LEA proteins are late embryogenesis abundant in the seeds of many higher plants and are probably universal in occurrence in plant seeds. LEA mRNAs and proteins can be induced to appear at other stages in the plant's life by desiccation stress and/or treatment with the plant hormone abscisic acid (ABA). A role in protecting plant structures during water loss is likely for these proteins, with ABA functioning in the stress transduction process. Presented here are conserved tracts of amino acid sequence among LEA proteins from several species that may represent domains functionally important in desiccation protection. Curiously, an 11 amino acid sequence motif is found tandemly repeated in a group of LEA proteins of vastly different sizes. Analysis of this motif suggests that it exists as an amphiphilic α helix which may serve as the basis for higher order structure.

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 (-) ...

Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 μg d(-1) during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10(-6) M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 μg embryo(-1) d(-1)) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.

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The development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without high osmoticum (0.48 and 0.69 molar sorbitol) was compared with normal development in situ to investigate the role of a drying environment in embryo maturation. Sensitivity to osmoticum was assayed in terms of its ability to mimic normal development, i.e. to both suppress germination and maintain 12 S storage protein (cruciferin) synthesis at levels comparable to those seen in the developing seed. Beversdorf, University of Guelph, Ont., Canada) were planted in plastic flats in a 2:1:1 (by volume) mixture of soil, vermiculite, and perlite (Krum, Silbrico Corp., Hodgkins, IL) and grown in a constant environment room (13°C nights, 18°C days) for 2 weeks. The seedlings were then transplanted to 15.2-cm pots and grown to maturity. Light was supplied by a combination of fluorescent (cool-white, very high output, Sylvania) and incandescent (40W, Sylvania, Seneca Falls, NY) lamps to give 16-h d. On the d of anthesis flowers were pollinated and tagged.Embryo Culture. Embryos were dissected aseptically, using tungsten knives (6) and placed immediately either in liquid N2 or in a Petri dish containing Monnier's embryo culture medium (17) with appropriate modifications (described below). Embryos were selected on the basis of age (dpa)2 and pooled embryos of each stage were used for parallel measurements of endogenous ABA, storage protein, and storage protein mRNA levels. Mature dry seeds were harvested from fully desiccated pods of plants grown in a constant environment room. Dry seeds were soaked in 95% ethanol to dissolve the waxy coats, surface-sterilized in commercial bleach diluted 1:4 (1% v/v NaOCl) for 20 min, and rinsed several times with sterile H20 before removing the embryos for culture. Ten to 15 embryos were placed in each Petri dish (6 x 2 cm). Duplicate plates constituted each sample. Embryos were harvested after 3 d of culture. Tissue for ABA and protein determinations was washed three times by filtration in 1% (w/v) sucrose, blotted, weighed and stored at -70°C. Tissue 2 Abbreviation: dpa, days post anthesis. 907www.plantphysiol.org on May 9, 2018 -Published by Downloaded from