SummaryArabidopsis thaliana expresses four nitrilases, three of which (NIT1, NIT2 and NIT3) are able to convert indole-3-acetonitrile to indole-3-acetic acid (IAA), the plant growth hormone, while the isozyme NIT4 is a b-cyano-L-alanine hydratase/nitrilase. NIT3 promoter activity is marginal in leaves or roots of vegetative plants and undetectable in bolting and¯owering plants, but its level increases strongly when plants experience sulphur deprivation. No other nitrilase genes respond to sulphur supply/de®ciency. Neither N-nor P-deprivation cause detectable changes in NIT3 promoter activity. In transgenic plants expressing uidA under the control of the NIT3 promoter (NIT3p::uidA), sulphate deprivation leads to the appearance of b-glucuronidase activity in shoots and particularly in roots, most strongly in the conductive tissues and lateral root primordia. Deletion analysis allowed localization of the sulphur-responsive element to a 317 bp segment of the NIT3 promoter encompassing nt ±2151 to ±1834 upstream of the transcriptional start point. Both nitrilase polypeptide and nitrilase activity were also induced by sulphur starvation. NIT3 promoter activity was strongly induced by O-acetylserine, suggesting that, as is the case with enzymes of sulphate assimilation, sulphate de®ciency may be communicated to NIT3 via an increase in the level of the cysteine precursor, O-acetylserine. During sulphur deprivation, a preferential depletion of the pool of the indole-3-acetonitrile precursor glucobrassicin compared with that of total glucosinolates was noticed. In the absence of an external sulphate supply, plants developed longer roots with a higher number of lateral roots. The increased growth of the root system occurred at the expense of shoot growth which was retarded under conditions of sulphur starvation. Taken together, these results suggest that a regulatory loop appears to exist by which sulphate de®ciency, through an increase in glucobrassicin turnover and nitrilase 3 accumulation, initiates the production of extra auxin leading to increased root growth and branching, thus allowing the root system to penetrate new areas of soil effectively to gain access to fresh supplies of sulphur.

The cyclic derivative of 13(S)-hydroperoxolinolenic acid, 12-oxophytodienoic acid, serves as a signal transducer in higher plants, mediating mechanotransductory processes and plant defenses against a variety of pathogens, and also serves as a precursor for the biosynthesis of jasmonic acid, a mediator of plant herbivore defense. Biosynthesis of 12-oxophytodienoic acid from ␣-linolenic acid occurs in plastids, mainly in chloroplasts, and is thought to start with free linolenic acid liberated from membrane lipids by lipase action. In Arabidopsis thaliana, the glycerolipid fraction contains esterified 12-oxophytodienoic acid, which can be released enzymatically by sn1-specific, but not by sn2-specific, lipases. The 12-oxophytodienoyl glycerolipid fraction was isolated, purified, and characterized. Enzymatic, mass spectrometric, and NMR spectroscopic data allowed us to establish the structure of the novel oxylipin as sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride. The novel class of lipids is localized in plastids. Purified monogalactosyl diglyceride was not converted to the sn1-(12-oxophytodienoyl) derivative by the combined action of (soybean) lipoxygenase and (A. thaliana) allene oxide synthase, an enzyme ensemble that converts free ␣-linolenic acid to free 12-oxophytodienoic acid. When leaves were wounded, a significant and transient increase in the level of (12-oxophytodienoyl)-monogalactosyl diglyceride was observed. In A. thaliana, the major fraction of 12-oxophytodienoic acid occurs esterified at the sn1 position of the plastid-specific glycerolipid, monogalactosyl diglyceride.

The reduction of 12-oxophytodienoic acid (OPDA) to 3-oxo-2(2[Z]-pentenyl)-cyclopentane-1-octanoic acid is catalyzed by 12-oxophytodienoate-10,11-reductase (OPR). Analysis of the isomer preference of OPR has indicated that the activity is composed of two isoenzymes exhibiting different stereoselectivities. The two isoforms of OPR have been separated, using protein extracts of Rock Harlequin (Corydalis sempervirens) as the starting material. OPRI, the enzyme reported earlier from the same species and corresponding to the cloned OPR from Arabidopsis, utilized 9R,13R-OPDA >> 9S,13R-OPDA but not the 13S-configured isomers, whereas the new activity, OPRII, effectively reduced all four OPDA isomers, including the natural 9S,13S-OPDA (cis-[؉]-OPDA). OPRII activity is characterized in detail. The enzyme's enzymatic, biochemical, and immunological properties prove that it is a close relative of OPRI. The roles of OPRI and OPRII in octadecanoid biology are discussed.

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