Biomechanical, biochemical, and morphological mechanisms of heat shock-mediated germination in Carica papaya seed

Abstract

Carica papaya (papaya) seed germinate readily fresh from the fruit, but desiccation induces a dormant state. Dormancy can be released by exposure of the hydrated seed to a pulse of elevated temperature, typical of that encountered in its tropical habitat. Carica papaya is one of only a few species known to germinate in response to heat shock (HS) and we know little of the mechanisms that control germination in tropical ecosystems. Here we investigate the mechanisms that mediate HS-induced stimulation of germination in pre-dried and re-imbibed papaya seed. Exogenous gibberellic acid (GA₃ ≥250 µM) overcame the requirement for HS to initiate germination. However, HS did not sensitise seeds to GA₃, indicative that it may act independently of GA biosynthesis. Seed coat removal also overcame desiccation-imposed dormancy, indicative that resistance to radicle emergence is coat-imposed. Morphological and biomechanical studies identified that neither desiccation nor HS alter the physical structure or the mechanical strength of the seed coat. However, cycloheximide prevented both seed coat weakening and germination, implicating a requirement for de novo protein synthesis in both processes. The germination antagonist abscisic acid prevented radicle emergence but had no effect on papaya seed coat weakening. Desiccation therefore appears to reduce embryo growth potential, which is reversed by HS, without physically altering the mechanical properties of the seed coat. The ability to germinate in response to a HS may confer a competitive advantage to C. papaya, an opportunistic pioneer species, through detection of canopy removal in tropical forests.

Keywords: Carica papaya; dormancy; germination; heat shock protein; seed coat; tropical crop..

Fig. 1.

Effects of gibberellic acid and heat shock on germination of C. papaya seeds. (A) Effects of gibberellic acid on germination of C. papaya seeds. Seeds were sown directly onto agar containing up to 1 mM GA₃ and imbibed at 25 °C. Error bars show SE, n=3. (B). Combined effects of gibberellic acid and heat shock on germination of C. papaya seeds. Seeds imbibed at 25 °C with 0.1 or 0.25 mM GA₃ in 1% agar-water; controls are no GA₃. Seeds were surface-disinfected after 5 d of imbibition at 25 °C and given a heat shock at 35 °C for 0, 1, 2, or 4 h before returning to 25 °C. Seeds were sown directly onto GA₃ agar. Error bars show SE, n=3. (C, D) The effect of the the GA biosynthesis inhibitor tetcyclasis on heat-shock stimulated germination of C. papaya seeds. Mean percentage germination calculated at two stages: (C) initiation of germination (seed-coat cracking), and (D) radicle emergence (completion of germination). Standard 4-h heat shocks (HS) at 35°C were provided to seeds treated either with water, 0.1% acetone (v/v in water) or 0–100 µM tetcylcasis in 0.1% acetone. Error bars give 95% confidence inertvals. One-way ANOVA shows significant differences between treatments for both initiation (F _7,16=48.66, P<0.0001) and completion (F _7,16= 50.90, P<0.0001) of germination. (A–D) Final germination was scored at 28 d imbibition.

Fig. 2.

Effects of seed-coat removal on germination of C. papaya seed. (A) Germination of intact seeds, seeds with seed coats cracked using a vice (as described in the Methods), and de-coated seeds. Seeds were imbibed for 5 d at 25 °C and given a 0- or 4-h heat shock at 35 °C before returning to 25 °C. Data are shown with 95% confidence intervals. Final germination was scored at 28 d imbibition. (B, C). Rate of germination of naked C. papaya seeds. Intact seeds and de-coated seeds were surface-disinfected after 5 d of imbibition at 25 °C and given a 0-, 2-h or 4-h heat shock at 35 °C before returning to 25 °C. Mean germination is adjusted to account for damage caused during seed-coat removal. (B) Intact seeds (filled symbols) and (C) naked seeds (open symbols). Symbols denote heat shock treatments: (■ □), 0 h control; (● ○), 2 h; and (▲ △), 4 h. Data are shown with 95% confidence intervals. Arrows signify the timing of the heat shock application.

Fig. 3.

Effects of desiccation and heat-shock mediated germination on the morphology of the C. papaya seed coat. Intact seeds were surface-disinfected and imbibed for 5 d at 25 ° C before either no treatment or 4 h heat shock at 35°C and subsequent return to 25 °C. Transverse sections of dormant and germinating seeds. (A, C) Imbibed dormant seed (no heatshock); (B, D) germinating seed showing cracking of the seed coat (T: tegmen and endotesta) at 24 h post-heat shock; incomplete cracks are indicated by arrows. (A) and (B) are sectioned near the micropyle through the radicle; (C) and (D) are sectioned through the hypocotyl. Scale bars are 1 mm; 20-µm thick sections are magnified 20×. Em, embryo; En, endosperm; T, tegmen and endotesta; Im, inner mesotesta; Om, outer mesotesta; Rb, ovuolar vascular bundle contained within the raphe.

