Homologous electron transport components fail to increase fatty acid hydroxylation in transgenic Arabidopsis thaliana

Plant materials and growth conditions

Seeds from Arabidopsis (ecotype Columbia-0) containing the RcFAH12 transgene line CL37¹³ were stratified for 2–3 d at 4°C and germinated on 1× MS medium (Sigma-Aldrich) supplemented with 1% (w/v) sucrose and 0.75% agar. Seeds were germinated with a 16 h day/8 h night cycle at 22°C with 80 µmol m^-2 s^-1 light. Ten to 12 day old seedlings were then transferred to soil and grown under 24 h light at 22°C with 200 µmol m^-2 s^-1 light at rosette height in a growth chamber or under a 16 h day/8 h night cycle at 22°C with greater than 300 µmol m^-2 s^-1 light in the greenhouse.

Real-time PCR

RNA was previously extracted by Dr. Chaofu Lu of Montana State University from castor developing endosperm, when the RcFAH12 is most highly expressed¹⁹. This RNA was then used to synthesize cDNA using Superscript III (Life Technologies) reverse transcriptase. Quantitative reverse transcription PCR (RT-PCR) was then performed with a MX3005P QPCR System (Stratagene) using the DNA binding fluorescent dye SYBR Green I (Life Technologies) and five sets of primers, amplifying full-length cDNA from each gene, see Supplementary Table 1 for list of primers. The collected data was then normalized to the RcACTIN control gene and the relative fold change was calculated (=1/(2^(experimental-control))).

Identification of castor genes and cloning procedures

Castor genes were subcloned from a castor seed cDNA library¹⁹ or cloned using castor endosperm RNA extracted by Dr. Lu. Reverse transcriptase (Superscript™ III First-Strand Synthesis System; Life Technologies) was used to make cDNA from the castor RNA. Full-length transcripts were amplified from cDNA with KOD polymerase (Takara). The 5´ start primer contained the CACC sequence for directional topoisomerase cloning into pENTR-D-Topo (Life Technologies) see Supplementary Table 1 for the list of primers. The castor genes were then sequenced with vector primers (M13 forward and M13 reverse, Life Technologies) and compared with the castor genome at The Institute of Genomic Research (TIGR) castor genome database (http://castorbean.tigr.org/)²⁰. These castor genes were renamed as follows: RcCb5-1, 28014.m000117; RcCb5-2, 30213.m000673; RcCb5-3, 29904.m002991; RcCb5-4, 30204.m001761; RcCb5-5, 29912.m005430; and RcCb5-6, 30174.m009087. From the entry vectors, these genes were subcloned into gateway compatible plant transformation vectors containing the seed specific β-phaseolin promoter, pGate-Phas-Basta (pGPB) or pGate-Phas-dsRed (pGPD), using LR Clonase I (Life Technologies). These constructs were then transformed into Agrobacterium GV3101 and used for plant transformations.

Dr. Edgar Cahoon (University of Nebraska-Lincoln) graciously provided a dual-gene plant transformation vector (renamed pEC-dsRed) along with a cassette vector pKMS2, which contains the seed-specific oleosin promoter. The pEC-dsRed vector contains two multiple cloning sites and the dsRed selection marker. This pEC-dsRed vector was made gateway compatible at the multiple cloning site containing the storage protein promoter glycinin. For cloning into pEC-dsRed, the RcCBR1 gene was subcloned from pENTR into pCR-Script (Stratagene) with NotI restriction sites on both primers. RcCBR1 gene out of pCR-Script and subclone it into the NotI site of pKMS2, which contains the oleosin promoter. The AscI sites were used to transfer the oleosin-RcCBR1 cassette from pKMS2 and into pEC-dsRed. The pEC-dsRed-RcCBR1 was then used for three individual gateway reactions with each of the three RcCb5 entry vectors using LR Clonase I (Life Technologies), so that the oleosin promoter controlled the RcCBR1 expression and the glycinin promoter controlled the expression of the RcCb5 genes. This cloning resulted in the three binary plant transformation vectors pEC-dsRed-RcCBR1-RcCb5-2, pEC-dsRed-RcCBR1-RcCb5-3, and pEC-dsRed-RcCBR1-RcCb5-4 that were transformed into the Agrobacterium strain GV3101.

