Plant materials and growth condition
Arabidopsis Col-0 was grown on soil with sand. Seeds of the homozygous sdp1-5 mutant (Salk_076697, Arabidopsis Biological Resource Center (ABRC), Ohio State University, Columbus, OH, USA) were stratified at 4°C for 3 days on soil and germinated, then seedlings were grown on in a growth chamber under 16 hour light/8 hour dark cycles, at 23°C?±?3°C under white light (100 to 150 μE/m/s photosynthetically active radiation). Rosette leaves were harvested from 3 to 4-week-old plants, and other organs such as inflorescence stems, cauline leaves, flowers, siliques, and developing seeds were harvested from 6-week-old plants. We considered seeds from siliques at 3 to 5 days after pollination) to be at the early stage of seed maturation in Arabidopsis. We collected leaves and developing fruits (S1: 1 week after fertilization (WAF) S2: 2 to 3 WAF, S3: 4 to 6 WAF, and S4: 7 to 8 WAF) from Jatropha plants grown in a greenhouse in Singapore.
RNA isolation and quantitative real-time PCR
Total RNA was isolated from plant samples using TRIzol reagent (Invitrogen, Carlsbad, CA USA) in accordance with the manufacturer’s instructions. cDNA was synthesized with 1 μg total RNA using MMLV Superscript II (Promega, Madison, WI, USA) after DNase I treatment (Roche Applied Science, Mannheim, Germany). Quantitative real-time PCR assay was performed on an ABI 7900 sequence Detection System (Applied Biosystems, Foster City, CA, USA). We used power SYBR Green PCR Master Mix (Applied Biosystems), using the manufacturer’s reagent protocol, but reducing the volume to 10 μl per reaction. As controls, we used the species-specific tubulin and actin primer sets for J. curcas and A. thaliana, respectively. Fold change values of the target gene transcripts were subsequently normalized by dividing the ∆Ct values by the ∆Ct values of each control gene transcript. All real-time PCR experiments were performed in triplicate using different biological samples. Sequences of primers used in PCR procedures are listed in Additional file 7.
Isolation of JcSDP1 full-length cDNA and its promoter
We found a partial SDP1 sequence in our J. curcas expressed sequence tag database. Using this partial sequence, we designed 5′ or 3′ cDNA RACE primers and performed RACE experiments using BD SMART™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA). We used 1 μg of total RNA derived from developing seeds (S2 to S3) for generation of 5′ or 3′ RACE pools. The 5′ and 3′ cDNA ends were obtained by touchdown PCR with the Advantage 2 PCR Enzyme System (Clontech), following the manufacturer’s instructions. To amplify the products specifically, the primary RACE products were diluted and mixed with a nested gene-specific primer (GSP2) and nested universal primer mixture (AP2). The PCR products were gel-purified and cloned into pDrive Cloning vector (Qiagen, Düsseldorf, Germany) to obtain full-length JcSDP1 cDNA (2577-bp). A Universal GenomeWalker Kit (Clontech) was used to isolate the promoter fragment of the JcSDP1 gene. Genomic DNA was digested with DraI, EcoRV, PvuII, SspI, and StuI endonucleases, and five libraries of adaptor-ligated genomic fragments were constructed. These genomic DNA libraries were used as templates for the PCR for promoter isolation. For each round of genome walking, the primary PCR products were amplified by a gene-specific primer (GSP1) and the outer adaptor primer (AP1). For the second PCR mix, the primary products were diluted and used as templates with a nested gene-specific primer (GSP2) and the nested adaptor primer (AP2). The secondary PCR products were then separated in agarose gels, and the relevant DNA fragment purified with QIAEXII Gel Extraction Kit (Qiagen), cloned into pDrive Cloning vector, and sequenced. Potential cis elements in the promoter region were analyzed using computational analytical methods available on two public websites, PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PLACE (http://www.dna.affrc.go.jp/PLACE/).
Construction of the ProJcSDP1:GUS fusion gene and analysis of promoter
We used the pKGWFS7 destination vector including GFP or GUS reporter gene for promoter analysis. The 5′-flanking region of JcSDP1 was amplified using two specific primers, JcSDP1-PF1T and JcSDP1-PR1. This promoter was 873 bp in length, including 149 bp of 5′-UTR. The amplified PCR product was inserted into a TOPO donor vector using the pENTRTM/D-TOPO Cloning Kit (Invitrogen), and then inserted into the destination vector, pKGWFS7, using the Gateway LR Clonase™ II enzyme mix (Invitrogen). For plant transformation, the constructs were introduced by electroporation into Agrobacterium tumefaciens strain AGL1. Constructs were transformed into WT (Col-0) or sdp1-5 mutant (Salk_076697) background through the floral dip method described by Clough and Bent .