Fig. 4.

Biomechanical weakening of C. papaya seed coats prior to germination. Intact seeds were imbibed at 25 °C for 5 d, then a 4-h heat shock at 35 °C was applied, before returning the seeds to 25 °C. The 6-d imbibition treatment is equivalent to a non-heat-shock control. Seed-coat strength was measured by compression tests recording the maximum load carried before breakage. Error bars are SE; n=30. Seed-coat cracking associated with the progression of germination was observed at 48 h after heat shock.

Fig. 5.

Effects of ABA on germination and mechanical strength of the seed coat in C. papaya. (A) Inhibition of C. papaya seed germination by ABA. Unless otherwise stated intact seeds were imbibed at 25 °C with up to 100 µM ABA for 5 d, then a 4-h heat shock at 35°C was applied, before returning the seeds to 25 °C. Seeds were imbibed continuously with ABA. Data show initiation of germination (coat cracking), completion of germination (radicle emergence), and early seedling growth. Error bars are SE; n=30. (B) Changes in strength of ABA-treated C. papaya seed coats prior to germination. Unless otherwise stated intact seeds were imbibed at 25 °C with up to 100 µM ABA for 5 d, then a 4-h heat shock at 35 °C treatment was applied, before returning the seeds to 25 °C. Seed-coat strength was measured by compression tests recording the maximum load carried before breakage. Key to symbols: (■ □), seeds imbibed on water agar; (▲△), seeds imbibed on 100 μM ABA agar; open symbols, no heat shock controls; filled symbols, seeds received 4-h heat shock at 35 °C. Error bars are SE; n=30. Seed-coat cracking was observed at 48 h after the heat shock in the water and positive heat-shock control; no germination was observed in other treatments.

Fig. 6.

Inhibitory effects of the protein synthesis inhibitor cycloheximide on seed-coat cracking and biomechanical weakening of C. papaya seed. A. The inhibitory effect of cycloheximide application on C. papaya seed-coat cracking. Unless otherwise stated intact seeds were imbibed at 25 °C for 5 d, given a 4-h heat shock at 35 °C before returning to 25 °C. Symbols denote: (■), seeds imbibed on water that received a standard 4-h heat shock; (□), seeds imbibed on water with no heat shock treatment; (∆), seeds treated with 25 µg ml^–1 cycloheximide (CHX) and given a standard heat shock; (◊), seeds treated with 50 µg ml^–1 cycloheximide and given a standard heat shock. Treatments were applied at 5 d. Cycloheximide was provided 2 h prior to heat shock treatment to maximise uptake and throughout the remainder of the experimental period. Error bars give 95% confidence intervals; n=3. Biomechanical tests were conducted between days 5 and 7, as indicated by the vertical dashed lines. (B). The strength of the cycloheximide-treated C. papaya seed coat during germination. Key to symbols: (■ □), water controls; (● ○), seeds treated with 25 µg ml^–1 cycloheximide; open symbols, no heat shock controls; filled symbols, seeds received 4-h HS at 35 °C. Treatments were given after 5 d of imbibition at 25 °C on water-agar. Cycloheximide was provided on filter paper 2 h before the standard heat shock treatment to ensure uptake into the seed and was also subsequently provided throughout the remainder of the experimental period. Seed-coat strength measured by compression tests recording the maximum load carried before breakage. Error bars give SE; n=30. Seed-coat cracking was observed at 48 h after the heat shock in the water and heat shock positive control; no germination was observed in other treatments.

Fig. 7.

Rapid changes in de novo protein synthesis occur upon heat-shock treatment of C. papaya seed. (A) De-coated C. papaya seed were labelled with 150 µCi Trans³⁵S-Label™ for 2 h (as described in the Methods) and extracted proteins were analysed by SDS-PAGE (4–12%) and autoradiography (100 000 cpm loaded per lane): Lane 1, protein extract from seeds imbibed for 5 d in water (no heat shock control); Lane 2, 1-h heat shock at 35 °C; Lane 3, during a 4-h heat shock at 35 °C seeds were incubated with label for the last 2 h of the heat shock; Lane 4, immediately after a 4-h heat shock at 35 °C seeds were incubated with label after completion of the heat shock; Lane 5, 24 h post-heat shock (which was the standard 4 h at 35 °C). Arrows indicate bands that alter over the time points. (B) Comparative profiles of Trans³⁵S-Label™ incorporation into proteins separated by SDS-PAGE (4–12%) and analysed by autoradiography (100 000 cpm loaded per lane). 0h HS corresponds to Lane 1 in the gel presented in (A); 1 h HS corresponds to Lane 2; and 4 h HS corresponds to Lane 3. Arrows indicate bands that alter over the time points.

Fig. 8.

Model of germination stimulation and inhibition in imbibed C. papaya seeds subjected to heat shock and the biochemistry of the germination progress. Stimulatory treatments are indicated by →; inhibitory treatments are indicated by ┤.