Development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without abscisic acid (ABA) was compared with normal development in situ to investipte the role of ABA in embryo maturation. Endogenous ABA levels were measured by radioimmunoassay, and sensitivity to ABA was assayed in terms of its ability to suppress precocious germination and stimulate accumulation of storage protein and storage protein mRNA. During development in situ, the levels of endogenous ABA and 12S storage protein mRNA both reach their peaks just before the embryos begin to desiccate. The ABA levels during this phase of development also correlate with the time required in culture before germination is evident. Following these peaks, increasing concentrations of exogenous ABA are required to both suppress germination and continue storage protein accumulation in vitro. Thus, both endogenous ABA and ABA sensitivity decline during maturation. The concentrations of exogenous ABA required to suppress germination at these later stages result in abnormally high levels of endogenous ABA and appear to be toxic. These results are consistent with the hypothesis that in maturing rapeseeds, low water content rather than ABA prevents germination during the later stages of development.Embryogenic development ceases during maturation, the final phase of seed development in most angiosperms. During this phase, the seeds desiccate and enter a period of developmental arrest which prevents them from germinating before environmental conditions favor seedling growth. Precocious DMSO (50% v/v), was added to the basal medium to give final concentrations of 1 and10 uM. In all cases, the media ingredients were mixed, pH was adjusted to 5.5 with 1.0 N KOH, and powdered agar (Difco-Bacto; Difco Laboratories) was added to 0.7% (w/v). The medium was autoclaved and dispensed 10 ml/ dish. The dishes were sealed with Parafilm (American Can Co.) and cultured at 28°C in continuous light from cool white fluorescent bulbs (General Electric).Extraction of ABA. Crude extracts for use in radioimmunoassay were prepared as described below, based on the method of Weiler (30). A conical sintered-glass homogenizer (Duall, Kontes of Illinois, Evanston, IL) attached to a Tri-R stirrer (Tri-R Instruments, Rockville Centre, NY) was used to grind 50 to 150 mg of tissue in 2.5 ml of 90% (v/v) methanol containing 10 mg/ L 2,6 di-t-butyl-4-methylphenol (BHT). Each sample was divided into two tubes, and 150 ng of ABA (mixed isomers, grade IV, Sigma) were added to one tube as an internal standard of recovery efficiency. Samples were stored in the dark at 4°C for 48 h, with intermittent shaking. Extracts were cleared by centrifugation for 3 min at 12,500 g, diluted 5-fold with H20, and immunoassayed within 2 d.Radioimmunoassay. Endogenous ABA was measured by radioimmunoassay according to the procedure of Weiler (30) using rabbit anti-ABA-human serum albumin serum (Miles-Yeda Ltd., Naperville, IL). This serum does not distinguish between the (+) and (-) ...

The most abundant protein in seeds of Brassica napus (L.) is cruciferin, a legumin-like 12S storage protein. By in vitro translation of embryo RNA, and pulse-chase labelling of cultured embryos with (14)C-leucine, we have shown that the 30 kd α polypeptides and 20 kd β polypeptides of cruciferin are synthesized as a family of 50 kd precursors which are cleaved post-translationally. One member of the cruciferin family was cloned from embryo cDNA and sequenced. The nucleotide sequence of the cruciferin cDNA clone, pC1, contains one long open reading frame, which originates in a hydrophobic signal peptide region. Therefore, the complete sequence of the cruciferin mRNA was obtained by primer extension of the cDNA. The predicted precursor polypeptide is 488 amino acids long, including the 22 amino acids of the putative signal sequence. The amino acid composition of cruciferin protein is very similar to the predicted composition of the precursor. Comparison with an amino acid sequence of legumin from peas, deduced from the nucleotide sequence of a genomic clone, shows that the α polypeptide precedes the β polypeptide on the precursor. Cruciferin and legumin share 40% homology in the regions which can be aligned. However, cruciferin contains a 38 amino acid region high in glutamine and glycine in the middle of the α subunit, which is absent in legumin. Legumin has a highly charged region, 57 amino acids long, at the carboxyl-end of the α subunit, which is not found in cruciferin. Both of these regions appear to have originated by reiteration of sequences. re]19850513 ac]19850715.

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An IgG fraction prepared from an antiserum against a holoenzyme preparation of (Na+ + K+)-ATPase precipitated a single antigen when samples of holoenzyme were subjected to crossed immunoelectrophoresis but precipitated an additional, immunochemically-related antigen when a plasma membrane-enriched fraction was subjected to crossed immunoelectrophoresis under the same conditions. The immunochemically-related antigen could be extracted from the plasma membrane fraction with CHCl3:CH3OH.