Immunohistochemistry of Active Gibberellins and Gibberellin-Inducible α-Amylase in Developing Seeds of Morning Glory

Abstract

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A1-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA1 and/or GA3 were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible α-amylase in this digestion. We isolated an α-amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA3. RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA1/3. An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA1/3, was also localized around starch grains in the integument of developing young seeds. The localization of GA1/3 in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds.

AtFLA11, a Fasciclin-Like Arabinogalactan-Protein, Specifically Localized in Screlenchyma Cells

“…The protocol for immunostaining was that of Nakayama et al 21) Signals were detected with an ECL Plus western blotting detection system (Amersham Biosciences) according to the manufacturer's protocol.…”

“…The recombinant fasciclin-like domain was expressed as a fusion product with GST in E. coli according to the previous method. 21) The fasciclin-like domain was PCR-amplified using a forward primer of 5 0 -ACGGATCC(BamHI)CCAAC-GAACATAACCGCAATCC-3 0 and a reverse primer of 5 0 -CGGAATTC(EcoRI)CCAAAACTTGATCAACC-TGATAA-3 0 , digested with BamHI and EcoRI, and cloned into the same restriction sites of the pGEX-4T-2 vector (Amersham Biosciences). The recombinant fusion protein was affinity-purified in a column of glutathione-Sepharose 4B (Amersham Biosciences) and used to immunize New Zealand white rabbits.…”

Distribution of Gibberellins and Expressional Analysis of GA 20-oxidase Genes of Morning Glory during Fruit Maturation

“…cDNA from the developing fruit of I. nil was prepared according to the method described previously. 7,8) PCR reaction was performed using cDNA as template, using the following degenerate primers synthesized based on the well-conserved sequence of known GA 20-oxidases: forward primer, 5 0 - GGPP, trans-genanylgeranyldiphosphate; CDP, ent-copalyl diphosphate; CPS, CDP synthase; KS, ent-kaurene synthase; KO, ent-kaurene oxidase; KAO, ent-kaurenoic acid oxidase; GA20ox, GA 20-oxidase; GA3ox, GA 3-oxidase.…”

“…Â 150 mm, Senshu, Tokyo, Japan) according to the previous method. 7) Each fraction was methylated with diazomethane, dried in vacuo, dissolved in 1 ml of 5% (v/v) methanol, and subjected to radioimmunoassay (RIA). The procedure for RIA was as follows: (i) 100 ml of fractionated solution described above was added to the glass tube for RIA; (ii) 400 ml of 1/10-diluted bovine serum solution in phosphate-buffered saline (PBS) containing tritium-labeled GA 4 -Me or tritium-labeled GA 20 -Me (166 bq per assay, tritium-labeled GA 4 -Me was used for the quantification of active GA 1=3 and tritium-labeled GA 20 -Me for that of GA 20 ) was added to the tube; (iii) 100 ml of 1/20,000-diluted anti-GA 1 -Me antiserum 14) in PBS or 1/5,000-diluted anti-GA 20 -Me antiserum 15) in PBS was added to the tube; (iv) incubation at 4 C overnight after vigorous mixing; (v) 750 ml of saturated ammonium sulfate was added to the tube and mixing followed by incubation at 4 C for 30 min; (vi) centrifugation (2;000g, 20 min, 4 C) and discarding of the supernatant; (vii) 1 ml of 50% (v/v)-saturated ammonium sulfate was added to the tube and mixing; (viii) centrifugation (2;000g, 20 min, 4 C) and discarding of the supernatant; (ix) 100 ml of water and 1 ml of scintillator (ACSII scintillation cocktail, Amersham Biosciences, NJ) were added to the tube and evaluation of radioactivity with a scintillation counter (Aloka, Tokyo, Japan).…”

“…7) We showed that both InAmy1 and InGA3ox2, a GA 3-oxidase gene, were expressed periodically, and sitespecifically overlapped in the seed coat, an outer tissue adjacent to the integument, by in situ hybridization experiments, 8) and suggested that GA 1=3 , which are major active GAs in the seeds, were synthesized in the seed coat and induced GA-responsible InAmy1 there, and both GAs and InAmy1 were secreted to the integument. In the in situ experiments, we noticed that expression of the InGA3ox2 and InAmy1 started at the hilum, where the seed attaches to the peduncle, and that they gradually spread over the seed coat.…”