The plant growth regulator abscisic acid (ABA) is formed by the oxidative cleavage of an epoxy-carotenoid. The synthesis of other apocarotenoids, such as vitamin A in animals, may occur by a similar mechanism. In ABA biosynthesis, oxidative cleavage is the first committed reaction and is believed to be the key regulatory step. A new ABA-deficient mutant of maize has been identified and the corresponding gene, Vp14, has been cloned. The recombinant VP14 protein catalyzes the cleavage of 9-cis-epoxy-carotenoids to form C25 apo-aldehydes and xanthoxin, a precursor of ABA in higher plants.

We show that reproductively mature male sea lampreys release a bile acid that acts as a potent sex pheromone, inducing preference and searching behavior in ovulated female lampreys. The secreted bile acid 7alpha,12alpha,24-trihydroxy-5alpha-cholan-3-one 24-sulfate was released in much higher amounts relative to known vertebrate steroid pheromones and may be secreted through the gills. Hence, the male of this fish species signals both its reproductive status and location to females by secreting a pheromone that can act over long distances.

We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance (∼45 pS at −100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of ∼630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces ∼40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14. point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GAS locus encodes GA 20-oxidase. Expression of the GAS gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.The gibberellins (GAs) are tetracylic diterpenoid compounds that play an important role in many aspects of plant growth and development, such as promotion of cell division and extension, seed germination, stem growth, and fruit set (1). The biosynthesis of GAs has been studied extensively in developing seeds (2, 3), but their function in immature seeds, if any, is unknown. In contrast, relatively little is known about GA biosynthesis in vegetative tissues, because GA levels in these tissues are low compared with levels in immature seeds, and active enzyme preparations for GA conversions are difficult to obtain. It is, therefore, desirable to extend studies on regulation of GA biosynthesis to green plants in which GAs have definite roles. One of these roles is to mediate photoperiodic control of stem elongation in rosette plants (4-6). Therefore, to understandThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6640the mechanism by which long days (LD) cause stem elongation, it is necessary to elucidate how daylength regulates GA biosynthesis at the biochemical and molecular levels.In Arabidopsis thaliana, a long-day plant (LDP) suitable for molecular genetic studies (7), a number of GA-responsive dwarf mutants have been isolated (8). It has been demonstrated that one of these mutants carrying thegaS mutation has reduced levels of Cl-GAs, indicating that the product of the GAS gene catalyzes elimination of C-20 at the aldehyde level in the GA biosynthetic pathway. The gaS dwarf mutant also has increased levels of certain C20-GAs, indicating existence of an additional control, possibly hydroxylation of C-20 (9). This suggests that the GAS locus encodes a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-]. In pumpkin endosperm, the enzyme activities for the steps between GA53 and GA20 ( Fig. 1) reside on a single polypeptide (10), indicating that GA 20-oxidase is a multifunctional enzyme that catalyzes the oxidation and elimination of C-20 and thus plays a pivotal role in the conversion of C20-to C19-GAs. Recently, cDNA clones encoding GA 20-oxidases that differ by one amino acid have been isolated from developing cotyledons (11) and liquid endosperm (12) of devel...

With the completion of the sequencing of the Arabidopsis genome and with the significant increase in the amount of other plant genome and expressed sequence tags (ESTs) data, plant proteomics is rapidly becoming a very active field. We have pursued a high-throughput mass spectrometry-based proteomics approach to identify and characterize membrane proteins localized to the Arabidopsis thaliana chloroplastic envelope membrane. In this study, chloroplasts were prepared from plate- or soil-grown Arabidopsis plants using a novel isolation procedure, and "mixed" envelopes were subsequently isolated using sucrose step gradients. We applied two alternative methodologies, off-line multidimensional protein identification technology (Off-line MUDPIT) and one-dimensional (1D) gel electrophoresis followed by proteolytic digestion and liquid chromatography coupled with tandem mass spectrometry (Gel-C-MS/MS), to identify envelope membrane proteins. This proteomic study enabled us to identify 392 nonredundant proteins.

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P Ϫ 3, P Ϫ 4, and P ϩ 5 (relative to the phosphorylated Ser at P ϭ 0).

No abstract

SummaryCertain plants produce glycine betaine (GlyBet) in the chloroplast by a two-step oxidation of choline. Introducing GlyBet accumulation into plants that lack it is a wellestablished target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by choline monooxygenase (CMO), a stromal enzyme with a Rieske-type [2Fe-2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet-deficient plants such as tobacco, but the endogenous choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe-2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post-transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO ⍣ plants were very low (0.02-0.05 µmol g -1 fresh weight) in both unstressed and salinized conditions. Experiments with [ 14 C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO ⍣ plants were supplied in culture with 5 mM choline or phosphocholine, their choline and GlyBet levels increased by at least 30-fold. The choline precursors monoand dimethylethanolamine also enhanced choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet-deficient plants engineered with choline-oxidizing genes, the size of the free choline