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Cold Responsive Gene Expression Profiling of Sugarcane and Saccharum spontaneum with Functional Analysis of a Cold Inducible Saccharum Homolog of NOD26-Like Intrinsic Protein to Salt and Water Stress.

Park JW, Benatti TR, Marconi T, Yu Q, Solis-Gracia N, Mora V, da Silva JA - PLoS ONE (2015)

Bottom Line: Salinity stress test on SspNIP2 transgenic tobacco plants resulted in more vigorous transgenic lines than the non-transgenic tobacco plants, suggesting some degree of tolerance to salt stress conferred by SspNIP2.SspNIP2-transgenic plants, exposed to 2 weeks of water stress without irrigation, developed various degrees of water stress symptom.The water stress test confirmed that the SspNIP2 transgenic lines had lower evapotranspiration rates than non-transgenic lines, suggesting that SspNIP2 transgenic lines showed a slight tolerance to the early water stress compared to wild type plants.

View Article: PubMed Central - PubMed

Affiliation: Texas A&M AgriLife Research, The Texas A&M System, Weslaco, Texas, United States of America.

ABSTRACT
Transcriptome analysis of sugarcane hybrid CP72-1210 (cold susceptible) and Saccharum spontaneum TUS05-05 (cold tolerant) using Sugarcane Assembled Sequences (SAS) from SUCEST-FUN Database showed that a total of 35,340 and 34,698 SAS genes, respectively, were expressed before and after chilling stress. The analysis revealed that more than 600 genes are differentially expressed in each genotype after chilling stress. Blast2Go annotation revealed that the major difference in gene expression profiles between CP72-1210 and TUS05-05 after chilling stress are present in the genes related to the transmembrane transporter activity. To further investigate the relevance of transmembrane transporter activity against abiotic stress tolerance, a S. spontaneum homolog of a NOD26-like major intrinsic protein gene (SspNIP2) was selected for functional analysis, of which expression was induced after chilling stress in the cold tolerant TUS05-05. Quantitative real-time PCR showed that SspNIP2 expression was increased ~2.5 fold at 30 minutes after cold treatment and stayed induced throughout the 24 hours of cold treatment. The amino acid sequence analysis of the cloned SspNIP2 confirmed the presence of six transmembrane domains and two NPA (Asn-Pro-Ala) motifs, signature features of major intrinsic protein families. Amino acid analysis confirmed that four amino acids, comprising the ar/R (aromatic residue/arginine) region responsible for the substrate specificity among MIPs, are conserved among monocot silicon transporters and SspNIP2. Salinity stress test on SspNIP2 transgenic tobacco plants resulted in more vigorous transgenic lines than the non-transgenic tobacco plants, suggesting some degree of tolerance to salt stress conferred by SspNIP2. SspNIP2-transgenic plants, exposed to 2 weeks of water stress without irrigation, developed various degrees of water stress symptom. The water stress test confirmed that the SspNIP2 transgenic lines had lower evapotranspiration rates than non-transgenic lines, suggesting that SspNIP2 transgenic lines showed a slight tolerance to the early water stress compared to wild type plants.

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Venn diagram of differentially expressed genes in CP72-1210 and TUS05-05.A. Venn diagram of differentially expressed SAS sequences before gene annotation. B. Venn diagram of annotated genes differentially expressed in each genotype. The number of uniquely and commonly expressed genes in each genotype is indicated in numbers in the diagram. Up and Down indicate up-regulation and down-regulation, respectively.
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pone.0125810.g001: Venn diagram of differentially expressed genes in CP72-1210 and TUS05-05.A. Venn diagram of differentially expressed SAS sequences before gene annotation. B. Venn diagram of annotated genes differentially expressed in each genotype. The number of uniquely and commonly expressed genes in each genotype is indicated in numbers in the diagram. Up and Down indicate up-regulation and down-regulation, respectively.

Mentions: We compared the transcriptomes of a cold susceptible sugarcane CP72-1210 and a cold tolerant S. spontaneum TUS05-05, before and after chilling treatment at 1.5°C for 20 hours. A total of 179.8 million reads were obtained by quality filtering, and of those, 88.4 million reads (49.2%) were mapped to the sugarcane assembled sequences (SAS) from SUCEST-FUN Database (http://sucest-fun.org) (Table 1). The RNA-Seq analysis revealed that each treatment generated a list of 35,340 and 34,698 SAS sequences, expressed before and after chilling stress in CP72-1210 and TUS05-05, respectively, which were further analyzed to determine the SAS sequences that were differentially expressed between treatments (Table 2). The analysis showed that ~80% of the 43,141 SAS sequences were expressed before and after chilling treatment in each genotype (Table 2). The number of differentially expressed SAS sequences showing fold change greater than ±2 after chilling stress was 611 in CP72-1210 and 655 in TUS05-05 (Table 2). The analysis confirmed that 447 SAS sequences were differentially expressed commonly in both CP72-1210 (73%) and TUS05-05 (68%) (Fig 1A). To validate the transcriptome data, quantitative real-time PCR (qRT-PCR) was conducted with six SAS sequences that were up-regulated in TUS05-05 (S1 Fig). The relative expression levels of four of six SAS sequences in qRT-PCR were ranged from 37% to 72% of their fold increases in RNA-Seq analysis (S1 Fig), and the other two qRT-PCR data were less than 20% of those obtained from RNA-Seq analysis (S1 Fig). Although variation was observed in the relative gene expression levels obtained from qRT-PCR and RNA-Seq, the up-regulation of six SAS sequences was confirmed by qRT-PCR (S1 Fig). However, these results also indicated that the validation of RNA-Seq data by qRT-PCR should be performed before genes of interest are further investigated.


