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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

sdp1-5 mutant accumulates higher triacylglycerol (TAG) levels than wild type (Columbia-0; Col-0) in mature seeds. (A) Scanning electron microscopy (SEM) showing the seed surface structure of WT (Col-0) and sdp1-5; (B) comparative dry seed weight of WT (Col-0) and sdp1-5; (C) total amount of FAs per seed of WT (Col-0) and sdp1-5; (D) relative FAs of dried seeds of WT (Col-0) and sdp1-5; (E) FA profile of WT (Col-0) and sdp1-5 seeds. (F) Seed eicosenoic acid (20:1) content; (G) thin layer chromatography (TLC) separation of neutral lipid fractions from WT (Col-0) and three lines of sdp1-5  mutant; 300 μg of neutral lipids were fractionated by TLC on silica gel plates. DAG, diacylglycerol, FA, fatty acid; FFA, free fatty acid (oleic acid), Mix, mixture of TAG and FFA; SE, sterol ester; TAG, Triacylglycerol (triolein). (H) Profiling of relative amounts of FFA and TAG by GC/MS. The absolute amount was calculated using C15:0 as an internal control by comparing their peak areas. *P?<?0.05 or **P?<?0.01 versus WT (Col-0). Each experiment was performed with 100 seeds per line with 5 biological replicates. Error bar shows standard deviation (SD) (n?=?5). DW, dry weight.
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Figure 1: sdp1-5 mutant accumulates higher triacylglycerol (TAG) levels than wild type (Columbia-0; Col-0) in mature seeds. (A) Scanning electron microscopy (SEM) showing the seed surface structure of WT (Col-0) and sdp1-5; (B) comparative dry seed weight of WT (Col-0) and sdp1-5; (C) total amount of FAs per seed of WT (Col-0) and sdp1-5; (D) relative FAs of dried seeds of WT (Col-0) and sdp1-5; (E) FA profile of WT (Col-0) and sdp1-5 seeds. (F) Seed eicosenoic acid (20:1) content; (G) thin layer chromatography (TLC) separation of neutral lipid fractions from WT (Col-0) and three lines of sdp1-5 mutant; 300 μg of neutral lipids were fractionated by TLC on silica gel plates. DAG, diacylglycerol, FA, fatty acid; FFA, free fatty acid (oleic acid), Mix, mixture of TAG and FFA; SE, sterol ester; TAG, Triacylglycerol (triolein). (H) Profiling of relative amounts of FFA and TAG by GC/MS. The absolute amount was calculated using C15:0 as an internal control by comparing their peak areas. *P?<?0.05 or **P?<?0.01 versus WT (Col-0). Each experiment was performed with 100 seeds per line with 5 biological replicates. Error bar shows standard deviation (SD) (n?=?5). DW, dry weight.

Mentions: We first examined the effect of SDP1 deficiency on seed development, total FA content, and FA profile. Using scanning electron microscopy (SEM), we found that sdp1-5 seeds were slightly larger than those of WT (Col-0) in length and width (Figure 1A; see Additional file 2). In addition, sdp1-5 seeds displayed an increase in dry weight of around 11.5% compared with WT. To investigate the role of SDP1 in lipid accumulation in seeds, total FA content and FA composition in dried seeds were compared between WT (Col-0) and sdp1-5 mutant. The average dry seed weight of WT (Col-0) was about 19 μg, containing approximately 5.54 μg of total FAs (Figure 1B, C). The seed lipid content obtained for the WT was very similar to those reported by others, which is around 30 to 35% of dry seed weight. However, sdp1-5 seeds had an average dry weight of around 22 μg per seed, containing 7.17 μg of total FAs. Therefore, the levels of FAs in sdp1-5 seeds were about 10% higher than found in WT (Col-0) seeds (Figure 1D). In addition, in sdp1-5 seeds, there was a clear increase in the relative proportion of unsaturated FAs, such as eicosenoic acid (C20:1) (Figure 1E, F). To characterize TAG accumulation in mature dried seeds, we analyzed total neutral lipid from WT (Col-0) and three homozygous lines of sdp1-5 allele by thin layer chromatography (TLC) on silica gel plates, and found reduced levels of FFAs in sdp1-5 compared with WT (Col-0) (Figure 1G). Triolein and oleic acid were used as standards of TAG and FFA, respectively. To obtain quantitative data, we analyzed the samples by gas chromatography and mass spectrometry (GC/MS), using pentadecanoic acid (C15:0) as an internal control for quantification. The sdp1-5 mutant had about 4.25% FFA and 95.75% TAG, compared with 13.35% FFA and 86.65% TAG in WT (Col-0) (Figure 1H).


