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Identification of sense and antisense transcripts regulated by drought in sugarcane.

Lembke CG, Nishiyama MY, Sato PM, de Andrade RF, Souza GM - Plant Mol. Biol. (2012)

Bottom Line: We validated the results obtained using quantitative real-time PCR (qPCR).Our custom sugarcane oligonucleotide array provides sensitivity and good coverage of sugarcane transcripts for the identification of a representative proportion of natural antisense transcripts (NATs) and sense-antisense transcript pairs (SATs).The antisense transcriptome showed, in most cases, co-expression with respective sense transcripts.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Transdução de Sinal, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, São Paulo, SP, 05508-000, Brazil.

ABSTRACT
Sugarcane is an important sugar and energy crop that can be used efficiently for biofuels production. The development of sugarcane cultivars tolerant to drought could allow for the expansion of plantations to sub-prime regions. Knowledge on the mechanisms underlying drought responses and its relationship with carbon partition would greatly help to define routes to increase yield. In this work we studied sugarcane responses to drought using a custom designed oligonucleotide array with 21,901 different probes. The oligoarrays were designed to contain probes that detect transcription in both sense and antisense orientation. We validated the results obtained using quantitative real-time PCR (qPCR). A total of 987 genes were differentially expressed in at least one sample of sugarcane plants submitted to drought for 24, 72 and 120 h. Among them, 928 were sense transcripts and 59 were antisense transcripts. Genes related to Carbohydrate Metabolism, RNA Metabolism and Signal Transduction were selected for gene expression validation by qPCR that indicated a validation percentage of 90%. From the probes presented on the array, 75% of the sense probes and 11.9% of the antisense probes have signal above background and can be classified as expressed sequences. Our custom sugarcane oligonucleotide array provides sensitivity and good coverage of sugarcane transcripts for the identification of a representative proportion of natural antisense transcripts (NATs) and sense-antisense transcript pairs (SATs). The antisense transcriptome showed, in most cases, co-expression with respective sense transcripts.

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qPCR of sense transcripts regulated by drought stress. The y axis is the normalized relative expression ratio between stressed versus irrigated samples. qPCR reactions were done only for experimental points differentially expressed in microarray experiments. Reactions were done in triplicates and on a third biological replicate. Error bars were calculated as in Rocha et al. (2007). **p = 0.95; ***p = 0.99; *p = 1.00 for control versus drought sample
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Fig4: qPCR of sense transcripts regulated by drought stress. The y axis is the normalized relative expression ratio between stressed versus irrigated samples. qPCR reactions were done only for experimental points differentially expressed in microarray experiments. Reactions were done in triplicates and on a third biological replicate. Error bars were calculated as in Rocha et al. (2007). **p = 0.95; ***p = 0.99; *p = 1.00 for control versus drought sample

Mentions: It is known that some of the pathways associated with sucrose content may overlap with drought stress signaling pathways (Papini-Terzi et al. 2009). This work shows that different aspects of carbohydrate metabolism were down regulated in drought stress. We observed that the Pyrophosphate-fructose 6-phosphate 1-phosphotransferase alpha subunit (SCEPRT2048D06.g) is repressed after 120 h of drought (Fig. 4). This alpha subunit is involved in the regulation of the enzyme which catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis (Buchanan et al. 2002). A phosphoglycerate kinase (SCEZLB1006F11.g), which catalyzes the formation of 3-phosphoglycerate from 1,3-bisphosphoglycerate in glycolysis is also repressed after 120 h of drought (Fig. 4). An Aconitate hydratase (SCACAD1037B06.g), overexpressed after 72 h of drought (Fig. 4), was the only carbohydrate metabolism gene that we selected for qPCR analysis that was overexpressed; the majority of genes involved in carbohydrate metabolism were down-regulated. The formation and mobilization of starch may also be altered in sugarcane leaves without irrigation. An ADP-glucose pyrophosphorylase small subunit (SCCCFL4002D04.g) involved in the biosynthesis of alpha 1,4-glucans (glycogen or starch) in bacteria and plants was repressed after 120 h of water deprivation (Fig. 4). It was already observed that the large subunit of ADP-glucose pyrophosphorylase was repressed in RNA from epidermal fragments of potato leaves after potato plants had been submitted to water deprivation (Kopka et al. 1997). A beta-amylase (SCUTAM2089E05.g) involved in the cleavage of maltose residues from the non-reducing end of starch was repressed after 72 and 120 h and of drought stress (Fig. 4).Fig. 4


Identification of sense and antisense transcripts regulated by drought in sugarcane.