For transient assay of JcSDP1 promoter expression, 10 μg of ProJcSDP1:GUS in pKGWFS7 plasmid DNA was coated with 1 μm diameter gold particles (2.5 mg gold particles, 200 μl of 2.5 M CaCl2 and 100 μl of 0.1 M spermidine). After being incubated on ice for 30 minutes, the pellet of DNA/gold particle was washed twice with 70% ethanol and resuspended in 100% ethanol. Jatropha fruits and leaves were centered in a petri dish containing MS agar medium . Fruits and leaves were bombarded at 1,350 psi with a biolistic helium gun device (PDS-1000/He, Bio-rad, Hercules, CA, USA). After incubation for 2 days at 25°C, the bombarded tissues were analyzed by histochemical assays as described previously by Jefferson et al. . Tissues were incubated in GUS staining buffer (0.1 M Sodium phosphate pH 7.0, 1 mM 5-bromo-4-chloro-3-indolyl-D-glucuronide (Sigma-Aldrich, St Louis, MO, USA), 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM Na2EDTA, and 0.1% Triton X-100) for 20 hours at 37°C. Stained tissues were rinsed with 70% to 80% ethanol until pigments had been cleared. Selected organs transgenic Arabidopsis lines expressing ProJcSDP1:GUS were analyzed for GUS expression. To investigate sugar-responsive expression of JcSDP1 promoter, we used 14-day-old seedlings of T2 lines, seedlings were incubated for 24 hours in an MS media with 1% or 3% of a sugar source: sucrose, fructose, and glucose. Mannitol was used as a control for osmotic stress.
Complementation of the JcSDP1/sdp1-5 mutation in Arabidopsis
Full-length JcSDP1 cDNA was cloned into a vector harboring a tandem CaMV35S promoter and a Nos polyA addition sequence from pCAMBIA 1300, and transformed into Arabidopsis sdp1-5 mutant (Salk_076697). T1 transformants were selected on 40 μg/ml hygromycin, and T2 seedlings were assayed for sugar responsiveness on MS agar plates with or without sucrose.
Construction of inducible marker-free ProJcSDP1:JcSDP1-RNAi vector and transformation of Jatropha plants
We used the pCCreloxP (pCCLB3) inducible marker-free vector system as described by Qiu et al. . The transfer DNA (T-DNA) of pCCreloxP vector harbors a loxP fragment that consists of CRE-int, HPT and XVE genes (Figure 5A). To silence JcSDP1 expression using RNAi, we selected a 373-bp fragment that includes 290 bp of the 3′ coding region of JcSDP1 cDNA with a stop codon and 83 bp of the 3′ UTR region. The 373-bp JcSDP1 fragment was amplified with two primer sets (JcSDP1-RNAiF-XhoI and JcSDP1-RNAiR2-HindIII or JcSDP1-RNAiF-BamHI and JcSDP1-RNAiR2-PstI, used for the sense orientation or anti-sense orientation, respectively), and the fragments were then cloned into pBluescript-SK intron vector (pBS-SKi) which has an intron (156 bp) to generate pJcSDP1-RNAi as previously described . ProJcSDP1 was amplified with two primers, JcSDP1-PF1-ApaI and JcSDP1-PR1-XhoI, and then restricted with ApaI/XhoI. To insert the ProJcSDP1 (JcSDP1 promoter) fragment into the pJcSDP1-RNAi construct, we used ApaI and XhoI restriction enzyme sites in the construct. A 226-bp fragment containing the CaMV35S polyA addition sequence (T35S) was amplified with T35S-F-XbaI and T35S-R-Pml-SacII primers. The amplified T35S fragment was cleaved with XbaI/SacII restriction enzymes, and then cloned into ProJcSDP1-RNAi. Finally, the ProJcSDP1:JcSDP1-RNAi:T
fragment from the ProJcSDP1-RNAi construct was inserted into the pCCreloxP marker-free vector at the ApaI and PmlI sites. All constructs were introduced into A. tumefaciens strain AGL1 using electroporation, and then transformed into Jatropha using cotyledon explants .
