The beta subunit of tomato fruit polygalacturonase isoenzyme 1: isolation, characterization, and identification of unique structural features.

We have purified and isolated cDNAs encoding the beta subunit of tomato fruit polygalacturonase isoenzyme 1 (PG1), a cell wall protein that associates with, and apparently regulates, the catalytic PG2 polypeptides. Expression of the beta subunit is fruit specific and temporally separated from the expression of PG2 during fruit development. The 37- to 39-kD beta subunit is encoded as a 69-kD precursor protein containing a signal sequence and two propeptide domains. The mature protein is composed almost entirely of the novel 14-amino acid motif FTNYGxxGNGGxxx in which many of the phenylalanine residues are post-translationally modified. The unique structural features of the motif suggest an important role in the function of the protein and hence in the activity of PG1. The beta subunit may represent a class of bifunctional plant proteins that interact both with structural components of the cell wall and catalytic proteins to localize and/or regulate metabolic activities within the cell wall.

Expression of a Brassica napus Malate Synthase Gene in Transgenic Tomato Plants during the Transition from Late Embryogeny to Germination.

To study gene regulation during the transition from late embryogeny to germination, we have analyzed the expression of a gene encoding the glyoxylate cycle enzyme malate synthase in transgenic tomato (Lycopersicon esculentum) plants. We have shown that although there are at least four classes of malate synthase genes in Brassica napus L., one gene is expressed at a high level during both late embryogeny and postgermination. Analyses of transgenic tomato plants containing the expressed B. napus gene along with 4.7 and 1.0 kilobase pairs of 5' and 3' flanking sequences, respectively, confirmed that a single gene is expressed at both stages of development. Furthermore, localization studies have shown that mRNA encoded by the B. napus gene is distributed throughout the tissues of a mature embryo but is not detected in the vascular cylinder of a seedling. We conclude that the sequences required to qualitatively regulate the gene correctly over the plant life cycle are present within the transferred gene and/or flanking regions. Moreover, the malate synthase gene is regulated differently during late embryogeny and postgermination in the developing vascular cylinder.

Spatial patterns of gene expression in Brassica napus seedlings: identification of a cortex-specific gene and localization of mRNAs encoding isocitrate lyase and a polypeptide homologous to proteinases.

We investigated the spatial expression of three genes that are expressed during seed germination and postgerminative development in Brassica napus L. using in situ hybridization procedures. Two of the mRNAs encode isocitrate lyase and a predicted polypeptide that is homologous to cysteine proteinases. We reported previously that the mRNAs are prevalent primarily in cotyledons of seedlings and accumulate with similar kinetics during postgerminative growth. Here, we show that the two mRNAs are detected in several seedling tissues, but they display different distribution patterns in both cotyledons and root-shoot axes. The third mRNA is abundant in seedling axes and accumulates specifically in the ground meristem and mature cortex of hypocotyls and roots. Distribution of the mRNA in root meristems suggests that the gene product participates in an early event in cortical cell differentiation. Our results provide insight into the physiological processes that characterize seedlings.

Physiological requirement for biosynthesis of multiple 24 beta-methyl sterols in Gibberella fujikuroi.

Studies with Gibberella fujikuroi have been designed to examine the relationship between the biosynthesis and function of fungal sterols. Evidence was obtained through appropriate feeding and trapping experiments for the existence of multiple end products which are produced by separate routes in the later stages of sterol biosynthesis. The three end products, ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol), brassicasterol (24 beta-methylcholesta-5,22E-dien-3 beta-ol), and 22(23)-dihydrobrassicasterol (24 beta-methyl-cholesterol), were found to be non-interconvertible during logarithmic phase growth; thus the metabolic route delta 5,7,22-24 beta-CH3----delta 5,22-24 beta-CH3----delta 5-24 beta-CH3 was ruled out. Ergosterol can be further metabolized, viz., to 24 beta-methylcholesta-5,7,9(11),22-tetraen-3 beta-ol, but only as the culture enters into the stationary phase. In the presence of growth inhibitory concentrations of 2,3-iminosqualene, a partial reversal of growth cessation was obtained when all three sterols were concurrently supplied to the medium. Since neither ergosterol nor the other two sterols added individually to the medium was able to overcome the inhibitor's deleterious effect, ergosterol cannot play a dual architectural role (bulk and regulatory) in this fungus as it apparently can do in other fungal systems, i.e., yeast. For G. fujikuroi each sterol end product appears to possess a unique physiological role. Mycelial growth requires more than simply ergosterol.

Sterol composition and biosynthesis in sorghum: Importance to developmental regulation.

Sterol composition and biosynthesis have been examined in seeds, germinating seeds and blades from fally matured leaves ofSorghum bicolor in various stages of development'from seedlings (seven-day plants) to flowering (66-day) plants. The profile of the dominant free sterols of seeds was similar to that of leaf blades; both contained cholesterol, 24α-methylcholesterol (campesterol), 24ß-methylcholesterol (dihydrobrassicasterol), 24α-ethylcholesterol (sitosterol) and 24α-ethylcholesta-5,22-dienol (stigmasterol). Sufficient sterol intermediates were identified in the plant to indicate separate post-cycloartenol pathways to sterolic end products. The total free sterol content of the seed (µg/seed) increased somewhat during the 20 hr germination period. However, as the plant developed (seven to 48 days), there was a logarithmic increase in the leaf blade sterol content (µg/leaf blade) which plateaued at the onset of floral differentiation (ca. day 41). Over the next 18 days (48 to 66 days-period of inflorescense development), the sterol content rapidly decreased. In the early stages of plant development, the leaf blade pentacyclic triterpenoid (PT) content was negligible. With the onset of floral differentiation, PT content increased logarithmically, reaching a plateau level that surpassed the sterol content as flowering progressed. These results imply that a critical mass of sterol is associated with sorghum for floral induction. Sterol loss from the leaves of the flowering plants presumably was compensated for by the diversion of 2,3-oxidosqualene (SO) from sterol synthesis to PT production. Additional feeding and trapping experiments with [2-(14)C]mevalonic acid, [2-(3)H]cycloartenol, [24-(3)H]lanosterol [4-(14)C]sitosterol and [4-(14)C]cholesterol fed to germinating seeds and leaves from flowering plants demonstrated that sorghum possessed a cycloartenolbased pathway; germinating seeds synthesized 24-alkylsterols but not cholesterol, although cholesterol was identified in both dry and germinating seeds by gas chromatography-mass spectroscopy (GC-MS); and mature leaves synthesized cholesterol and 24α-alkylsterols but not 24ß-methylcholesterol.