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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

Production ofProJcSDP1:JcSDP1-RNAi transgenic Jatropha plants and analysis of seed oils. (A) Structure of the inducible ProJcSDP1:JcSDP1-RNAi marker-free construct. (B)ProJcSDP1:JcSDP1-RNAi marker-free transgenic plants: (a-c) T1 generation of dried seeds; (d) overall phenotype of T1 transgenic plants generated from T1 embryos; (e) size comparison of the fifth leaf between control (CK; 35S:GFP) plants and the JcSDP1-RNAi transgenic line #158. (C) Analysis of total lipid content in mature endosperm of T1 seed. (D) Fatty acid profile in JcSDP1-RNAi transgenic lines. (E)JcSDP1 transcript levels in transgenic Jatropha lines. JcTubulin expression levels were used as an internal control. Numbers refer to transgenic line numbers. Asterisks indicate a statistically significant difference compared with the control, *P?<?0.05, **P?<?0.01, ***P?<?0.001 versus control (CK; 35S:GFP) plants, in different biological replicates.
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Figure 5: Production ofProJcSDP1:JcSDP1-RNAi transgenic Jatropha plants and analysis of seed oils. (A) Structure of the inducible ProJcSDP1:JcSDP1-RNAi marker-free construct. (B)ProJcSDP1:JcSDP1-RNAi marker-free transgenic plants: (a-c) T1 generation of dried seeds; (d) overall phenotype of T1 transgenic plants generated from T1 embryos; (e) size comparison of the fifth leaf between control (CK; 35S:GFP) plants and the JcSDP1-RNAi transgenic line #158. (C) Analysis of total lipid content in mature endosperm of T1 seed. (D) Fatty acid profile in JcSDP1-RNAi transgenic lines. (E)JcSDP1 transcript levels in transgenic Jatropha lines. JcTubulin expression levels were used as an internal control. Numbers refer to transgenic line numbers. Asterisks indicate a statistically significant difference compared with the control, *P?<?0.05, **P?<?0.01, ***P?<?0.001 versus control (CK; 35S:GFP) plants, in different biological replicates.

Mentions: We generated JcSDP1-RNAi transgenic Jatropha plants to see if a reduction of SDP1 expression in Jatropha would also lead to increased seed oil accumulation. As we wanted to specifically control lipid accumulation in the mature seeds without ectopic effects, the JcSDP1-RNAi transgene was placed under the control of the native JcSDP1 promoter, which is seed-specific. Through a two-step selection process using hygromycin and β-estradiol, marker-free or non-marker-free transgenic plants were screened. These plants were further confirmed by genotyping with specific primer sets for HygF and HygR for the hygromycin-resistance gene, and P1 and R16 or P1 and T35S-R for the marker-free transgene. Based on the genotyping results, we obtained chimeric (heterozygote) marker-free plants in which the antibiotic selection marker was partially removed by homologous recombination. We recovered several independent lines in which growth patterns, growth rates, leaf number, and leaf size were all normal (Figure 5B–E). In contrast to the enlarged seed size found in Arabidopsis sdp1-5, T1 mature seeds from JcSDP1-RNAi plants were normal in size (Figure 5B-a to -c). To investigate total lipid content in T1 mature seeds, we carefully separated endosperms from embryos. The endosperms were used for further molecular and biochemical analysis, while the embryos were regenerated to maintain the transgenic lines.


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)

Production ofProJcSDP1:JcSDP1-RNAi transgenic Jatropha plants and analysis of seed oils. (A) Structure of the inducible ProJcSDP1:JcSDP1-RNAi marker-free construct. (B)ProJcSDP1:JcSDP1-RNAi marker-free transgenic plants: (a-c) T1 generation of dried seeds; (d) overall phenotype of T1 transgenic plants generated from T1 embryos; (e) size comparison of the fifth leaf between control (CK; 35S:GFP) plants and the JcSDP1-RNAi transgenic line #158. (C) Analysis of total lipid content in mature endosperm of T1 seed. (D) Fatty acid profile in JcSDP1-RNAi transgenic lines. (E)JcSDP1 transcript levels in transgenic Jatropha lines. JcTubulin expression levels were used as an internal control. Numbers refer to transgenic line numbers. Asterisks indicate a statistically significant difference compared with the control, *P?<?0.05, **P?<?0.01, ***P?<?0.001 versus control (CK; 35S:GFP) plants, in different biological replicates.
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Figure 5: Production ofProJcSDP1:JcSDP1-RNAi transgenic Jatropha plants and analysis of seed oils. (A) Structure of the inducible ProJcSDP1:JcSDP1-RNAi marker-free construct. (B)ProJcSDP1:JcSDP1-RNAi marker-free transgenic plants: (a-c) T1 generation of dried seeds; (d) overall phenotype of T1 transgenic plants generated from T1 embryos; (e) size comparison of the fifth leaf between control (CK; 35S:GFP) plants and the JcSDP1-RNAi transgenic line #158. (C) Analysis of total lipid content in mature endosperm of T1 seed. (D) Fatty acid profile in JcSDP1-RNAi transgenic lines. (E)JcSDP1 transcript levels in transgenic Jatropha lines. JcTubulin expression levels were used as an internal control. Numbers refer to transgenic line numbers. Asterisks indicate a statistically significant difference compared with the control, *P?<?0.05, **P?<?0.01, ***P?<?0.001 versus control (CK; 35S:GFP) plants, in different biological replicates.
Mentions: We generated JcSDP1-RNAi transgenic Jatropha plants to see if a reduction of SDP1 expression in Jatropha would also lead to increased seed oil accumulation. As we wanted to specifically control lipid accumulation in the mature seeds without ectopic effects, the JcSDP1-RNAi transgene was placed under the control of the native JcSDP1 promoter, which is seed-specific. Through a two-step selection process using hygromycin and β-estradiol, marker-free or non-marker-free transgenic plants were screened. These plants were further confirmed by genotyping with specific primer sets for HygF and HygR for the hygromycin-resistance gene, and P1 and R16 or P1 and T35S-R for the marker-free transgene. Based on the genotyping results, we obtained chimeric (heterozygote) marker-free plants in which the antibiotic selection marker was partially removed by homologous recombination. We recovered several independent lines in which growth patterns, growth rates, leaf number, and leaf size were all normal (Figure 5B–E). In contrast to the enlarged seed size found in Arabidopsis sdp1-5, T1 mature seeds from JcSDP1-RNAi plants were normal in size (Figure 5B-a to -c). To investigate total lipid content in T1 mature seeds, we carefully separated endosperms from embryos. The endosperms were used for further molecular and biochemical analysis, while the embryos were regenerated to maintain the transgenic lines.

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