Transformations

Line CL37¹³ was transformed with constructs mentioned above using the floral dip method²¹. In brief, a 72 h 500 mL culture of Agrobacterium containing the plasmid was resuspended in 5% sucrose and 0.05% Silwet L-77 (Lehle Seeds), flowers were dipped in the solution for 30 seconds, and plants were covered with plastic wrap over-night. For constructs containing the dsRed selection marker (pGPD and pEC-dsRed), T₁ and T₂ selection was conducted using a green LED light with a red filter¹³. For the pGPB constructs, T₁ transformants were selected for Basta® (Bayer) resistance on 1× MS-agar supplemented with 1% sucrose, containing 20 µg/mL glufosinate-ammonium.

Fatty acid analysis

Fatty acid methyl esters were prepared from 20–50 seeds and analyzed by gas chromatography²². Statistical analyses were conducted by an unpaired two-tailed t-test in Excel, with a 95% confidence (P < 0.05).

Gene expression analysis

Ten to 12 developing siliques from select lines were frozen in liquid nitrogen and used for RNA extraction according to the protocol described by Onate-Sanchez and Vicente-Carbajosa²³, with minor modifications: tissue was ground in liquid nitrogen with a mortar and pestle and transferred into a pre-chilled microcentrifuge tube where 550 µL of extraction buffer (0.4 M LiCl, 0.2 M Tris pH 8, 25 mM EDTA, 1% SDS) and 550 µL of chloroform were added²³. The tubes were vortexed and then centrifuged for 3 min at 14,000 × g at 4°C. 500 µL of water-saturated acidic phenol, 200 µl of chloroform, and 8 µL of iso-amyl alcohol were added to the supernatant and the tubes were vortexed followed by centrifugation for 3 min. The supernatant was extracted twice with 500 µL of chloroform and 1/3 of a volume of 8 M LiCl was added to the resulting supernatant. The tubes were incubated at 20°C for 1.5 h and then centrifuged for 30 min at 14,000 × g at 4°C. The pellet was dissolved in 20 µL DEPC-water and subjected to DNaseI treatment according to the DNA-free RNA kit (Zymo). Complementary DNA was synthesized using Superscript III reverse transcriptase (Life Technologies) and the full-length castor genes were amplified using KOD polymerase (Takara), see Supplementary Table 1 for the list of primers.

Identification of castor cytochrome b5 genes

In Arabidopsis, five cytochrome b5 proteins (AtCb5-A to AtCb5-E) and one AtCb5-like protein receive electrons from NADH:cytochrome b5 reductase encoded by AtCBR1^24,25. In developing Arabidopsis seeds, the most highly expressed genes encode AtCb5-E (At5g53560) and AtCb5-D (At5g48810); AtCb5-B (At2g32720) is also strongly expressed, but there is only weak expression of the remaining three genes^26,27. The AtCb5-E and AtCb5-D genes are also highly expressed in other tissues of the plant. Proteins encoded by these three strongly expressed isoforms are predicted to localize to the endoplasmic reticulum, based on their homology to Cb5 proteins from tung tree (Vernicia fordii) VfCb5-A, VfCb5-B and VfCb5-C that have been shown to be targeted exclusively to the endoplasmic reticulum²⁸.

The Arabidopsis and Vernicia protein sequences were used to search the castor genome²⁰ and identify likely RcCb5 orthologues. Sequences from all three species were used to derive an unrooted dendrogram (Figure 1) showing the phylogenetic relationships among the proteins. The castor isoforms designated RcCb5-2, RcCb5-3 and RcCb5-4 (see Materials and methods for accession numbers) fall into a distinct clade with the Vernicia and Arabidopsis endoplasmic reticulum Cb5 proteins. Quantitative PCR (qPCR) was performed to measure the expression of RcCb5 genes in developing endosperm of castor seeds during the period of HFA synthesis in this tissue¹⁹. We found that RcCb5-2, RcCb5-3 and RcCb5-4 transcripts were all strongly expressed, with highest transcript levels found for RcCb5-2 (Figure 2). By contrast, the RcCb5-1 and RcCb5-6 genes showed very low expression. Data from a transcript profiling database with ~10⁶ sequences covering five stages of endosperm development²⁹ includes 21 reads for RcCb5-2 and 26 reads for RcCb5-4, while RcCb5-3, RcCb5-1 and RcCb5-6 were not represented in this data set. Taken together, these results indicate that RcCb5-2, RcCb5-3 and RcCb5-4 likely have redundant roles in providing electrons to the FAH12 hydroxylase enzyme in the endoplasmic reticulum of endosperm cells of developing castor seeds.

Figure 1. Phylogenetic relationships among 16 Cb5 proteins.

Protein sequences from Arabidopsis thaliana (At), Ricinus communis (Rc), and Vernicia fordii (Vf) were used to build an unrooted dendrogram in Geneious v5.1 (www.geneious.com). Size bar indicates genetic distance between proteins using the Neighbor-Joining method.