Cold Responsive Gene Expression Profiling of Sugarcane and Saccharum spontaneum with Functional Analysis of a Cold Inducible Saccharum Homolog of NOD26-Like Intrinsic Protein to Salt and Water Stress.

Park JW, Benatti TR, Marconi T, Yu Q, Solis-Gracia N, Mora V, da Silva JA - PLoS ONE (2015)

Venn diagram of differentially expressed genes in CP72-1210 and TUS05-05.A. Venn diagram of differentially expressed SAS sequences before gene annotation. B. Venn diagram of annotated genes differentially expressed in each genotype. The number of uniquely and commonly expressed genes in each genotype is indicated in numbers in the diagram. Up and Down indicate up-regulation and down-regulation, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418668&req=5

pone.0125810.g001: Venn diagram of differentially expressed genes in CP72-1210 and TUS05-05.A. Venn diagram of differentially expressed SAS sequences before gene annotation. B. Venn diagram of annotated genes differentially expressed in each genotype. The number of uniquely and commonly expressed genes in each genotype is indicated in numbers in the diagram. Up and Down indicate up-regulation and down-regulation, respectively.
Mentions: We compared the transcriptomes of a cold susceptible sugarcane CP72-1210 and a cold tolerant S. spontaneum TUS05-05, before and after chilling treatment at 1.5°C for 20 hours. A total of 179.8 million reads were obtained by quality filtering, and of those, 88.4 million reads (49.2%) were mapped to the sugarcane assembled sequences (SAS) from SUCEST-FUN Database (http://sucest-fun.org) (Table 1). The RNA-Seq analysis revealed that each treatment generated a list of 35,340 and 34,698 SAS sequences, expressed before and after chilling stress in CP72-1210 and TUS05-05, respectively, which were further analyzed to determine the SAS sequences that were differentially expressed between treatments (Table 2). The analysis showed that ~80% of the 43,141 SAS sequences were expressed before and after chilling treatment in each genotype (Table 2). The number of differentially expressed SAS sequences showing fold change greater than ±2 after chilling stress was 611 in CP72-1210 and 655 in TUS05-05 (Table 2). The analysis confirmed that 447 SAS sequences were differentially expressed commonly in both CP72-1210 (73%) and TUS05-05 (68%) (Fig 1A). To validate the transcriptome data, quantitative real-time PCR (qRT-PCR) was conducted with six SAS sequences that were up-regulated in TUS05-05 (S1 Fig). The relative expression levels of four of six SAS sequences in qRT-PCR were ranged from 37% to 72% of their fold increases in RNA-Seq analysis (S1 Fig), and the other two qRT-PCR data were less than 20% of those obtained from RNA-Seq analysis (S1 Fig). Although variation was observed in the relative gene expression levels obtained from qRT-PCR and RNA-Seq, the up-regulation of six SAS sequences was confirmed by qRT-PCR (S1 Fig). However, these results also indicated that the validation of RNA-Seq data by qRT-PCR should be performed before genes of interest are further investigated.

Bottom Line: Salinity stress test on SspNIP2 transgenic tobacco plants resulted in more vigorous transgenic lines than the non-transgenic tobacco plants, suggesting some degree of tolerance to salt stress conferred by SspNIP2.SspNIP2-transgenic plants, exposed to 2 weeks of water stress without irrigation, developed various degrees of water stress symptom.The water stress test confirmed that the SspNIP2 transgenic lines had lower evapotranspiration rates than non-transgenic lines, suggesting that SspNIP2 transgenic lines showed a slight tolerance to the early water stress compared to wild type plants.

View Article: PubMed Central - PubMed

Affiliation: Texas A&M AgriLife Research, The Texas A&M System, Weslaco, Texas, United States of America.

ABSTRACT
Transcriptome analysis of sugarcane hybrid CP72-1210 (cold susceptible) and Saccharum spontaneum TUS05-05 (cold tolerant) using Sugarcane Assembled Sequences (SAS) from SUCEST-FUN Database showed that a total of 35,340 and 34,698 SAS genes, respectively, were expressed before and after chilling stress. The analysis revealed that more than 600 genes are differentially expressed in each genotype after chilling stress. Blast2Go annotation revealed that the major difference in gene expression profiles between CP72-1210 and TUS05-05 after chilling stress are present in the genes related to the transmembrane transporter activity. To further investigate the relevance of transmembrane transporter activity against abiotic stress tolerance, a S. spontaneum homolog of a NOD26-like major intrinsic protein gene (SspNIP2) was selected for functional analysis, of which expression was induced after chilling stress in the cold tolerant TUS05-05. Quantitative real-time PCR showed that SspNIP2 expression was increased ~2.5 fold at 30 minutes after cold treatment and stayed induced throughout the 24 hours of cold treatment. The amino acid sequence analysis of the cloned SspNIP2 confirmed the presence of six transmembrane domains and two NPA (Asn-Pro-Ala) motifs, signature features of major intrinsic protein families. Amino acid analysis confirmed that four amino acids, comprising the ar/R (aromatic residue/arginine) region responsible for the substrate specificity among MIPs, are conserved among monocot silicon transporters and SspNIP2. Salinity stress test on SspNIP2 transgenic tobacco plants resulted in more vigorous transgenic lines than the non-transgenic tobacco plants, suggesting some degree of tolerance to salt stress conferred by SspNIP2. SspNIP2-transgenic plants, exposed to 2 weeks of water stress without irrigation, developed various degrees of water stress symptom. The water stress test confirmed that the SspNIP2 transgenic lines had lower evapotranspiration rates than non-transgenic lines, suggesting that SspNIP2 transgenic lines showed a slight tolerance to the early water stress compared to wild type plants.

Show MeSH
Related in: MedlinePlus