Does the endosperm pose a mechanical resistance on embryonic axis (radicle) growth for lettuce seed (achene) germination? To aid answering this question, the cell wall degrading enzyme, carboxymethylcellul a se (CMCase) was extracted and assayed from lettuce seeds imbibed for 0 to 12 h, prior to germination. Measuring the loss of viscosity of carboxymethylcellulose, CMCase activity was high in dry seeds, low after 6 h of imbibition, high after 9 and 10 h, and then reduced again after 12 h. Fractions from Sephadex columns showed CMCase activity in three peaks labeled E1, E2, E3. The greatest change in CMCase activity during imbibition was with E3 (molecular weight of about 40,000 Daltons) and some reduction in E2 (molecular weight about 280,000). The RNA synthesis inhibitor, 6-methyl purine, eliminated CMCase activity when present from 4.5 to 7 h of imbibition and the protein synthesis inhibitor, cycloheximide, eliminated CMCase activity when present between 5.5 and 9 h. Imbibition in darkness lowered CMCase activity while 15 min of light at 3.5 h restored it and 30 min of far-red light at 3 h eliminated it. Increasing the imbibition temperature to 35°C under light reduced activity while under darkness, activity was eliminated under 24 °C and 35°C. CMCase activity was localized in the endosperm surrounding the embryonic axis (micropylar end) of 9 h imbibed seeds. These observations showed that CMCase was active in degrading the cell wall in the endosperm surrounding the radicle, weakening it, prior to radicle protrusion so that the radicle remains undamaged.
The embryo in the lettuce seed is surrounded by an endosperm that is two-cell layers thick at the cotyledon end and up to four-cell thick at the radicle or micropylar end [
In contrast, the light requirement for germination is reinstated in half seeds and embryos sown in growth inhibiting osmoticum [
The mechanical restraint of the lettuce endosperm may be overcome by two mechanisms: force of the growing embryo against it [
Cellulases are able to act on plant cell walls and play a role in loosening and separating cells in the abscission zone in plants [
The primary objective of this study was to measure cellulase activity over the time course of imbibition prior to radicle protrusion in lettuce. Molecular weight of the cellulase was estimated, as well as the location of its activity within the seed, and activity under inhibiting conditions of synthesis inhibitors, light and temperature.
Lettuce seeds (Lactuca sativa L.) cultivar Grand Rapids were obtained from Vaughan’s Seed Co. (Downer’s Grove IL) and vernalized in brown bottles wrapped in AL foil stored in a desiccator at 4˚C. Prior to use, seeds were acclimated to room temperature and surface sterilized with 5% Clorox dissolved in 5% ethanol followed by 70% ethanol. Seeds were then rinsed four times with sterile distilled water and allowed to air dry. For germination, one gram of seed was placed in each of five sterile Petri dishes containing 10 ml of sterile distilled water and placed in a Sherer-Gillette controlled environment chamber set at 20˚C. Seeds were exposed to continuous white light, a mixture of fluorescent and incandescent light, which was 750 foot-candles at seed level. Germination was scored when the radicle first appeared as seed under a magnification of 30×.
From 0 to 12 h of imbibition, a Petri dish with seeds was removed from the environment chamber. One gram seed was ground in 10 ml of cold homogenizing solution consisting of 0.05 M potassium phosphate buffer at pH 5.9, 0.003 M (0.02%) NaN3, and 0.025 M Na2S2O5. This coarse ground seed mixture was further homogenized with a drill press with an additional 5 ml of homogenizing solution used to wash the press. The finely ground seed mixture was placed in an ultracentrifuge and spun at 10,000 g for 10 min. at 4˚C. The supernatant was then re-centrifuged at 37,000 g for 20 min. at 4˚C. The resulting supernatant was aspirated through a milli-pore filter, 0.22 um pore size. The filtrate was the extraction for assaying carboxymethylcellulase (CMCase) activity. This procedure as well as the assay described in 2.3 were based on that reported for bean and pea [
The substrate solution was 1.5% carboxymethylcellulose (CMC) (7HS or 7H, Hercules Powder Co. dissolved in 0.05 M potassium phosphate buffer (pH 5.9) containing 0.003 M (0.02%) NaN3. Equal quantities of the enzyme extract and substrate solution were combined and placed in a water bath set at 41˚C. The resulting concentration of CMC was 0.75%. The pH of the assay mixture was 5.8; preliminary tests determined that optimal pH was between 5.2 and 6.2. After a 2-h reaction time, 1 ml aliquots were removed and placed in the measuring chamber of a Wells-Brookfield Rotary Spindle Micro-viscometer kept at 41˚C; preliminary tests determined that optimal temperature was between 40 and 50˚C. Viscosity was measured within 5 min after mixture was added to chamber. A reaction time longer than 2 h did not change viscosity loss. In the special case when germination-inhibiting conditions were imposed, a reaction time of 24 h was used. CMCase activity was expressed as the percent decrease in viscosity (−%Δµ, reaction time) of enzyme extract/substrate solution compared to the viscosity of boiled enzyme extract plus substrate solution, the no activity control. Viscosity of the control was 41 ± 0.6 cps. Data were obtained from five experimental runs, and means with standard errors were calculated for enzyme activity at each time interval of imbibition and graphed.
