Gatlin and Barrows are Chair and Vice-chair, respectively, of the Plant Products in Aquafeeds Working Group, and coordinated the development of this document; all other authors are listed in alphabetical order. AbstractContinued growth and intensi¢cation of aquaculture production depends upon the development of sustainable protein sources to replace ¢sh meal in aquafeeds. This document reviews various plant feedstu¡s, which currently are or potentially may be incorporated into aquafeeds to support the sustainable production of various ¢sh species in aquaculture. The plant feedstu¡s considered include oilseeds, legumes and cereal grains, which traditionally have been used as protein or energy concentrates as well as novel products developed through various processing technologies. The nutritional composition of these various feedstu¡s are considered along with the presence of any bioactive compounds that may positively or negatively a¡ect the target organism. Lipid composition of these feedstu¡s is not speci¢cally considered although it is recognized that incorporating lipid supplements in aquafeeds to achieve proper fatty acid pro¢les to meet the metabolic requirements of ¢sh and maximize human health bene¢ts are important aspects. Speci¢c strategies and techniques to optimize the nutritional composition of plant feedstu¡s and limit potentially adverse e¡ects of bioactive compounds are also described. Such information will provide a foundation for developing strategic research plans for increasing the use of plant feedstu¡s in aquaculture to reduce dependence of animal feedstu¡s and thereby enhance the sustainability of aquaculture.

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Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes1. However, the lineage of the enzyme chalcone isomerase (CHI) remained a quandary. In vascular plants, CHI-catalyzed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defense2, and development3. CHI operates near the diffusion limit with stereospecific control4,5. While associated primarily with plants, the CHI-fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties, and in vivo functional characterization of a non-catalytic CHI-fold family from plants. A. thaliana contains five actively transcribed CHI-fold genes, three of which additionally encode amino-terminal chloroplast-transit sequences (cTP). These three CHI-fold proteins localize to plastids, the site of de novo fatty acid (FA) biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core FA biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased FA storage in the developing embryo. In vitro, these proteins are Fatty Acid-binding Proteins (FAP). FAP knockout A. thaliana plants exhibit elevated alpha-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically ‘perfected’ enzyme6 from a non-enzymatic ancestor over a defined period of plant evolution.

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Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A 4 B 4 configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by the ACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of the ACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom.Acetyl-coenzyme A (CoA) is an intermediate metabolite that is juxtaposed between catabolic and anabolic processes. As the entry point for the tricarboxylic acid (TCA) cycle, acetyl-CoA can be considered the gateway in the oxidation of carbon derived from the catabolism of fatty acids, certain amino acids (e.g. Leu, Ile, Lys, and Trp), and carbohydrates. Furthermore, acetyl-CoA is the intermediate precursor for the biosynthesis of a wide variety of phytochemicals. Because membranes are impermeable to CoA derivatives, it can be inferred that acetyl-CoA is generated in at least four distinct metabolic pools representing the four subcellular compartments where acetyl-CoA metabolism occurs: plastids, mitochondria, peroxisomes, and the cytosol (Fig. 1). Therefore, plants should have distinct acetyl-CoA-generating systems in mitochondria (for the TCA cycle), in plastids (for de novo fatty acid biosynthesis), in peroxisomes (the product of ␤-oxidation of fatty acids), and in the cytosol (for the biosynthesis of isoprenoids, flavo...

Acetyl-CoA provides organisms with the chemical flexibility to biosynthesize a plethora of natural products that constitute much of the structural and functional diversity in nature. Recent studies have characterized a novel ATP-citrate lyase (ACL) in the cytosol of Arabidopsis thaliana. In this study, we report the use of antisense RNA technology to generate a series of Arabidopsis lines with a range of ACL activity. Plants with even moderately reduced ACL activity have a complex, bonsai phenotype, with miniaturized organs, smaller cells, aberrant plastid morphology, reduced cuticular wax deposition, and hyperaccumulation of starch, anthocyanin, and stress-related mRNAs in vegetative tissue. The degree of this phenotype correlates with the level of reduction in ACL activity. These data indicate that ACL is required for normal growth and development and that no other source of acetyl-CoA can compensate for ACL-derived acetyl-CoA. Exogenous malonate, which feeds into the carboxylation pathway of acetyl-CoA metabolism, chemically complements the morphological and chemical alterations associated with reduced ACL expression, indicating that the observed metabolic alterations are related to the carboxylation pathway of cytosolic acetyl-CoA metabolism. The observations that limiting the expression of the cytosolic enzyme ACL reduces the accumulation of cytosolic acetyl-CoA-derived metabolites and that these deficiencies can be alleviated by exogenous malonate indicate that ACL is a nonredundant source of cytosolic acetyl-CoA.

SUMMARYLittle is known about the role of proteins that lack primary sequence homology with any known motifs (proteins with unknown functions, PUFs); these comprise more than 10% of all proteins. This paper offers a generalized experimental strategy for identifying the functions of such proteins, particularly in relation to metabolism. Using this strategy, we have identified a novel regulatory function for Arabidopsis locus At3g30720 (which we term QQS for qua-quine starch). QQS expression, revealed through global mRNA profiling, is up-regulated in an Arabidopsis Atss3 mutant that lacks starch synthase III and has increased leaf starch content. Analysis of public microarray data using MetaOmGraph (metnetdb.org), in combination with transgenic Arabidopsis lines containing QQS promoter-GUS transgenes, indicated that QQS expression responds to a variety of developmental/genetic/environmental perturbations. In addition to the increase in the Atss3 mutant, QQS is up-regulated in the carbohydrate mutants mex1 and sis8. A 586 nt sequence for the QQS mRNA was identified by 5¢ and 3¢ RACE experiments. The QQS transcript is predicted to encode a protein of 59 amino acids, whose expression was confirmed by immunological Western blot analysis. The QQS gene is recognizable in sequenced Arabidopsis ecotypes, but is not identifiable in any other sequenced species, including the closely related Brassica napus. Transgenic RNA interference lines in which QQS expression is reduced show excess leaf starch content at the end of the illumination phase of a diurnal cycle. Taken together, the data identify QQS as a potential novel regulator of starch biosynthesis.