Figure 2. Quantitative PCR of RcCb5 transcripts from developing castor endosperm.

Transcripts were normalized to RcACTIN. Error bars represent standard error of the mean of three independent experiments with three replicates for each experiment.

Transformation of the CL37 line with three RcCb5 proteins

The cDNAs encoding the three RcCb5 isoforms that are strongly expressed in seeds (RcCb5-2, RcCb5-3 and RcCb5-4) were each cloned under control of the phaseolin promoter in vector pGate-Phas-Basta and transformed into CL37 plants expressing RcFAH12¹³. Putative T₁ transformants were selected for Basta resistance. Analysis of the bulk T₂ seed from 35 independent RcCb5-2 CL37 primary transformants showed no significant difference in total HFA (18:1-OH plus 18:2-OH) levels compared to the control CL37 plants grown at the same time, with a mean of 19.5% HFA (P value = 0.5979) for the 35 RcCb5-2 events (Figure 3A). There were also no significant differences in HFA levels for the 82 independent T₁ events for RcCb5-3 CL37 compared to untransformed CL37, with a mean of 18.3% HFA (P value = 0.2155) (Figure 3B), nor for the 44 independent T₁ events of RcCb5-4 CL37 (mean of 18.6% HFA) compared to untransformed CL37 (P value = 0.2470; Figure 3C).

Figure 3. Total HFA accumulation in seed of individual RcCb5 CL37 primary transformants.

Total HFA (18:1-OH+18:2-OH) contents of mature T₂ seeds of individual transformants expressing (A) RcCb5-2, (B) RcCb5-3, or (C) RcCb5-4 in the CL37 line. Control at left is mean HFA in untransformed CL37 plants grown alongside T₁ plants. For controls, error bars indicate standard deviation; n=28 in A, n=11 in B, and n=15 in C.

This analysis of T₂ seed samples indicates that there is no dramatic increase in HFA accumulation through the co-expression of any of the three RcCb5 isoforms with the RcFAH12 in Arabidopsis. However, gene expression is typically variable across a population of individual T₁ transformants and T₂ seeds are segregating for the newly introduced transgene. We therefore identified five T₁ events that produced seed with relatively high HFA and that contained a single RcCb5 transgene insert, based on segregation of the Basta-resistance marker in the T₂ generation. For each of the chosen lines, RcCb5-2 #34, RcCb5-2 #27, RcCb5-2 #33, RcCb5-3 #43, and RcCb5-4 #40, T₂ progeny were grown to maturity and nine to 10 plants homozygous for the RcCb5 transgenes, together with four to nine segregants lacking the transgene from each event, were identified by pedigree analysis of T₃ progeny for Basta resistance. Comparisons of the total HFA content between these two sets of sibling segregants (Figure 4) failed to identify any statistically significant increase in HFA as a result of RcCb5 expression. Thus, plants from the three lines of RcCb5-2 CL37, #34, #27, and #33, were not significantly different from their segregating untransformed CL37 counterparts, with P = 0.2204, 0.0942, and 0.3965 respectively (Figure 4). The RcCb5-3 CL37 #43 was not significantly different from segregating untransformed CL37 (P = 0.4211), and the RcCb5-4 CL37 #40 was also not significantly different from segregating untransformed CL37 (P = 0.2102) (Figure 4). Although no changes were observed in the total HFA levels from these lines, we did observe some minor changes in the fatty acid profile; there were increases in the level of 18:1 and minor decreases in 18:2 and 18:3 (Supplementary Table 2).

Figure 4. Total HFA accumulation in RcCb5 CL37 T₃ seed from selected T₂ lines.

Total HFA (18:1-OH+18:2-OH) contents of T₃ seed of selected homozygous lines expressing RcCb5-2, RcCb5-3, or RcCb5-4 in CL37 were compared with segregating untransformed CL37 siblings. Error bars represent standard error of the mean for four to ten individual plants.

Since it is assumed that the RcFAH12 in the CL37 line is receiving reductant via the endogenous Arabidopsis CBR1 (AtCBR1) and an AtCb5, we attempted to increase reductant supply to the AtCb5 proteins via overexpressing AtCBR1. However, again we found no changes in total HFA in the T₂ seeds (Supplementary Figure 1). Taken together, these results indicate that none of the three RcCb5 proteins or any of the AtCBR1 electron acceptors can provide increased RcFAH12 activity in CL37.

