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Gene silencing of Sugar-dependent 1 (JcSDP1), encoding a patatin-domain triacylglycerol lipase, enhances seed oil accumulation in Jatropha curcas.

Kim MJ, Yang SW, Mao HZ, Veena SP, Yin JL, Chua NH - Biotechnol Biofuels (2014)

Bottom Line: We cloned Jatropha JcSDP1, and verified its function by complementation of the Arabidopsis sdp1-5 mutant.Taking advantage of the observation with Arabidopsis, we used RNAi technology to generate JcSDP1 deficiency in transgenic Jatropha.Based on this result, we generated SDP1-deficient transgenic Jatropha plants using by RNAi technology with a native JcSDP1 promoter to silence endogenous JcSDP1 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA. chua@mail.rockefeller.edu.

ABSTRACT

Background: Triacylglycerols (TAGs) are the most abundant form of storage oil in plants. They consist of three fatty acid chains (usually C16 or C18) covalently linked to glycerol. SDP1 is a specific lipase for the first step of TAG catabolism in Arabidopsis seeds. Arabidopsis mutants deficient in SDP1 accumulate high levels of oils, probably due to blockage in TAG degradation. We applied this knowledge from the model plant, Arabidopsis thaliana, to engineer increased seed oil content in the biodiesel plant Jatropha curcas using RNA interference (RNAi) technology.

Results: As Jatropha is a biodiesel crop, any significant increase in its seed oil content would be an important agronomic trait. Using A. thaliana as a model plant, we found that a deficiency of SDP1 led to higher TAG accumulation and a larger number of oil bodies in seeds compared with wild type (Columbia-0; Col-0). We cloned Jatropha JcSDP1, and verified its function by complementation of the Arabidopsis sdp1-5 mutant. Taking advantage of the observation with Arabidopsis, we used RNAi technology to generate JcSDP1 deficiency in transgenic Jatropha. We found that Jatropha JcSDP1-RNAi plants accumulated 13 to 30% higher total seed storage lipid, along with a 7% compensatory decrease in protein content, compared with control (CK; 35S:GFP) plants. Free fatty acid (FFA) content in seeds was reduced from 27% in control plants to 8.5% in JcSDP1-RNAi plants.

Conclusion: Here, we showed that SDP1 deficiency enhances seed oil accumulation in Arabidopsis. Based on this result, we generated SDP1-deficient transgenic Jatropha plants using by RNAi technology with a native JcSDP1 promoter to silence endogenous JcSDP1 expression. Seeds of Jatropha JcSDP1-RNAi plants accumulated up to 30% higher total lipid and had reduced FFA content compared with control (CK; 35S:GFP) plants. Our strategy of improving an important agronomic trait of Jatropha can be extended to other oil crops to yield higher seed oil.

No MeSH data available.


Related in: MedlinePlus

JcSDP1promoter and its expression analysis inProJcSDP1:GUStransgenic plants. (A) Composition of putative cis elements in JcSDP1 promoter. (B) Transient ProJcSDP1:GUS expression in Jatropha fruit and leaf. (C) Heterologous expressions of ProJcSDP1:GUS in transgenic Arabidopsis plants: (a) 14-day-old seedling, (b, c) inflorescence, (d, e) rosette leaf, (f) siliques, (g) seeds at different developing stages, (h) young seeds (3 to 4 DAP, globular-stage embryo), (i) mid-stage seeds (9 to 10 DAP, mature green embryo). DAP, days after pollination. (D) Sugar-dependent expression of ProJcSDP1:GUS transgenic plants (14?=?day-old seedling). Different sugar sources (1% or 3% fructose, glucose, or sucrose) were used, and mannitol was used as a control for osmotic stress. (E)JcSDP1 gene expression profile using real-time quantitative PCR at different seed development stages (S1, 1 WAF; S2. 2 to 3 WAF; S3; 4 to 6 WAF; and S4; 7 to 8 WAF) JcTubulin expression levels were used as an internal control. Values are given as mean?±?SD (n?=?3). WAF, weeks after fertilization.
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Figure 4: JcSDP1promoter and its expression analysis inProJcSDP1:GUStransgenic plants. (A) Composition of putative cis elements in JcSDP1 promoter. (B) Transient ProJcSDP1:GUS expression in Jatropha fruit and leaf. (C) Heterologous expressions of ProJcSDP1:GUS in transgenic Arabidopsis plants: (a) 14-day-old seedling, (b, c) inflorescence, (d, e) rosette leaf, (f) siliques, (g) seeds at different developing stages, (h) young seeds (3 to 4 DAP, globular-stage embryo), (i) mid-stage seeds (9 to 10 DAP, mature green embryo). DAP, days after pollination. (D) Sugar-dependent expression of ProJcSDP1:GUS transgenic plants (14?=?day-old seedling). Different sugar sources (1% or 3% fructose, glucose, or sucrose) were used, and mannitol was used as a control for osmotic stress. (E)JcSDP1 gene expression profile using real-time quantitative PCR at different seed development stages (S1, 1 WAF; S2. 2 to 3 WAF; S3; 4 to 6 WAF; and S4; 7 to 8 WAF) JcTubulin expression levels were used as an internal control. Values are given as mean?±?SD (n?=?3). WAF, weeks after fertilization.

