New Insights in the Sugarcane Transcriptome Responding to Drought Stress as Revealed by Supersage

In the scope of the present work, four SuperSAGE libraries have been generated, using bulked root tissues from four drought-tolerant accessions as compared with four bulked sensitive genotypes, aiming to generate a panel of differentially expressed stress-responsive genes. Both groups were submitted to 24 hours of water deficit stress. The SuperSAGE libraries produced 8,787,315 tags (26 bp) that, after exclusion of singlets, allowed the identification of 205,975 unitags. Most relevant BlastN matches comprised 567,420 tags, regarding 75,404 unitags with 164,860 different ESTs. To optimize the annotation efficiency, the Gene Ontology (GO) categorization was carried out for 186,191 ESTs (BlastN against Uniprot-SwissProt), permitting the categorization of 118,208 ESTs (63.5%). In an attempt to elect a group of the best tags to be validated by RTqPCR, the GO categorization of the tag-related ESTs allowed the in silico identification of 213 upregulated unitags responding basically to abiotic stresses, from which 145 presented no hits after BlastN analysis, probably concerning new genes still uncovered in previous studies. The present report analyzes the sugarcane transcriptome under drought stress, using a combination of high-throughput transcriptome profiling by SuperSAGE with the Solexa sequencing technology, allowing the identification of potential target genes during the stress response

Validation of Novel Reference Genes for Reverse Transcription Quantitative Real-Time PCR in Drought-Stressed Sugarcane

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches

Characterization of the subcellular localization of arabidopsis thaliana thip.

O produto do gene thi1 de Arabidopsis thaliana está provavelmente envolvido na biossíntese de tiamina (vitamina B1) e na proteção do DNA organelar contra danos. Estudos sobre a biossíntese da tiamina em plantas sugerem uma localização plastidial para este mecanismo, o que está de acordo com a existência de um peptídeo de trânsito cloroplástico (TP) na região N-terminal de THI1. Por outro lado, em leveduras a tiamina é sintetizada em mitocôndrias. Interessantemente, o cDNA de thi1 de A. thaliana complementa uma cepa de levedura com disrupção no gene homólogo thi4. A análise da seqüência de aminoácidos de THI1 revelou a presença de uma região capaz de formar uma estrutura do tipo a-hélice anfifílica, freqüentemente encontrada em preseqüências mitocondriais, localizada logo após o peptídeo de trânsito cloroplástico. O papel desta região na localização da proteína THI1 nas mitocôndrias foi comprovado a partir de ensaios envolvendo construções de genes quiméricos (contendo ou não a putativa seqüência de direcionamento mitocondrial) e um gene repórter (uidA). Estas construções foram introduzidas em plantas de tabaco e a localização da atividade GUS foi determinada nas frações subcelulares das plantas transgênicas. Análise direta da presença de THI1 nas mitocôndrias e cloroplastos de Arabidopsis foi realizada via imunolocalização. Também foram fornecidas evidências que as duas isoformas organelares são codificadas por um único transcrito nuclear. Experimentos de transcrição/tradução in vitro indicaram a ocorrência de dois produtos da tradução a partir de códons de iníciação em fase de leitura. Mutações sítio-específicas na seqüência de thi1 acoplados à experimentos usando a proteína fluorescente verde (GFP) mostraram que a tradução no primeiro AUG determina a localização da proteína nos cloroplastos, enquanto que a tradução no segundo AUG é responsável pelo endereçamento da proteína às mitocôndrias. A análise do contexto para início da tradução revelou que a região em torno do primeiro AUG é mais favorável para a tradução do mRNA de thi1. Além disso, observou-se a presença de uma forte estrutura em "grampo de cabelo" próximo ao segundo códon AUG, indicando um contexto subótimo capaz de interferir na tradução. Estas observações confirmaram os dados obtidos a partir da tradução in vitro na qual a iniciação se dá preferencialmente no primeiro AUG o que pode sugerir uma maior necessidade da proteína nos plastídeos.Arabidopsis thaliana thi1 gene product is probably involved in both thiamine biosynthesis as well as protection of organellar DNA from damage. Studies of thiamine biosynthesis in plants suggest a plastid location for the pathway, which is in agreement with the predicted THI1 N-terminal chloroplastic transit peptide (TP). On the other hand, thiamine is synthesized in mitochondria in yeast cells. Interestingly, A. thaliana thi1 cDNA complements a yeast strain disrupted for the homologous thi4 gene. Analysis of THI1 amino acid sequence revealed the presence of a putative amphiphilic a-helix, which is typical for mitochondrial presequences, located downstream of the chloroplast transit peptide. The role of this sequence on mitochondrial import has been shown by chimeric gene constructs (carrying or not the putative mitochondrial presequence) and the uidA reporter gene. These constructions have been introduced into tobacco plants and the GUS activity has been measured in subcellular fractions of transgenic plants. Direct analysis of THIp in mitochondria and chloroplasts has been done via ImmunoGold labelling experiments. Additional evidence suggested that the two organellar isoforms were encoded by a single nuclear transcript. In vitro transcription/translation experiments revealed the presence of two translational products by a differential usage of two in-frame translational start codons. Coupling site-specific mutations on THI1 encoding sequence with green fluorescent protein (GFP) gene fusions showed that translation initiation in the first AUG directs translocation of THI1 to plastids. However, when translation initiates from the second AUG THI1 is addressed to mitochondria. Analysis of the translation efficiency of thi1 mRNA revealed that the best context for translation initiation is present at the first AUG. In addition, it has been shown a suboptimal context at the second AUG and a strong stem-and-loop structure which is likely to slow translation. These observation confirm the in vitro translation data in which translation occurs preferentially in the first AUG, what could suggest a higher requirement of the protein in plastids

Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches

Expression analysis of sugarcane aquaporin genes under water deficit

The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific

Relative expression profile of miRNA targets.

<p>Analysis of relative expression of miR159 target (GAMYB), miR164 target (NAC1), miR168 target (AGO1) and miR397 target (laccase) by qRT-PCR using samples of three sugarcane cultivar submitted to water depletion. In each case, control condition had relative expression equal 1 (dotted line). *represent significantly changing of miRNA expression between control and treatment samples (p-value <0.05).</p

Regulation of miRNAs whether by genotype or/and water depletion.

<p>Comparison of miRNA regulation identified in individual cultivar libraries and pool libraries.</p

Relative expression of sRNA pathway genes.

<p>Analysis by qRT-PCR using samples of three sugarcane cultivar submitted to water depletion. *represent significantly changing of miRNA expression between analyzed samples (p-value <0.05).</p

Summary of results obtained after computational data mining of small RNA libraries.

a<p>Filtering for tRNA, rRNA, low-complexity sequence and trimming for “N” bases and 3′ adapters.</p>b<p>Conserved miRNA deposited at miRBase database identified by miRProf pipeline.</p