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Transcriptome profiling identifies ABA mediated regulatory changes towards storage filling in developing seeds of castor bean (Ricinus communis L.).

Chandrasekaran U, Xu W, Liu A - Cell Biosci (2014)

Bottom Line: Exogenous ABA (10 μM) enhanced the accumulation of soluble sugar content (6.3%) followed by deposition of total lipid content (4.9 %).These genes were involved in sugar metabolism (such as glucose-6-phosphate, fructose 1,6 bis-phosphate, glycerol-3-phosphate, pyruvate kinase), lipid biosynthesis (such as ACS, ACBP, GPAT2, GPAT3, FAD2, FAD3, SAD1 and DGAT1), storage proteins synthesis (such as SGP1, zinc finger protein, RING H2 protein, nodulin 55 and cytochrome P450), and ABA biosynthesis (such as NCED1, NCED3 and beta carotene).Further, we confirmed the validation of RNA-Sequencing data by Semi-quantitative RT-PCR analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Tropical Plant Resource and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 88 Xuefu Road, Kunming 650223, China ; University of Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT

Background: The potential biodiesel plant castor bean (Ricinus communis) has been in the limelight for bioenergy research due to the availability of its genome which raises the bar for genome-wide studies claiming advances that impact the "genome-phenome challenge". Here we report the application of phytohormone ABA as an exogenous factor for the improvement of storage reserve accumulation with a focus on the complex interaction of pathways associated with seed filling.

Results: After the application of exogenous ABA treatments, we measured an increased ABA levels in the developing seeds cultured in vitro using the ELISA technique and quantified the content of major biomolecules (including total lipids, sugars and protein) in treated seeds. Exogenous ABA (10 μM) enhanced the accumulation of soluble sugar content (6.3%) followed by deposition of total lipid content (4.9 %). To elucidate the possible ABA signal transduction pathways towards overall seed filling, we studied the differential gene expression analysis using Illumina RNA-Sequencing technology, resulting in 2568 (1507-up/1061-down regulated) differentially expressed genes were identified. These genes were involved in sugar metabolism (such as glucose-6-phosphate, fructose 1,6 bis-phosphate, glycerol-3-phosphate, pyruvate kinase), lipid biosynthesis (such as ACS, ACBP, GPAT2, GPAT3, FAD2, FAD3, SAD1 and DGAT1), storage proteins synthesis (such as SGP1, zinc finger protein, RING H2 protein, nodulin 55 and cytochrome P450), and ABA biosynthesis (such as NCED1, NCED3 and beta carotene). Further, we confirmed the validation of RNA-Sequencing data by Semi-quantitative RT-PCR analysis.

Conclusions: Taken together, metabolite measurements supported by genes and pathway expression results indicated in this study provide new insights to understand the ABA signaling mechanism towards seed storage filling and also contribute useful information for facilitating oilseed crop functional genomics on an aim for utilizing castor bean agricultural and bioenergy use.

No MeSH data available.


Related in: MedlinePlus

RT-sqPCR validation of differentially expressed genes (DGEs) identified from high throughput RNA-sequencing analysis [left]. Total RNA used for RNA-Seq (21 DAP) was used for sqRT-PCR. Expression profiles during different developmental stages (7-63 DAP) [right]. The PCR primers are shown in Additional file 5: Table S2.
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Figure 5: RT-sqPCR validation of differentially expressed genes (DGEs) identified from high throughput RNA-sequencing analysis [left]. Total RNA used for RNA-Seq (21 DAP) was used for sqRT-PCR. Expression profiles during different developmental stages (7-63 DAP) [right]. The PCR primers are shown in Additional file 5: Table S2.

Mentions: In order to confirm the ABA regulatory changes validated by DGE analysis, sqRT-PCR analysis was performed with lipid and ABA biosynthesis genes. We randomly selected seven lipid genes encoding the key enzymes in lipid biosynthesis pathway which were differentially expressed along with two ABA biosynthesis genes. Acyl coA synthase (RcACS), Acyl coA binding protein (RcACBP), ER glycerol-3-phophate acyltransferase (RcGPAT2 and RcGPAT3), type 1 Diacylglycerol acyltransferase (RcDGAT1), Omega-6 desaturase (RcFAD2) and the two ABA biosynthesis genes (RcNCED1 and RcNCED3) showed positive response to ABA treatment with a negative response being observed for biotin carboxylase subunit of Het-Accase (RcBACC) gene (Figure 5). In order to broaden our knowledge on expression profile ration of these genes after ABA treatment, we also studied the expression patterns at different seed developmental stages (7-63 DAP) (Figure 5). From our results, genes like RcDGAT, RcFAD2, RcNCED1 and RcNCED3 were expressed at all the stages with exceptions of genes like RcGPAT2 expressed in early to mid developmental stages. From these results it was apparent that sqRT-PCR results were consistent with the DGE data and showed up regulation of all the six lipid genes that were up regulated in DGE analysis (Figure 5).


