Role of ABA in Maturation of Rapeseed Embryos

Abstract

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 (-) ...

Identification and characterization of the mRNA for major storage proteins from radish

“…We have confirmed the recent observation [7] that napin mRNA appears a few days before cruciferin mRNA. But, in addition, the possibility of distinguishing between two subfamilies of differently sized napin precursors allowed us to demonstrate that their respective genes are not expressed simultaneously at their maximum level.…”

“…Our results with radish storage protein mRNA confirm and extent the data recently obtained by studying rapeseed cruciferin and napin mRNA [6,7,191. Rapeseed cruciferin is made from a series of precursors ranging from 43 kDa to 51 kDa as shown by immunoprecipitation of translation products, The rapeseed pattern is more complex than the radish one, as expected from an allotetraploid compared to a diploid.…”

Gene expression during plant embryogenesis and germination: an overview.

“…ABA is required for the accumulation of the 12s seed protein cruciferin in Brassica embryos (Finkelstein et al, 1985), for the continued synthesis of the p subunit of 8-conglycinin in soybean cotyledons (Bray and Beachy, 1985), and the modulation of sunflower helianthinin gene expression (Thomas et al, 1991; A. Nunberg, Z. Li, M. Bogue, J. Vivekananda, A. Reddy, and T.L. Thomas, unpublished results).…”

Frost, Abscisic Acid, and Desiccation Hasten Embryo Development in Brassica napus

“…The increase in ABA was greater in seed containing 55% moisture relative to that in the 70% moisture range. (8,(11)(12)(13), with the change in direction occurring at approximately 55% seed moisture (13). Both ABA levels and gene transcripts have been shown to be good markers of seed developmental stage.…”

“…Both ABA levels and gene transcripts have been shown to be good markers of seed developmental stage. For example, Crouch and co-workers (8,(11)(12)(13) demonstrated that the levels of ABA and cruciferin mRNA peaked just before the onset of desiccation. Harada and coworkers similarly demonstrated increases in LEA.76 transcripts throughout the desiccation phase (16), and increases in ICL mRNA during the latter stages of desiccation (15).…”