Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

Abstract

Background and aims: α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone.

Methods: α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties.

Key results: The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population.

Conclusions: The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.

Fig. 1.

α-Amylase activity in developing and mature annual ryegrass seeds. α-Amylase activity in heated extracts of low-dormancy (A, C) and dormant (B, D) seeds was detected by native PAGE, using β-limit dextrin as a substrate. Developing seeds (A, B) were assayed at 15, 30, 45 and 58 days after anthesis (DAA) and after 3 d germination (Germ) of mature seeds. Mature seeds were also dissected (C, D) into embryo-enriched (+emb) and embryo-less (–emb) parts before being germinated for 3 d in the presence of 10 µm gibberellin A₄ (+GA) or 100 mm glucose (+gluc). Untreated dry and germinating seed parts were also included. Prior to germination in (B) and (D), dormant seeds were dark-stratified for 21 d to release dormancy. A commercial preparation of α-amylase from Aspergillus oryzeae was included as a positive control. Representative gels are shown from three (A) or two (B) independent seed collections/treatments, using approx. 50 seeds or seed parts per sample. Arrows indicate the position of the various α-amylase isoforms, numbered according to their migration speed.

Fig. 2.

Effect of plant growth regulators on seed germination and α-amylase activity. Seeds were incubated in the presence or absence of 50 µm abscisic acid (ABA) or 1 mm benzoxazolinone (BOA) under optimum germination conditions (25/15 °C, 12 h photoperiod). Germination was measured after 7 and 21 d exposure to the plant growth regulators (A), and α-amylase activity in heated seed extracts was measured after 3 d exposure (B). Dormant seeds were first dark-stratified in the absence of plant growth regulators for 21 d to release dormancy. In (B), α-amylase activity was quantified using the dinitrosalicylate-based detection of reducing sugars (Guglielminetti et al., 1995) released from the β-limit dextrin substrate. Values represent means ± s.e. (n = 4); different letters above columns denote significant differences between treatments (P < 0·05).

Fig. 3.

Purification of α-amylase from dry and germinating seeds. (A) The pH and (B) temperature optima of the ryegrass α-amylase activity eluted from an alginate affinity matrix were determined using the starch–iodine quantitative assay method. Values are means ± s.e. of three independent purifications. Proteins eluted from the alginate matrix following incubation with extracts from dry or germinating (germ) ryegrass and wheat seeds were analysed by SDS–PAGE (C), using 30 µg of protein per lane for ryegrass and 2 µg per lane for wheat. A representative gel is shown, using ryegrass proteins pooled from three independent purifications. Molecular mass standards are shown to the left of the gel.

Fig. 4.

Two-dimensional PAGE analysis of α-amylase preparations. Protein eluted from the alginate affinity matrix (60 µg of protein per gel, pooled from at least three independent purifications for the ryegrass samples) from (A) dry low-dormancy, (B) germinating low-dormancy, (C) dry dormant, (D) germinating dormant, (E) dry wheat and (F) germinating wheat seeds was analysed by 2-D-PAGE stained with colloidal Coomassie Brilliant Blue G-250. Spots that were used for peptide sequencing, based on their abundance and/or similarity to the expected size of α-amylase (approx. 45 kDa), are circled. Molecular mass standards are shown to the left of the gel, with pH at the bottom.