Steven H. Schwartz

Elucidation of the Indirect Pathway of Abscisic Acid Biosynthesis by Mutants, Genes, and Enzymes

Abscisic acid (ABA) was discovered independently by several groups in the early 1960s. Originally believed to be involved in the abscission of fruit and dormancy of woody plants, the role of ABA in these processes is still not clear. ABA is, however, necessary for seed development, adaptation to

Characterization of a Novel Carotenoid Cleavage Dioxygenase from Plants

The plant hormone abscisic acid is derived from the oxidative cleavage of a carotenoid precursor. Enzymes that catalyze this carotenoid cleavage reaction,nine- c is epoxy-carotenoid dioxygenases, have been identified in several plant species. Similar proteins, whose functions are not yet known, are present in diverse organisms. A putative cleavage enzyme from Arabidopsis thaliana contains several highly conserved motifs found in other carotenoid cleavage enzymes. However, the overall homology with known abscisic acid biosynthetic enzymes is low. To determine the biochemical function of this protein, it was expressed in Escherichia coli and used for in vitro assays. The recombinant protein was able to cleave a variety of carotenoids at the 9–10 and 9′-10′ positions. In most instances, the enzyme cleaves the substrate symmetrically to produce a C14 dialdehyde and two C13 products, which vary depending on the carotenoid substrate. Based upon sequence similarity, orthologs of this gene are present throughout the plant kingdom. A similar protein in beans catalyzes the same reaction in vitro. The characterization of these activities offers the potential to synthesize a variety of interesting, natural products and is the first step in determining the function of this gene family in plants.

Biochemical Characterization of the aba2 and aba3 Mutants in Arabidopsis thaliana

Abscisic acid (ABA)-deficient mutants in a variety of species have been identified by screening for precocious germination and a wilty phenotype. Mutants at two new loci, aba2 and aba3, have recently been isolated in Arabidopsis thaliana (L.) Heynh. (K.M. Leon-Kloosterziel, M. Alvarez-Gil, G.J. Ruijs, S.E. Jacobsen, N.E. Olszewski, S.H. Schwartz, J.A.D. Zeevaart, M. Koornneef [1996] Plant J 10: 655–661), and the biochemical characterization of these mutants is presented here. Protein extracts from aba2 and aba3 plants displayed a greatly reduced ability to convert xanthoxin to ABA relative to the wild type. The next putative intermediate in ABA synthesis, ABA-aldehyde, was efficiently converted to ABA by extracts from aba2 but not by extracts from aba3 plants. This indicates that the aba2 mutant is blocked in the conversion of xanthoxin to ABA-aldehyde and that aba3 is impaired in the conversion of ABA-aldehyde to ABA. Extracts from the aba3 mutant also lacked additional activities that require a molybdenum cofactor (Moco). Nitrate reductase utilizes a Moco but its activity was unaffected in extracts from aba3 plants. Moco hydroxylases in animals require a desulfo moiety of the cofactor. A sulfido ligand can be added to the Moco by treatment with Na2S and dithionite. Treatment of aba3 extracts with Na2S restored ABA-aldehyde oxidase activity. Therefore, the genetic lesion in aba3 appears to be in the introduction of S into the Moco.

The genetic and molecular dissection of abscisic acid biosynthesis and signal transduction in Arabidopsis

Abstract The role of the plant hormone abscisic acid (ABA) has been studied in Arabidopsis by using mutants affected in either the biosynthesis or the mode of action of this hormone. Mutants have been isolated mainly by altered germination characteristics and seedling growth. The biochemical lesions of the aba1, aba2 and aba3 mutants have been identified and the zeaxanthin epoxidase gene encoded by ABA1 has been cloned by homology with a Nicotiana plumbaginifolia gene blocked at the same biosynthetic step. ABA-insensitive mutants have either a phenotype affecting several ABA processes ( abi1 and abi2 ) and therefore were suggested to encode early steps in ABA signal transduction, or they affect specific steps (e.g. abi3, abi4, abi5 ). The ABI1 and ABI2 genes encode protein phosphatase 2C enzymes and ABI3 a transcription factor with seed-specific expression. The ABA hypersensitive era1 mutant is impaired in a farnesyl transferase. The various mutants have been used to analyse the role of ABA in seed development and seed germination, stress tolerance, and stomatal closure.

Substrate specificity and kinetics for VP14, a carotenoid cleavage dioxygenase in the ABA biosynthetic pathway

The plant growth regulator, abscisic acid (ABA), is synthesized via the oxidative cleavage of an epoxy-carotenoid. Specifically, a double bond is cleaved by molecular oxygen and an aldehyde is formed at the site of cleavage in both products. The Vp14 gene from maize encodes an oxidative cleavage enzyme for ABA biosynthesis and the recombinant VP14 protein catalyzes the cleavage reaction in vitro. The enzyme has a strict requirement for a 9-cis double bond adjacent to the site of cleavage (the 11-12 bond), but shows some plasticity in other features of carotenoids that are cleaved. A kinetic analysis with the 9-cis isomer of five carotenoids displays several substrate activity relationships. One of the carotenoids was not readily cleaved, but inhibited the cleavage of another substrate in mixed assays. Of the remaining four carotenoids used in this study, three of the substrates have similar V(max) values. The V(max) for the cleavage of one carotenoid substrate was significantly higher. Molecular modeling and several three-dimensional quantitative substrate-activity relationship programs were used to analyze these results. In addition to a 9-cis double bond, the presence and orientation of the ring hydroxyl affects substrate binding or the subsequent cleavage. Additional variations that affect substrate cleavage are proposed.

Abscisic Acid Biosynthesis and Metabolism

Abscisic acid (ABA) (Fig. 1) was discovered independently by several groups in the early 1960s. Originally believed to be involved in the abscission of fruit and dormancy of woody plants, the role of ABA in these processes is still not clear. In later work it became evident, however, that ABA is necessary for seed development, adaptation to several abiotic stresses, and sugar sensing. The regulation of these processes is in large part mediated by changes in de novosynthesis of ABA.

Transposon insertion in genes coding for the biosynthesis of structural components of the Anabaena sp. phycobilisome

Anabaena sp. PCC 7120 mutants defective in phycobiliprotein biosynthesis or phycobilisome assembly were generated by transposon mutagenesis. Four mutants with grossly reduced content of the major phycobiliprotein, phycocyanin, were found to have insertions within the cpcBACDEFG1G2G3G4 operon coding for phycocyanin biosynthesis and assembly. The insertion in mutant B646 separated the promoter from the open reading frames and eliminated production of the phycocyanin α (CpcA) and β (CpcB) subunits. Insertion in cpcC in mutant B642 eliminated production of the L36Rlinker polypeptide required for assembly of phycocyanin into the distal discs of the phycobilisome rod substructures. Mutants B64328 and B64407 had insertions, respectively, in cpcE and cpcF, genes coding for the subunits of the heterodimeric lyase which catalyzes the attachment of phycocyanobilin to the phycocyanin apo-α subunit. Mutant SB12, often unable to survive under low light, was found to have an insertion in the apcE gene coding for the large core-membrane linker (L128CM) that provides the scaffold for assembly of the phycobilisome core. DNA sequencing 3′ of apcE revealed genes apcABC, coding for the α and β subunits of allophycocyanin and for the small core linker L7.8C. Amino acid sequence comparisons showed that the ApcA and ApcB proteins are 37% identical and that each of these polypeptides is highly similar to corresponding polypeptides from the distantly related filamentous strains Calothrix sp. PCC7601 and Mastigocladus laminosus.