in...
The dirigent proteins (DP) families and resembling the DP (DP-like) are exclusive in land plants ... more The dirigent proteins (DP) families and resembling the DP (DP-like) are exclusive in land plants and related to plants defense and forming phenylpropanoids com-pounds optically active, mainly pinoresinol [1]. Pinoresi-nol is converted into a variety of lignans. The dirigent protein involvement is not confirmed in lignin forma-tion, a biopolymer which is a negative factor for the ethanol cellulosic production. However, recently it was demonstrated Arabidopsis DP-like, AtDIR10, localization in the lignin polymerization site and a determinant role in the formation of a lignin specific root structure, named as Casparian strip [2]. Despite of the controversy of the DP forming-lignin its substrate diversity is consis-tent. Since DPs and DPs-like are represented by numer-ous members of gene families with high diversified
Although reference genes have previously been used in the expression analysis of genes involved i... more Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use ...
Sugarcane mosaic virus (SCMV) is the causal agent of sugarcane mosaic disease (SMD) in Brazil; it... more Sugarcane mosaic virus (SCMV) is the causal agent of sugarcane mosaic disease (SMD) in Brazil; it is mainly controlled by using resistant cultivars. Studies on the changes in sugarcane transcriptome provided the first insights about the molecular basis underlying the genetic resistance to SMD; nonetheless, epigenetic modifications such as cytosine methylation is also informative, considering its roles in gene expression regulation. In our previous study, differentially transcribed fragments (DTFs) were obtained using cDNA-amplified fragment length polymorphism by comparing mock- and SCMV-inoculated plants from two sugarcane cultivars with contrasting responses to SMD. In this study, the identification of unexplored DTFs was continued while the same leaf samples were used to evaluate SCMV-mediated changes in the cytosine methylation pattern by using methylation-sensitive amplification polymorphism. This analysis revealed minor changes in cytosine methylation in response to SCMV infec...
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