Coexpression of RcCBR1 and RcCb5s in CL37

We transformed CL37 plants with RcCb5 genes on the basis that the RcFAH12 hydroxylase enzyme may not interact optimally with endogenous AtCb5 proteins to receive electrons required for the hydroxylation reaction. However, it is also possible that interactions of the RcCb5 isoforms with the Arabidopsis cytochrome b5 reductase (AtCBR1) are weak, resulting in incomplete reduction of the RcCb5 proteins in the transgenic plants. To test this possibility, and to provide a fully compatible electron transport chain from NADH to the hydroxylase, we transformed CL37 plants with expression constructs containing both RcCBR1, encoding the castor cytochrome b5 reductase isozyme and a RcCb5 coding sequence, under control of strong, seed-specific promoters (see Materials and methods for details). The RcCBR1 and each of the top three RcCb5 genes were subcloned into pEC-dsRed plant transformation vector, resulting in three dual-gene vectors expressing RcCBR1+RcCb5-2, RcCBR1+RcCb5-3, and RcCBR1+RcCb5-4. The T₁ transformants were selected by screening for dsRed fluorescence in seeds and grown to maturity. Screening of individual transformation events for total HFA accumulation was performed by gas-chromatography of bulk T₂ seeds. The 45 RcCBR1+RcCb5-2 CL37 events generated were not statistically different (mean of 18.4% HFA) from the untransformed CL37 control lines (P = 0.6820) (Figure 5). For the 16 RcCBR1+RcCb5-3 CL37 events the mean of 18.5% HFA was not significantly changed in HFA accumulation compared to the CL37 control plants (P = 0.6834). Similarly, the 30 RcCBR1+RcCb5-4 CL37 events (mean of 17.4% HFA) also were not significantly different from the CL37 controls (P = 0.0898).

Figure 5. Total HFA accumulation in seed of individual RcCBR1+RcCb5 CL37 primary transformants.

Total HFA (18:1-OH+18:2-OH) contents of mature T₂ seeds from individual transformants expressing RcCBR1+RcCb5-2, RcCBR1+RcCb5-3 or RcCBR1+RcCb5-4 in the CL37 line. Results are compared with untransformed CL37 controls grown alongside T₁ plants. Red inverse triangles indicate events that were further analyzed. Error bars represent standard deviation; n=25 for control CL37, n=45 for RcCBR1+RcCb5-2, n=16 for RcCBR1+RcCb5-3, and n=30 for RcCBR1+RcCb5-4.

Three single-insert lines with relatively high HFA were selected from the T₁ events shown in Figure 5. Progeny from each of the three lines were grown to maturity and four to seven plants homozygous for the RcCBR1 and RcCb5 transgenes were identified by the presence of the DsRed marker in their seeds and were grown together with CL37 control plants. Analyses of total HFA content of the homozygous seed showed no statistically significant difference between RcCBR1+RcCb5-2 #24 double transgenic and seed of CL37 lacking these transgenes (P = 0.0571; Figure 6). Similarly, HFA in seed of homozygous plants of lines RcCBR1+RcCb5-3 #12 and RcCBR1+RcCb5-4 #20 were not statistically different from HFA in CL37 controls (P = 0.1388 and P = 0.5774 respectively; Figure 6).

Figure 6. Total HFA accumulation in RcCBR1+RcCb5 CL37 from selected lines.

Total HFA (18:1-OH+18:2-OH) contents of seed from selected lines expressing RcCBR1+RcCb5-2, RcCBR1+RcCb5-3 or RcCBR1+RcCb5-4 in CL37. Plants homozygous for each dual-gene construct were compared with untransformed CL37 controls. Error bars represent standard error of the mean; n=14 for CL37 controls, n=6 for #24, n=7 for #12, and n=4 for #20.

To confirm that the RcCBR1 and RcCb5 transgenes were being expressed, we isolated RNA from developing seeds of representative plants from these three homozygous transgenic lines, and from the untransformed CL37 siblings. The RNA samples were used as templates for RT-PCR using primers specific for RcCBR1 and each of the RcCb5 isoforms. Bands of the expected full-length sizes were observed for the transgenic plants but were not detected in the untransformed CL37 siblings (Figure 7). Successful amplification of a band using primers to the ACT8 gene indicated that none of the RNA samples was degraded.

Figure 7. Reverse transcriptase-PCR from representative RcCBR1+RcCb5 CL37 T₃ plants.

(A) RcCBR1+RcCb5-2 #24, (B) RcCBR1+RcCb5-3 #12 and (C) RcCBR1+RcCb5-4 #20. RNA prepared from developing siliques was subject to RT-PCR using gene-specific primers. In each panel, --- indicates no-template control. U indicates untransformed CL37, T indicates homozygous transformant. The ACTIN8 gene (ACT8, At1g49240) was used as a control to test RNA quality.