Modulating seed β-ketoacyl-acyl carrier protein synthase II level converts the composition of a temperate seed oil to that of a palm-like tropical oil

Abstract

␤-Ketoacyl-acyl carrier protein (ACP) synthase II (KASII) elongates 16:0-ACP to 18:0-ACP in the plastid, where it competes with three other enzymes at the first major branch point in fatty acid biosynthesis. Despite its key metabolic location, the influence of KASII in determining seed oil composition remains unclear, in part because the biochemical consequences of the fab1-1 mutation were unresolved. Thus, fab1-1, and a newly identified knockout allele, fab1-2, were analyzed in the context of the hypothesis that modulating KASII activity is sufficient to convert the composition of a temperate seed oil into that of a palm-like tropical oil. No homozygous fab1-2 individuals were identified in progeny of self-fertilized heterozygous fab1-2 plants, Ϸ1/4 of which aborted before the torpedo stage, suggesting that fab1-2 represents a complete loss of function and results in lethality when homozygous. Consistent with this hypothesis, homozygous fab1-2 plants were identified when a fab1-1 transgene was introduced, demonstrating that fab1-1 encodes an active KASII. Strong seed-specific hairpin-RNAi reductions in FAB1 expression resulted in abortion of Ϸ1/4 of the embryos in an apparent phenocopy of fab1-2 homozygosity. In less severe FAB1 hairpin-RNAi individuals, embryos developed normally and exhibited a 1:2:1 segregation ratio for palmitate accumulation. Thus, early embryo development appears sensitive to elevated 16:0, whereas at later stages, up to 53% of 16:0, i.e., a 7-fold increase over wild-type levels, is tolerated. These results resolve the role of KASII in seed metabolism and demonstrate that modulation of Arabidopsis KASII levels is sufficient to convert its temperate oilseed composition to that of a palm-like tropical oil.condensing enzyme ͉ fatty acid biosynthesis ͉ metabolic engineering ͉ plant oil T emperate crops such as canola, soybean, and sunflower contain predominantly unsaturated 18-carbon fatty acids in their seed oils, whereas tropical oils such as palm oil contain higher proportions (Ϸ50%) of 16-carbon saturated fatty acids (1). Whereas oils with high contents of long-chain or very-longchain polyunsaturated fatty acids are desirable for many purposes including human nutrition, oils with highly saturated 16-carbon-chain-length fatty acids, including palm oil, can provide the starting materials for many industrial applications. It is well documented that different membrane fatty acid compositions, i.e., the ratio of saturated to unsaturated fatty acids, play key roles in adaptation to ambient temperatures (2); however, we reasoned that this need not be the case for seed oils. Thus, we hypothesized that it should be possible to mimic a palm-like oil composition in a temperate crop by genetic manipulation of oilseed biosynthesis.The two-carbon elongation steps in fatty acid biosynthesis (3) are catalyzed by a small family of ␤-ketoacyl-acyl-carrier protein (ACP) synthases, commonly referred to as condensing enzymes of the KAS family (4). Three distinct plastidial KAS activities with characteristic c...

Expression and developmental function of the 3-ketoacyl-ACP synthase2 gene in Arabidopsis thaliana

“…Our alignment and phylogenetic analyses showed that the AtKAS2 (At1g74960) gene encodes a functional protein having KAS II activity, being consistent with the complementation experiment of leaky fab1-1 mutant (Carlsson et al, 2002) and with the recently reported fatty-acid composition of knockout fab1-2 mutant (Pidkowich et al, 2007). Thus, AtKAS2 catalyzes the elongation of 16:0-ACP to 18:0-ACP in plastids, and may have many important biological functions in various tissues in various developmental stages.…”

Metabolic Engineering of Seeds Can Achieve Levels of ω-7 Fatty Acids Comparable with the Highest Levels Found in Natural Plant Sources

“…The plants were transformed according to Clough and Bent's method (Clough and Bent, 1998) using Agrobacterium tumefaciens strain GV3101. We identified individual T1 seeds carrying the transgenes by the fluorescence emitted (Stuitje et al, 2003) upon illumination with green light from an X5 LED flashlight (Inova) in conjunction with a 25A red camera filter (Pidkowich et al, 2007). A WILD M3Z dissection microscope equipped with an Olympus U-LH100HG illumination system was used to discriminate between seeds carrying Zs-Green and Ds-Red markers with the use of filters fluorescein isothiocyanate 535 and fluorescein isothiocyanate 515, respectively.…”

“…Two such lines were recently reported in which fab1 was suppressed, one by hairpin RNA interference (RNAi; Pidkowich et al, 2007) and the other by a novel method of suppression termed hairpin-antisense (HPAS) RNAi (Nguyen and Shanklin, 2009). These lines contain strongly elevated seed 16:0 accumulation levels at 42% and 46%, respectively (Fig.…”

PEMANFAATAN TEKNOLOGI GENOMIKA DAN TRANSFORMASI GENETIK UNTUK MENINGKATKAN PRODUKTIVITAS KELAPA SAWIT / The Use of Genomic and Genetic Transformation Technologies for Oil Palm Productivity Improvement

“…Hasil studi pada tanaman model A. thaliana menunjukkan bahwa penurunan tingkat aktivitas enzim KASII sudah mencukupi untuk mengkonversi minyak pada biji Arabidopsis menyerupai karakteristik minyak kelapa sawit (Pidkowich et al, 2007). Penggunaan promoter yang diekspresikan spesifik pada mesocarp kelapa sawit (mesocarp-specific promoter) dan aplikasi metode antisense RNA dari gen KASII dan gen palmitoyl-ACP thio esterase diharapkan menghasilkan tanaman transgenik dengan komposisi minyak yang kaya akan asam lemak tak jenuh seperti asam oleat.…”