Fatty acid analysis in Arabidopsis and Jatropha
We used GC to analyze the FA profile of sdp1-5 as described by Li et al. . For this, 100 dried seeds of each line were weighed, and samples were transmethylated at 85°C for 2 hours in a reaction buffer (1 ml of 3 M HCl-methanol, 25 μl of butylhydroxytoluene solution, and 300 μl of toluene (all Sigma-Aldrich)]. As an internal standard, 50 μg of pentadecanoic acid (C15:0; Sigma-Aldrich) was added to each sample. After the samples had been cooled down to room temperature, 1.5 ml of 0.9% NaCl (w/v) was added to the mix, and the FA methyl esters (FAMEs) were extracted two times with 1 ml of hexane. Extracts were evaporated under nitrogen and then dissolved in 100 μl of hexane. The final extracts were analyzed with GC using a flame ionization detector (FID) on Agilent 6890 (Agilent, Santa Clara, CA, USA) employing helium as the carrier gas. Total FAs were estimated by comparing the total FAME peak area (pA*sec) to that of the C15:0.
To analyze total lipid content in Jatropha transgenic lines, we used three changes of hexane 3 to extract all lipid components in dried endosperm , which includes mainly TAG, and sterols, FFA, DAG, and monoacylglycerol (MAG) components. To profile FA composition, about 10 mg of total lipid were transmethylated at 70°C for 20 minutes in a reaction buffer [1 ml of 3 N methanolic-HCl, 400 μl of 2,2, dimethoxypropane (Sigma-Aldrich) and 50 μg of pentadecanoic acid (C15:0)]. After being cooled down to room temperature, the FAMEs were extracted by twice 1 ml of water and 1 ml of hexane extraction. Extracts were evaporated under nitrogen and then dissolved in 500 μl of hexane. The final samples were analyzed in a GCMS-QP2010 (Shimadzu, Japan). SD was calculated based on several different plants.
Analysis of lipids by TLC and quantification by GC/MS
Seed lipids were extracted with hexane three times [7, 46]. After determination of total lipid amount, 300 μg of neutral lipid were fractionated by TLC on silica gel plates in a running solvent mixture (hexane: ethyl acetic acid: acetic acid; 90:10:1, respectively, by volume). Triolein (T7140; Sigma-Aldrich) and oleic acid (75090; Sigma-Aldrich) were used as a standard of TAG and of FFA, respectively. TLC plates were exposed to iodine (I2) vapor for visualization. The separated neutral lipid species, including TAG and FFA, were recovered from the plates using hexane, and quantified by GC/MS after conversion to their corresponding methyl esters by the methanolic-HCl method as described by Li et al. . The absolute amount was calculated using C15:0 as an internal standard and by comparing the relative peak areas.
Protein and carbohydrate analysis
Protein content in Jatropha transgenic lines was analyzed as described by Focks and Benning , using 50 mg of dried endosperm. Protein amounts were measured by the Lowry DC protein assay (Bio-Rad) using γ-globulin as a standard. To analyze carbohydrate content, 50 mg of dried endosperm were homogenized in 200 μl of assay buffer and centrifuged at full speed. The extracted supernatant was used for carbohydrate quantification using a Total Carbohydrate Assay Kit (Sigma-Aldrich). D-glucose was used as a standard for calibration.
Genomic DNA isolation and Southern blotting
Total genomic DNA was isolated from leaves from transgenic or control (CK; 35S:GFP) plants grown in a greenhouse, using cetyltrimethylammonium bromide (Sigma-Aldrich) . Genomic DNA was digested with restriction enzymes and separated in 0.8% agarose gels. The gels were processed and blotted onto Hybond-N+ membranes (Roche Applied Science) following standard procedures . Probes were prepared with PCR DIG Labelling Mix using specific primer sets for the hygromycin phosphotransferase (HPT) gene and the JcSDP1 gene. Hybridization was performed using PCR DIG detection kit following the supplier’s instructions (Roche Applied Science).
TEM and SEM
TEM was performed with mature dried seeds of WT (Col-0) and sdp1-5 mutant. Seeds were embedded in resin and sectioned on an ultramicrotome (Leica Ultracut UCT; Leica, Wetzlar, Germany) set at 70 nm thickness. Sectioned samples were placed onto 300 mesh copper grids. Sections were examined, and pictures were taken with a TEM (JEM-1230; JEOL, Tokyo, Japan) at 120 kV. For SEM analysis, dried seeds were mounted directly and examined under a JSM-6360LV SEM (JEOL) with an acceleration voltage of 20 kV.
Seed weight and size measurement
Mature seeds were harvested from WT (Col-0) and sdp1-5 mutant grown under the same conditions. A sample size of 100 seeds per WT (Col-0) or sdp1-5 mutant was used to obtain an average seed weight with at least five biological replications. Values (n?=?5) are given as mean?±?SD. A DM5000B microscope (Leica) and ImageJ analysis software were used to measure seed sizes. Values (n?=?10) are given as mean?±?SD.