Lembke CG, Nishiyama MY, Sato PM, de Andrade RF, Souza GM - Plant Mol. Biol. (2012)

qPCR of sense transcripts regulated by drought stress. The y axis is the normalized relative expression ratio between stressed versus irrigated samples. qPCR reactions were done only for experimental points differentially expressed in microarray experiments. Reactions were done in triplicates and on a third biological replicate. Error bars were calculated as in Rocha et al. (2007). **p = 0.95; ***p = 0.99; *p = 1.00 for control versus drought sample
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Related In: Results  -  Collection

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Fig4: qPCR of sense transcripts regulated by drought stress. The y axis is the normalized relative expression ratio between stressed versus irrigated samples. qPCR reactions were done only for experimental points differentially expressed in microarray experiments. Reactions were done in triplicates and on a third biological replicate. Error bars were calculated as in Rocha et al. (2007). **p = 0.95; ***p = 0.99; *p = 1.00 for control versus drought sample
Mentions: It is known that some of the pathways associated with sucrose content may overlap with drought stress signaling pathways (Papini-Terzi et al. 2009). This work shows that different aspects of carbohydrate metabolism were down regulated in drought stress. We observed that the Pyrophosphate-fructose 6-phosphate 1-phosphotransferase alpha subunit (SCEPRT2048D06.g) is repressed after 120 h of drought (Fig. 4). This alpha subunit is involved in the regulation of the enzyme which catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis (Buchanan et al. 2002). A phosphoglycerate kinase (SCEZLB1006F11.g), which catalyzes the formation of 3-phosphoglycerate from 1,3-bisphosphoglycerate in glycolysis is also repressed after 120 h of drought (Fig. 4). An Aconitate hydratase (SCACAD1037B06.g), overexpressed after 72 h of drought (Fig. 4), was the only carbohydrate metabolism gene that we selected for qPCR analysis that was overexpressed; the majority of genes involved in carbohydrate metabolism were down-regulated. The formation and mobilization of starch may also be altered in sugarcane leaves without irrigation. An ADP-glucose pyrophosphorylase small subunit (SCCCFL4002D04.g) involved in the biosynthesis of alpha 1,4-glucans (glycogen or starch) in bacteria and plants was repressed after 120 h of water deprivation (Fig. 4). It was already observed that the large subunit of ADP-glucose pyrophosphorylase was repressed in RNA from epidermal fragments of potato leaves after potato plants had been submitted to water deprivation (Kopka et al. 1997). A beta-amylase (SCUTAM2089E05.g) involved in the cleavage of maltose residues from the non-reducing end of starch was repressed after 72 and 120 h and of drought stress (Fig. 4).Fig. 4

Bottom Line: We validated the results obtained using quantitative real-time PCR (qPCR).Our custom sugarcane oligonucleotide array provides sensitivity and good coverage of sugarcane transcripts for the identification of a representative proportion of natural antisense transcripts (NATs) and sense-antisense transcript pairs (SATs).The antisense transcriptome showed, in most cases, co-expression with respective sense transcripts.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Transdução de Sinal, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, São Paulo, SP, 05508-000, Brazil.

ABSTRACT
Sugarcane is an important sugar and energy crop that can be used efficiently for biofuels production. The development of sugarcane cultivars tolerant to drought could allow for the expansion of plantations to sub-prime regions. Knowledge on the mechanisms underlying drought responses and its relationship with carbon partition would greatly help to define routes to increase yield. In this work we studied sugarcane responses to drought using a custom designed oligonucleotide array with 21,901 different probes. The oligoarrays were designed to contain probes that detect transcription in both sense and antisense orientation. We validated the results obtained using quantitative real-time PCR (qPCR). A total of 987 genes were differentially expressed in at least one sample of sugarcane plants submitted to drought for 24, 72 and 120 h. Among them, 928 were sense transcripts and 59 were antisense transcripts. Genes related to Carbohydrate Metabolism, RNA Metabolism and Signal Transduction were selected for gene expression validation by qPCR that indicated a validation percentage of 90%. From the probes presented on the array, 75% of the sense probes and 11.9% of the antisense probes have signal above background and can be classified as expressed sequences. Our custom sugarcane oligonucleotide array provides sensitivity and good coverage of sugarcane transcripts for the identification of a representative proportion of natural antisense transcripts (NATs) and sense-antisense transcript pairs (SATs). The antisense transcriptome showed, in most cases, co-expression with respective sense transcripts.

Show MeSH
Related in: MedlinePlus