Metabolic engineering of seeds can achieve levels of omega-7 fatty acids comparable with the highest levels found in natural plant sources.

Plant oils containing ω-7 fatty acids (FAs; palmitoleic 16:1Δ(9) and cis-vaccenic 18:1Δ(11)) have potential as sustainable feedstocks for producing industrially important octene via metathesis chemistry. Engineering plants to produce seeds that accumulate high levels of any unusual FA has been an elusive goal. We achieved high levels of ω-7 FA accumulation by systematic metabolic engineering of Arabidopsis (Arabidopsis thaliana). A plastidial 16:0-ACP desaturase has been engineered to convert 16:0 to 16:1Δ(9) with specificity >100-fold than that of naturally occurring paralogs, such as that from cat's claw vine (Doxantha unguis-cati). Expressing this engineered enzyme (Com25) in seeds increased ω-7 FA accumulation from <2% to 14%. Reducing competition for 16:0-ACP by down-regulating the ß-ketoacyl-ACP synthase II 16:0 elongase further increased accumulation of ω-7 FA to 56%. The level of 16:0 exiting the plastid without desaturation also increased to 21%. Coexpression of a pair of fungal 16:0 desaturases in the cytosol reduced the 16:0 level to 11% and increased ω-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.

The Arabidopsis BEL1-LIKE HOMEODOMAIN proteins SAW1 and SAW2 act redundantly to regulate KNOX expression spatially in leaf margins.

In Arabidopsis thaliana, the BEL1-like TALE homeodomain protein family consists of 13 members that form heterodimeric complexes with the Class 1 KNOX TALE homeodomain proteins, including SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). The BEL1-like protein BELLRINGER (BLR) functions together with STM and BP in the shoot apex to regulate meristem identity and function and to promote correct shoot architecture. We have characterized two additional BEL1-LIKE HOMEODOMAIN (BLH) proteins, SAWTOOTH1 (BLH2/SAW1) and SAWTOOTH2 (BLH4/SAW2) that, in contrast with BLR, are expressed in lateral organs and negatively regulate BP expression. saw1 and saw2 single mutants have no obvious phenotype, but the saw1 saw2 double mutant has increased leaf serrations and revolute margins, indicating that SAW1 and SAW2 act redundantly to limit leaf margin growth. Consistent with this hypothesis, overexpression of SAW1 suppresses overall growth of the plant shoot. BP is ectopically expressed in the leaf serrations of saw1 saw2 double mutants. Ectopic expression of Class 1 KNOX genes in leaves has been observed previously in loss-of-function mutants of ASYMMETRIC LEAVES (AS1). Overexpression of SAW1 in an as1 mutant suppresses the as1 leaf phenotype and reduces ectopic BP leaf expression. Taken together, our data suggest that BLH2/SAW1 and BLH4/SAW2 establish leaf shape by repressing growth in specific subdomains of the leaf at least in part by repressing expression of one or more of the KNOX genes.

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

beta-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, approximately 1/4 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 approximately 1/4 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.

Synthesis of enzymatically active human alpha-L-iduronidase in Arabidopsis cgl (complex glycan-deficient) seeds.

As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human alpha-l-iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T(2) seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 microg IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 microg IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans.

Switching desaturase enzyme specificity by alternate subcellular targeting.

The functionality, substrate specificity, and regiospecificity of enzymes typically evolve by the accumulation of mutations in the catalytic portion of the enzyme until new properties arise. However, emerging evidence suggests enzyme functionality can also be influenced by metabolic context. When the plastidial Arabidopsis 16:0Delta7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, Delta7 to Delta9. Conversely, retargeting of two related cytoplasmic 16:0Delta9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity approximately 25-fold, Delta9 to Delta7. All three desaturases exhibited Delta9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine. Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred Delta7 desaturation, with 16:1Delta7 accumulating specifically on the plastidial lipid MGDG. Positional analysis is consistent with ADS desaturation of 16:0 on MGDG. The lipid headgroup acts as a molecular switch for desaturase regiospecificity. FAD5 Delta7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic Delta9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes. The MGDG-dependent desaturase activity enabled plants to synthesize 16:1Delta7 and its abundant metabolite, 16:3Delta(7,10,13). Bioinformatics analysis of the Arabidopsis genome identified 239 protein families that contain members predicted to reside in different subcellular compartments, suggesting alternative targeting is widespread. Alternative targeting of bifunctional or multifunctional enzymes can exploit eukaryotic subcellular organization to create metabolic diversity by permitting isozymes to interact with different substrates and thus create different products in alternate compartments.