Mentions: To specifically silence JcSDP1 gene expression in Jatropha, we isolated a native JcSDP1 promoter fragment from Jatropha genomic DNAs using a GenomeWalker kit. We cloned an approximately 0.7 kb fragment of JcSDP1 proximal genomic locus to the 5′-untranslated region (UTR), which contained several putative cis elements for gene expression and regulation. This region of the JcSDP1 promoter contains a TATA box and a CAAT box, located at positions −13 to −16 and −66 to −69, respectively. The promoter region also contains two putative sugar-responsive elements, TATCCA and TAACAAA, found in the α- amylase gene [20,21], located at positions −154 to −159 and −139 to −145, respectively. In addition, the promoter fragment includes four E-box motifs, CANNTG [22,23], which are likely to be involved in seed-specific expression (Figure 4A).


Gene silencing of Sugar-dependent 1 (JcSDP1), encoding a patatin-domain triacylglycerol lipase, enhances seed oil accumulation in Jatropha curcas.

Kim MJ, Yang SW, Mao HZ, Veena SP, Yin JL, Chua NH - Biotechnol Biofuels (2014)

JcSDP1promoter and its expression analysis inProJcSDP1:GUStransgenic plants. (A) Composition of putative cis elements in JcSDP1 promoter. (B) Transient ProJcSDP1:GUS expression in Jatropha fruit and leaf. (C) Heterologous expressions of ProJcSDP1:GUS in transgenic Arabidopsis plants: (a) 14-day-old seedling, (b, c) inflorescence, (d, e) rosette leaf, (f) siliques, (g) seeds at different developing stages, (h) young seeds (3 to 4 DAP, globular-stage embryo), (i) mid-stage seeds (9 to 10 DAP, mature green embryo). DAP, days after pollination. (D) Sugar-dependent expression of ProJcSDP1:GUS transgenic plants (14?=?day-old seedling). Different sugar sources (1% or 3% fructose, glucose, or sucrose) were used, and mannitol was used as a control for osmotic stress. (E)JcSDP1 gene expression profile using real-time quantitative PCR at different seed development stages (S1, 1 WAF; S2. 2 to 3 WAF; S3; 4 to 6 WAF; and S4; 7 to 8 WAF) JcTubulin expression levels were used as an internal control. Values are given as mean?±?SD (n?=?3). WAF, weeks after fertilization.
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Related In: Results  -  Collection

