Identification and characterization of an mRNA encoding a proline-rich protein that rapidly declines in abundance in the tips of harvested asparagus spears.

We previously isolated a cDNA clone, pTIP13, whose homologous mRNA rapidly declined in abundance in the tips of harvested asparagus (Asparagus officinalis L.) spears [King and Davies (1992) Plant Physiol. 100: 1661]. In order to identify factors regulating the postharvest deterioration of asparagus, we have now sequenced the pTIP13 cDNA, derived the encoded amino acid sequence and determined the cellular location of pTIP13 mRNA by in situ hybridization. pTIP13 encodes a derived protein that is rich in proline (22.3%), but also has a high content of lysine (15.2%) and threonine (14.1%). The proline residues are located in motifs at the amino-terminal region of the protein. The carboxyl-terminal region of the derived protein has a high leucine content and shares > 64% amino acid identity with derived proteins identified from cDNA clones to cell wall protein precursor mRNAs obtained from soybean hypocotyls, alfalfa roots, and tomato fruit. Genomic Southern analysis suggests that pTIP13 is encoded by a single-copy gene in asparagus. pTIP13 mRNA was localized to specific cell types in the young bracts of the asparagus spear tip. The results provide new information on the complexity of tissue responses in the tips of asparagus spears following harvest.

Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening.

Avocado (Persea americana) fruit experience a rapid and extensive loss of firmness during ripening. In this study, we examined whether the chelator solubility and molecular weight of avocado polyuronides paralleled the accumulation of polygalacturonase (PG) activity and loss in fruit firmness. Polyuronides were derived from ethanolic precipitates of avocado mesocarp prepared using a procedure to rapidly inactivate endogenous enzymes. During ripening, chelator (cyclohexane-trans-1,2-diamine tetraacetic acid [CDTA])-soluble polyuronides increased from approximately 30 to 40 [mu]g of galacturonic acid equivalents (mg alcohol-insoluble solids)-1 in preripe fruit to 150 to 170 [mu]g mg-1 in postclimacteric fruit. In preripe fruit, chelator-extractable polyuronides were of high molecular weight and were partially excluded from Sepharose CL- 2B-300 gel filtration media. Avocado polyuronides exhibited marked downshifts in molecular weight during ripening. At the postclimacteric stage, nearly all chelator-extractable polyuronides, which constituted from 75 to 90% of total cell wall uronic acid content, eluted near the total volume of the filtration media. Rechromatography of low molecular weight polyuronides on Bio-Gel P-4 disclosed that oligomeric uronic acids are produced in vivo during avocado ripening. The gel filtration behavior and pattern of depolymerization of avocado polyuronides were not influenced by the polyuronide extraction protocol (imidazole versus CDTA) or by chromatographic conditions designed to minimize interpolymeric aggregation. Polyuronides from ripening tomato (Lycopersicon esculentum) fruit extracted and chromatographed under conditions identical with those used for avocado polyuronides exhibited markedly less rapid and less extensive downshifts in molecular weight during the transition from mature-green to fully ripe. Even during a 9-d period beyond the fully ripe stage, tomato fruit polyuronides exhibited limited additional depolymerization and did not include oligomeric species. A comparison of the data for the avocado and tomato fruit indicates that downshifts in polyuronide molecular weight are a prominent feature of avocado ripening and may also explain why molecular down-regulation of PG (EC 3.2.1.15) in tomato fruit has resulted in minimal effects on fruit performance until the terminal stages of ripening.

Identification of peptides from autolysates of Saccharomyces cerevisiae that exhibit glucose tolerance factor activity in a yeast assay.

1. Cationic fractions were isolated from a low chromium (less than 0.2 ppm) commercial yeast extract in an attempt to purify the material responsible for glucose tolerance factor (GTF) activity observed in a standard yeast assay system. 2. Following previously described procedures a fraction with GTF activity but containing negligible chromium was isolated, which on further purification was found to be composed of many separate small basic peptides. 3. Much of the activity of the yeast GTF material in the yeast assay could be attributed to the presence of basic peptides and free amino acids acting as nitrogen sources for the yeast. 4. Additional activity was present in the yeast GTF sample, which was not due to a synergistic effect of the mixed amino acids and peptides although the component of the yeast extract responsible for this activity was not identified. 5. The results show that the GTF fractions isolated according to most previously published procedures are highly impure, and conclusions drawn about the nature of GTF based on these isolates must remain open to question. 6. The activity due to the presence of peptides and amino acids is a major cause of lack of specificity of the yeast systems as an assay for GTF.