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)

sdp1-5 mutant accumulates higher triacylglycerol (TAG) levels than wild type (Columbia-0; Col-0) in mature seeds. (A) Scanning electron microscopy (SEM) showing the seed surface structure of WT (Col-0) and sdp1-5; (B) comparative dry seed weight of WT (Col-0) and sdp1-5; (C) total amount of FAs per seed of WT (Col-0) and sdp1-5; (D) relative FAs of dried seeds of WT (Col-0) and sdp1-5; (E) FA profile of WT (Col-0) and sdp1-5 seeds. (F) Seed eicosenoic acid (20:1) content; (G) thin layer chromatography (TLC) separation of neutral lipid fractions from WT (Col-0) and three lines of sdp1-5  mutant; 300 μg of neutral lipids were fractionated by TLC on silica gel plates. DAG, diacylglycerol, FA, fatty acid; FFA, free fatty acid (oleic acid), Mix, mixture of TAG and FFA; SE, sterol ester; TAG, Triacylglycerol (triolein). (H) Profiling of relative amounts of FFA and TAG by GC/MS. The absolute amount was calculated using C15:0 as an internal control by comparing their peak areas. *P?<?0.05 or **P?<?0.01 versus WT (Col-0). Each experiment was performed with 100 seeds per line with 5 biological replicates. Error bar shows standard deviation (SD) (n?=?5). DW, dry weight.
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Related In: Results  -  Collection

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Figure 1: sdp1-5 mutant accumulates higher triacylglycerol (TAG) levels than wild type (Columbia-0; Col-0) in mature seeds. (A) Scanning electron microscopy (SEM) showing the seed surface structure of WT (Col-0) and sdp1-5; (B) comparative dry seed weight of WT (Col-0) and sdp1-5; (C) total amount of FAs per seed of WT (Col-0) and sdp1-5; (D) relative FAs of dried seeds of WT (Col-0) and sdp1-5; (E) FA profile of WT (Col-0) and sdp1-5 seeds. (F) Seed eicosenoic acid (20:1) content; (G) thin layer chromatography (TLC) separation of neutral lipid fractions from WT (Col-0) and three lines of sdp1-5 mutant; 300 μg of neutral lipids were fractionated by TLC on silica gel plates. DAG, diacylglycerol, FA, fatty acid; FFA, free fatty acid (oleic acid), Mix, mixture of TAG and FFA; SE, sterol ester; TAG, Triacylglycerol (triolein). (H) Profiling of relative amounts of FFA and TAG by GC/MS. The absolute amount was calculated using C15:0 as an internal control by comparing their peak areas. *P?<?0.05 or **P?<?0.01 versus WT (Col-0). Each experiment was performed with 100 seeds per line with 5 biological replicates. Error bar shows standard deviation (SD) (n?=?5). DW, dry weight.
Mentions: We first examined the effect of SDP1 deficiency on seed development, total FA content, and FA profile. Using scanning electron microscopy (SEM), we found that sdp1-5 seeds were slightly larger than those of WT (Col-0) in length and width (Figure 1A; see Additional file 2). In addition, sdp1-5 seeds displayed an increase in dry weight of around 11.5% compared with WT. To investigate the role of SDP1 in lipid accumulation in seeds, total FA content and FA composition in dried seeds were compared between WT (Col-0) and sdp1-5 mutant. The average dry seed weight of WT (Col-0) was about 19 μg, containing approximately 5.54 μg of total FAs (Figure 1B, C). The seed lipid content obtained for the WT was very similar to those reported by others, which is around 30 to 35% of dry seed weight. However, sdp1-5 seeds had an average dry weight of around 22 μg per seed, containing 7.17 μg of total FAs. Therefore, the levels of FAs in sdp1-5 seeds were about 10% higher than found in WT (Col-0) seeds (Figure 1D). In addition, in sdp1-5 seeds, there was a clear increase in the relative proportion of unsaturated FAs, such as eicosenoic acid (C20:1) (Figure 1E, F). To characterize TAG accumulation in mature dried seeds, we analyzed total neutral lipid from WT (Col-0) and three homozygous lines of sdp1-5 allele by thin layer chromatography (TLC) on silica gel plates, and found reduced levels of FFAs in sdp1-5 compared with WT (Col-0) (Figure 1G). Triolein and oleic acid were used as standards of TAG and FFA, respectively. To obtain quantitative data, we analyzed the samples by gas chromatography and mass spectrometry (GC/MS), using pentadecanoic acid (C15:0) as an internal control for quantification. The sdp1-5 mutant had about 4.25% FFA and 95.75% TAG, compared with 13.35% FFA and 86.65% TAG in WT (Col-0) (Figure 1H).

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