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Figure 4: JcSDP1promoter and its expression analysis inProJcSDP1:GUStransgenic plants. (A) Composition of putative cis elements in JcSDP1 promoter. (B) Transient ProJcSDP1:GUS expression in Jatropha fruit and leaf. (C) Heterologous expressions of ProJcSDP1:GUS in transgenic Arabidopsis plants: (a) 14-day-old seedling, (b, c) inflorescence, (d, e) rosette leaf, (f) siliques, (g) seeds at different developing stages, (h) young seeds (3 to 4 DAP, globular-stage embryo), (i) mid-stage seeds (9 to 10 DAP, mature green embryo). DAP, days after pollination. (D) Sugar-dependent expression of ProJcSDP1:GUS transgenic plants (14?=?day-old seedling). Different sugar sources (1% or 3% fructose, glucose, or sucrose) were used, and mannitol was used as a control for osmotic stress. (E)JcSDP1 gene expression profile using real-time quantitative PCR at different seed development stages (S1, 1 WAF; S2. 2 to 3 WAF; S3; 4 to 6 WAF; and S4; 7 to 8 WAF) JcTubulin expression levels were used as an internal control. Values are given as mean?±?SD (n?=?3). WAF, weeks after fertilization.
Mentions: To specifically silence JcSDP1 gene expression in Jatropha, we isolated a native JcSDP1 promoter fragment from Jatropha genomic DNAs using a GenomeWalker kit. We cloned an approximately 0.7 kb fragment of JcSDP1 proximal genomic locus to the 5′-untranslated region (UTR), which contained several putative cis elements for gene expression and regulation. This region of the JcSDP1 promoter contains a TATA box and a CAAT box, located at positions −13 to −16 and −66 to −69, respectively. The promoter region also contains two putative sugar-responsive elements, TATCCA and TAACAAA, found in the α- amylase gene [20,21], located at positions −154 to −159 and −139 to −145, respectively. In addition, the promoter fragment includes four E-box motifs, CANNTG [22,23], which are likely to be involved in seed-specific expression (Figure 4A).

Bottom Line: We cloned Jatropha JcSDP1, and verified its function by complementation of the Arabidopsis sdp1-5 mutant.Taking advantage of the observation with Arabidopsis, we used RNAi technology to generate JcSDP1 deficiency in transgenic Jatropha.Based on this result, we generated SDP1-deficient transgenic Jatropha plants using by RNAi technology with a native JcSDP1 promoter to silence endogenous JcSDP1 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA. chua@mail.rockefeller.edu.

ABSTRACT

Background: Triacylglycerols (TAGs) are the most abundant form of storage oil in plants. They consist of three fatty acid chains (usually C16 or C18) covalently linked to glycerol. SDP1 is a specific lipase for the first step of TAG catabolism in Arabidopsis seeds. Arabidopsis mutants deficient in SDP1 accumulate high levels of oils, probably due to blockage in TAG degradation. We applied this knowledge from the model plant, Arabidopsis thaliana, to engineer increased seed oil content in the biodiesel plant Jatropha curcas using RNA interference (RNAi) technology.

Results: As Jatropha is a biodiesel crop, any significant increase in its seed oil content would be an important agronomic trait. Using A. thaliana as a model plant, we found that a deficiency of SDP1 led to higher TAG accumulation and a larger number of oil bodies in seeds compared with wild type (Columbia-0; Col-0). We cloned Jatropha JcSDP1, and verified its function by complementation of the Arabidopsis sdp1-5 mutant. Taking advantage of the observation with Arabidopsis, we used RNAi technology to generate JcSDP1 deficiency in transgenic Jatropha. We found that Jatropha JcSDP1-RNAi plants accumulated 13 to 30% higher total seed storage lipid, along with a 7% compensatory decrease in protein content, compared with control (CK; 35S:GFP) plants. Free fatty acid (FFA) content in seeds was reduced from 27% in control plants to 8.5% in JcSDP1-RNAi plants.

Conclusion: Here, we showed that SDP1 deficiency enhances seed oil accumulation in Arabidopsis. Based on this result, we generated SDP1-deficient transgenic Jatropha plants using by RNAi technology with a native JcSDP1 promoter to silence endogenous JcSDP1 expression. Seeds of Jatropha JcSDP1-RNAi plants accumulated up to 30% higher total lipid and had reduced FFA content compared with control (CK; 35S:GFP) plants. Our strategy of improving an important agronomic trait of Jatropha can be extended to other oil crops to yield higher seed oil.

No MeSH data available.


Related in: MedlinePlus