Transcriptome profiling identifies ABA mediated regulatory changes towards storage filling in developing seeds of castor bean (Ricinus communis L.).

Chandrasekaran U, Xu W, Liu A - Cell Biosci (2014)

RT-sqPCR validation of differentially expressed genes (DGEs) identified from high throughput RNA-sequencing analysis [left]. Total RNA used for RNA-Seq (21 DAP) was used for sqRT-PCR. Expression profiles during different developmental stages (7-63 DAP) [right]. The PCR primers are shown in Additional file 5: Table S2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109380&req=5

Figure 5: RT-sqPCR validation of differentially expressed genes (DGEs) identified from high throughput RNA-sequencing analysis [left]. Total RNA used for RNA-Seq (21 DAP) was used for sqRT-PCR. Expression profiles during different developmental stages (7-63 DAP) [right]. The PCR primers are shown in Additional file 5: Table S2.
Mentions: In order to confirm the ABA regulatory changes validated by DGE analysis, sqRT-PCR analysis was performed with lipid and ABA biosynthesis genes. We randomly selected seven lipid genes encoding the key enzymes in lipid biosynthesis pathway which were differentially expressed along with two ABA biosynthesis genes. Acyl coA synthase (RcACS), Acyl coA binding protein (RcACBP), ER glycerol-3-phophate acyltransferase (RcGPAT2 and RcGPAT3), type 1 Diacylglycerol acyltransferase (RcDGAT1), Omega-6 desaturase (RcFAD2) and the two ABA biosynthesis genes (RcNCED1 and RcNCED3) showed positive response to ABA treatment with a negative response being observed for biotin carboxylase subunit of Het-Accase (RcBACC) gene (Figure 5). In order to broaden our knowledge on expression profile ration of these genes after ABA treatment, we also studied the expression patterns at different seed developmental stages (7-63 DAP) (Figure 5). From our results, genes like RcDGAT, RcFAD2, RcNCED1 and RcNCED3 were expressed at all the stages with exceptions of genes like RcGPAT2 expressed in early to mid developmental stages. From these results it was apparent that sqRT-PCR results were consistent with the DGE data and showed up regulation of all the six lipid genes that were up regulated in DGE analysis (Figure 5).

Bottom Line: Exogenous ABA (10 μM) enhanced the accumulation of soluble sugar content (6.3%) followed by deposition of total lipid content (4.9 %).These genes were involved in sugar metabolism (such as glucose-6-phosphate, fructose 1,6 bis-phosphate, glycerol-3-phosphate, pyruvate kinase), lipid biosynthesis (such as ACS, ACBP, GPAT2, GPAT3, FAD2, FAD3, SAD1 and DGAT1), storage proteins synthesis (such as SGP1, zinc finger protein, RING H2 protein, nodulin 55 and cytochrome P450), and ABA biosynthesis (such as NCED1, NCED3 and beta carotene).Further, we confirmed the validation of RNA-Sequencing data by Semi-quantitative RT-PCR analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Tropical Plant Resource and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 88 Xuefu Road, Kunming 650223, China ; University of Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT

Background: The potential biodiesel plant castor bean (Ricinus communis) has been in the limelight for bioenergy research due to the availability of its genome which raises the bar for genome-wide studies claiming advances that impact the "genome-phenome challenge". Here we report the application of phytohormone ABA as an exogenous factor for the improvement of storage reserve accumulation with a focus on the complex interaction of pathways associated with seed filling.

Results: After the application of exogenous ABA treatments, we measured an increased ABA levels in the developing seeds cultured in vitro using the ELISA technique and quantified the content of major biomolecules (including total lipids, sugars and protein) in treated seeds. Exogenous ABA (10 μM) enhanced the accumulation of soluble sugar content (6.3%) followed by deposition of total lipid content (4.9 %). To elucidate the possible ABA signal transduction pathways towards overall seed filling, we studied the differential gene expression analysis using Illumina RNA-Sequencing technology, resulting in 2568 (1507-up/1061-down regulated) differentially expressed genes were identified. These genes were involved in sugar metabolism (such as glucose-6-phosphate, fructose 1,6 bis-phosphate, glycerol-3-phosphate, pyruvate kinase), lipid biosynthesis (such as ACS, ACBP, GPAT2, GPAT3, FAD2, FAD3, SAD1 and DGAT1), storage proteins synthesis (such as SGP1, zinc finger protein, RING H2 protein, nodulin 55 and cytochrome P450), and ABA biosynthesis (such as NCED1, NCED3 and beta carotene). Further, we confirmed the validation of RNA-Sequencing data by Semi-quantitative RT-PCR analysis.

Conclusions: Taken together, metabolite measurements supported by genes and pathway expression results indicated in this study provide new insights to understand the ABA signaling mechanism towards seed storage filling and also contribute useful information for facilitating oilseed crop functional genomics on an aim for utilizing castor bean agricultural and bioenergy use.

No MeSH data available.


Related in: MedlinePlus