Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

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 BREVIPE-DICELLUS (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.

␤-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...

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 16:0⌬ 7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, ⌬ 7 to ⌬ 9 . Conversely, retargeting of two related cytoplasmic 16:0⌬ 9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity Ϸ25-fold, ⌬ 9 to ⌬ 7 . All three desaturases exhibited ⌬ 9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine. Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred ⌬ 7 desaturation, with 16:1⌬ 7 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 ⌬ 7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic ⌬ 9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes. The MGDG-dependent desaturase activity enabled plants to synthesize 16:1⌬ 7 and its abundant metabolite, 16:3⌬ 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.M etabolic diversity in living systems arises primarily from biotransformations catalyzed by enzymes; indeed life itself depends on the specificity of enzymes. The unique functionality, substrate specificity, and regiospecificity of an enzyme typically evolves by the gradual accumulation of changes in the catalytic portion of the enzyme until new properties arise. However, there is an emerging body of evidence suggesting that an enzyme's functional characteristics can also be affected by its metabolic context and that temporal and spatial dynamics of enzyme interactions can be important determinants of enzyme functionality (1). Examples include the ''rewiring'' of mitogen-activated protein kinase pathways on alternative scaffolds (2); the localization-dependent interactions of transcription factors with RNA polymerase in the nucleus (reviewed in ref. 3); the interchangeable tissue-specific roles of the transcription factors WER and GL1 in plant epidermal hair development (4); the modification of laccase͞peroxidase activity in the extracellular matrix by specific dirigent proteins (reviewed in ref. 5); the altered gati...

Plant oils containing v-7 fatty acids (FAs; palmitoleic 16:1D 9 and cis-vaccenic 18:1D 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 v-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 16:1D 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 v-7 FA accumulation from ,2% to 14%. Reducing competition for 16:0-ACP by down-regulating the b-ketoacyl-ACP synthase II 16:0 elongase further increased accumulation of v-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 v-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.There is increasing interest in the use of plant oils as renewable sources of industrial chemical feedstocks Carlsson, 2009). Recent developments in olefin metathesis have demonstrated that long-chain monoene FA from vegetable oils can be efficiently split into the corresponding short-chain a-olefin and v-alkenoic acids (Rybak et al., 2008;Meier, 2009). Thus, ethenolytic metathesis of v-7 fatty acids (FAs), such as palmitoleic or cis-vaccenic acids, yields 1-octene and 9-decenoate. 1-Octene is a high-demand feedstock with a global consumption of over half a million tons per year that is primarily used as a comonomer in the expanding production of linear low density polyethylene. It is mainly synthesized from petroleumderived ethylene via oligomerization to yield a complex mixture of a-olefins or from coal-derived syngas (Anonymous, 2007).A plant oil containing high (.70%) content of v-7 FA would represent a new and sustainable feedstock for 1-octene production. Several natural plant oil sources of v-7 FA have been reported, e.g. milkweed (Asclepias syriaca) accumulates approximately 25% v-7 FA (comprising approximately 10% 16:1D 9 and approximately 15% 18:1D 11 ) and cat's claw vine (Doxantha unguis-cati) accumulates approximately 72% v-7 FA (comprising approximately 55% 16:1D 9 and approximately 17% 18:1D 11 ), but the low yields and poor agronomic properties of these plants preclude their commercial use for v-7 FA production. Isolation of the D 9 -16:0-ACP desaturases genes responsible for the production of v-7 FA from milkweed and Doxantha (Cahoon et al., 1997 presented an opportunity for their heterologous expression and v-7 FA production in transgenic crops. However, heterologous expression of the milkweed desaturase in Arabidopsis (Arabidopsis thaliana) failed to produce detectable v-7 FA, and when the Doxantha desaturase was expressed in Brassica napus, it resulted in the accumul...

No abstract

SummaryAs 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 α -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 µ g 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

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS ( STM ) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.