Arabidopsis GPAT9 contributes to synthesis of intracellular glycerolipids but not surface lipids

Highlight Arabidopsis glycerol-3-phosphate acyltransferase 9 (GPAT9) is an sn-1 specific acyl-CoA:GPAT that contributes to intracellular glycerolipid biosynthesis in seeds, developing leaves and pollen grains, but not to extracellular glycerolipid biosynthesis.

confirmed in developing siliques (14 DAF) from a selection of T1 GPAT9-SS-OE lines via quantitative real-time RT-PCR, whereby GPAT9 transcripts were detected at levels enhanced by 682.4% to 3137.7% compared to wild-type levels (Fig. S8A). As was the case for T2 GPAT9-OE seeds, T2 GPAT9-SS-OE seeds also exhibited a small but significant 3.0% relative increase in oil content compared to wild-type seeds (Fig. S4B).
To further verify these changes in seed oil content in GPAT9 over-expression lines, T3 seeds from two homozygous GPAT9-OE lines with confirmed over-expression of GPAT9 were similarly analyzed. Interestingly, while oil content analyses of these lines demonstrated small relative increases in seed oil content as a percentage of weight (1.6% and 2.9%, respectively), these changes were not significant (Fig. S5A); a result which could possibly be attributed to the relatively small sample size of T3 homozygous plants compared to T2 lines and the inherent variability of seed oil content in Arabidopsis, which can make detecting statistically valid changes in oil content difficult (Li et al. 2006). However, due to the increase in seed size in GPAT9-OE lines, when seed oil content was measured on a per seed basis rather than on a per weight basis, a significant increase in mean oil content was noted in both transgenic lines, corresponding to 11.7% (GPAT9-OE-6) and 12.2% (GPAT9-OE-7) relative increases compared to wild-type plants (Fig. S5B).
Significant alterations in the FA composition of seed oil from both GPAT9-OE and GPAT9-SS-OE lines were also evident. Overall, the seed oil of transgenic lines exhibited reductions in 20:1, as well as increases in 18:1, and 22:0 ( Fig. S4 and Fig. S5C).

Down-regulation of GPAT9 in Arabidopsis decreases seed oil content and alters the fatty acid composition of seed oil
Constitutive down-regulation of GPAT9 in Arabidopsis using RNAi resulted in a significant 5.3% relative decrease in oil content on a per weight basis in T2 transgenic seeds compared to wild type (Fig. S6A). Similarly, seed-specific GPAT9-SS-RNAi lines (Fig. S1), where GPAT9 transcripts were reduced in T1 developing siliques (14 DAF) in a selection of lines by between 32.3% and 29.6% compared to wild-type levels (Fig. S9B), exhibited a significant 8.9% relative decrease in oil content compared to wild-type lines (Fig. S6B).
To provide further verification of these reductions in seed oil content in GPAT9 RNAi lines, we also analyzed lipids from T3 seeds from two independent homozygous GPAT9-RNAi lines with confirmed down-regulation of GPAT9. As was the case for both constitutive GPAT9-RNAi and seed-specific GPAT9-SS-RNAi T2 seeds, oil content analyses of GPAT9-RNAi T3 seeds demonstrated a significant decrease in seed oil content (relative decreases of 7.3% and 4.2% for GPAT9-RNAi-10 and GPAT9-RNAi-21 lines, respectively, compared to wild type) as a percentage of weight (Fig. S7A). Similarly, when seed oil content was measured on a per seed basis rather than on a per weight basis, even greater decreases in mean oil content were noted in both transgenic lines, corresponding to 20.6% (GPAT9-RNAi-10) and 8.0% (GPAT9-  relative reductions compared to wild-type lines (Fig. S7B). RNAi lines compared to wild type. 25 Fig. S1. Arabidopsis GPAT9 expression analyses with quantitative real-time RT-PCR analysis of Arabidopsis Col-0 tissues at numerous developmental stages. Three technical replicates were carried out in each case. Blocks represent the mean GPAT9 transcript level of two biological replicates relative to levels of the internal control transcript, PP2AA3. Bars denote standard errors. DAF, days after flowering; fl, flower; mat, mature; sen, senescing; sil, siliques.   Fig. S4. Seed weight and seed area in homozygous GPAT9-OE, GPAT9-RNAi and wild-type lines. A, Seed weights of T 3 homozygous lines. Blocks indicate mean weights of seeds from wild-type (n=14), OE-6 (n=5), OE-7 (n=7); wild-type (n=15), RNAi-10 (n=5), and RNAi-21 (n=7) lines, with three technical replicates measured in each case. B, Seed areas of T 3 homozygous lines. Blocks indicate mean areas of wildtype (n=113), OE-6 (n=65), OE-7 (n=107); wild-type (n=127), RNAi-10 (n=59), and RNAi-21 (n=81) seeds. In all instances, bars denote standard errors. Significant increases and decreases compared to wild type (as measured by Student's t-test) are indicated by +/++ (P ≤ 0.05/P ≤ 0.01) and --(P ≤ 0.01). OE, GPAT9-OE lines, RNAi, GPAT9-RNAi lines, wt, wild type.  A, Seed oil content on a per weight basis. B, Seed oil content on a per seed basis. In the case of oil content analyses, blocks represent mean values from wild-type (n=14), GPAT9-OE-6 (n=5) and GPAT9-OE-7 (n=7) lines. C, Fatty acid composition of seed oil. In the case of fatty acid composition, blocks represent the mean values of wild-type and pooled GPAT9-OE-6 and GPAT9-OE-7 lines. Bars denote standard errors. Significant increases and decreases compared to wild type (as measured by Student's t-test) are indicated by +/++ (P ≤ 0.05/P ≤ 0.01) and -/--(P ≤ 0.05/P ≤ 0.01). OE, GPAT9-OE; wt, wild type. In the case of oil content, blocks represent mean values from wild-type (n=15), GPAT9-RNAi-10 (n=5) and GPAT9-RNAi-21 (n=7) lines. C, Fatty acid composition of seed oil. In the case of fatty acid composition, blocks represent the mean values of wild-type and pooled GPAT9-RNAi-10 and GPAT9-RNAi-21 lines. Bars denote standard errors. Significant increases and decreases compared to wild type (as measured by Student's t-test) are indicated by ++ (P ≤ 0.01) and -/--(P ≤ 0.05/P ≤ 0.01). RNAi, GPAT9-RNAi; wt, wild type; TFA, total fatty acids.