Summnary. Gibberellic acid enhances the synthesis of a-amylase in isolated aleurone layers of barley-seeds (Hordeumn vulgare var. Himalaya). In the presence of 20 mm calcium chloride the amount of enzyme obtained from isolated aleurone layers is quantitatively comparable to that of the half-seeds used in earlier studies. After a lag period of 6 to 8 hours enzyme is produced at a linear rajte. Gibberelflic acid does not merely trigger a-amylase synthesis, but it is continuously required during the period of enzyme formation. Enzyme synlthesis is inhibited by inhibitors of protein and RNA synthesis. Small amounts of actinomycin D differentially inhibit enzyme release and enzyme synithesis suggesting 2 distinct processes. Gibberellic acid similarly enhances the formation of ribonuclease which increases linearly over a 48 hotur period. During the first 24 hours the enzyme is retained by the aleurone cells and this is followed by a rapid release of ribonuclease during the next 24 hour period. The capacity to release the enzyme is generated between 20 and 28 hours after the addition of the hormone. Ribonuclease formation is inhibited by inhibitors o-f protein and RNA synthesis. These inhibitors also prevent the formation of the release mechanism if added at the appropriate moment.Haberlandt observed in 1890 (3) that the aleurone layers of rye produce a substance (or substances) which causes liquefaction of the starchy portion of the endosperm and dissoluition of the starch grains. This observation was confirmed by Brow-n and Escombe (2) who used barley seeds as their experimental material. Mlany years later Yomo (13) and Paleg (7) showed independently that when gibberellic acid is added to endosperm portions of barley seeds it initates the production of amylolytic enzymes and the release of stugars.Briggs (1) extended this observation to include several other hydrolytic enzymes (protease, phosphatase and t-glucanase).The only-living ce'lls in the endosperm portion of the seed, judged by presence of respiration andl amino acid incorporation are those of the aleturone lavers and it has been shown that all the a-amylase is prodtuced by the aleurone cells (6,9

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Extensins are hydroxyproline‐rich glycoproteins found in many plant cell walls as a major protein component. The peptide Ser‐Hyp‐Hyp‐Hyp‐Hyp is abundant in the extensins. Using extensin cDNA clones as probes, we isolated six different clones from carrot genomic libraries. One of the genomic clones, pDC5A1, was characterized and found to contain an open reading frame encoding extensin and a single intron in the 3′‐non‐coding region. The derived amino acid sequence contains a signal peptide sequence and 25 Ser‐Pro‐Pro‐Pro‐Pro repetitive sequences. Two extensin transcripts were found corresponding to pDC5A1 with different 5′ start sites. These transcripts increase in abundance after wounding. This is consistent with the reported extensin accumulation in the cell wall upon wounding.

Abstract. In soybean seed coats the accumulation of the hydroxyproline-rich glycoprotein extensin is regulated in a developmental and tissue-specific manner. The time course of appearance of extensin during seed development was studied by Western blot analysis and by immunogold-silver localization. Using these techniques extensin was first detected at 16-18 d after anthesis, increasing during development to high levels at 24 d after anthesis. Immunogold-silver localization of extensin in the seed coat showed marked deposition of the glycoprotein in the walls of palisade epidermal cells and hourglass cells. The immunolocalization of extensin in developing soybean seeds was also made by a new technique-tissue printing on nitrocellulose paper. It was found that extensin is primarily localized in the seed coat, hilum, and vascular elements of the seed.

The salt-extractable hydroxyproline-rich cell wall glycoprotein from carrot (Daucus carota L.) roots is composed of 35% (w/w) protein, 3% (w/w) galactose, and 62% (w/w) arabinose. The arabinose is attached to hydroxyproline as tetra-and trisaccharides. The circular dichroism of the glycoprotein shows that it is completely in the polyproline II conformation. After deglycosylation of the glycoprotein, the polyproline II conformation of the peptide backbone was lost. This indicates that the carbohydrate reinforces the polyproline II conformation.Hydroxyproline-containing insoluble cell wall glycoproteins (extensins) occur in a wide variety of plants. One of the main problems in studying these extensins has been the difficulty in extracting the proteins from the wall (see 22 for review). They are usually covalently linked in the wall, presumably by isodityrosine cross-links (7,14). Wounded carrots accumulate a hydroxyproline-rich cell wall glycoprotein (see 30 for review) that slowly becomes insolubilized (4, 7). A considerable amount of this protein not yet covalently linked to the wall can be extracted under nondenaturing conditions with salts (4, 31). The biosynthesis of this protein has been studied extensively (30) but a reliable amino acid composition was not available until 1980 (31). The amino acid composition is similar to that of the hydroxyproline-rich potato agglutinin (24) which is localized in the wall (25). The major amino acids found in the carrot glycoprotein are the same amino acids which preferentially accumulate in the cell walls of elongating pea and bean stems (19,34).The hydroxyproline-rich cell wall glycoprotein is thought to be a structural wall component (22). We studied the structure of the hydroxyproline-rich cell wall glycoprotein from carrot roots (which we propose to call 'extensin precursor' in concordance with Lamport's usage of the term).MATERIALS AND METHODS Plant Material. Carrot (Daucus carota L.) roots were obtained from a local merchant and stored at 5°C until needed. For incubation of carrot slices, the roots were washed, surface sterilized with 1% (w/w) NaClO for 10 min and sliced (about 1 mm thick in a food processor). The slices were put in racks in a plastic box and incubated for 5 d in moist air at room temperature.Cell Wall Isolation. The fresh or incubated tissue (about 1.5 kg) was homogenized in a blender in a solution containing 0.5% ' Supported by grants from the National Science Foundation (PCM 7923550 and PCM 8104516) and from the Monsanto Co. (an unrestricted postdoctoral fellowship grant).(v/v) Nonidet P-40 (Sigma) plus 2 mM Na2S205 and filtered through Miracloth (Calbiochem). The cell walls were extensively washed with 2 mm-Na2S205 and finally with 20 mM Tris-HCl buffer, pH 8.0, plus 2 mM Na2S20.Isolation of Hydroxyproline-Rich Cell Wall Glycoprotein. In the following fractionation procedures, we determined the salt concentration of the fractions with the refractometer, the protein content by light absorption at 280 nm, and the hydroxyproline content-aft...

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Hydroxyproline-rich glycoproteins (HRGPs) are important structural components ofplant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with 32P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

Simumary. Thle productiOn of protease by isolated aleurone layers of lbarley in response to gibberellic acid has beeni examiined. The protease arises in the aleurone layer and is mostly released from the aleurone cells. The courses of release of amylase and protease fronm aleurone layers, the dose responses to gibberellic acid and the effects of inhibitors on tlle Jproduction of both enzymes are parallel. As is the case for amylae.e, protease is iiade de noxo in responise to the hormiione. Tlhese data give some credence to the hypotlhesis that the effect of gibberellic acid is to promote the siniultalneouis synfthesis and -cretiom of a grout) of liydrolase>.