Using the micro-biuret method of Goa (1953), protein content of 1 ml of extract plus buffer (1:1) was measured spectrophotometrically at 330 mm with a Spectronic 20 (Bausch-Lomb) after 20 to 40 min. at room temperature. Bovine serum albumin was used as a standard. Protein content was calculated from a standard curve obtained from bovine serum albumin.
Sephadex columns, G-100 and G-200, were prepared with 0.05 M potassium phosphate buffer at pH 5.9 plus 0.003 M NaN3, and maintained at 8˚C. The column bed was 200 ml and the flow rates were 4 and 2.5 ml/cm2-h for G-100 and G-200 columns, respectively. Protein standards were aldolase, ovalbumin, chymotrypsinogen A, ribonuclease A, and bovine serum albumin (Pharmacia Calibration Kit). Two ml CMCase extracts were applied to the column through a 4-way valve followed by 1 ml of 10% sucrose. Sixty drop fractions were collected and assayed for CMCase activity. Viscosity was measured 24 h after reaction time and expressed as %Δµ, 24 h. The partitioning coefficient (Kav) of active fractions was calculated and plotted against the log molecular weight as determined by the standards. Molecular weights in Daltons were estimated from the plots.
Lettuce seeds imbibed in either sterile distilled water, cycloheximide (CH) at 100 mg/l or 6-methyl purine (MP) at 67 mg/l for varying periods in the first 9 h of imbibition. In the case of 6-methyl purine, seeds were punctured with a 22 G syringe needle at the cotyledonary end to allow penetration. Between treatments of inhibitor and sterile distilled water, seeds were rinsed with the appropriate next medium and returned to Petri dishes containing fresh treatment. CMCase was extracted and assayed after the 9 h imbibition period. Data from three replicated trials were averaged with standard errors and treatments were compared least significant differences at 95% confidence level.
To test the effects of light, seeds were imbibed in water for 9 h under three lighting conditions: continuous darkness, darkness with a 15-min white light exposure at 750 foot-candles after 3.5 h of darkness, and a 1 h exposure to far-red light after 3 h of darkness. Far-red light exposure was accomplished by using two incandescent 150 watt lamps underlaid with a 3 mm thick slab of Rohm-Haas 2025 black plexiglass and 3 cm of water. The effect of germination temperature was determined by germinating seeds under 20˚C, 24˚C and 35˚C in continuous white light and continuous darkness. CMCase was extracted and assayed after the 9 h imbibition period. Data from three replicated trials were averaged with standard errors and treatments were compared least significant differences at 95% confidence level.
Lettuce seeds imbibed in water for 1 and 9 h under standard conditions were dissected into three parts. The radicle end comprised one-third of the seed, and the cotyledonary end was the other two-thirds. The radicle end was further separated into the embryonic axis and the endosperm surrounding it. Separation of the cotyledons from the surrounding endosperm even after 1 h of imbibition was not successful without severely damaging both. One gram of seed was dissected into these parts, and CMCase was extracted, fractionated on a Sephadex G-100 column, and assayed as described earlier. Data from three replicated trials were averaged with standard errors and treatments were compared least significant differences at 95% confidence level.
Seed imbibition was rapid upon water exposure and reached maximum after three hours. Seed germination began after imbibing for 12 h with the rate of germination highest at 15 h - 16 h after imbibition, reaching 74% and 78% germination after 18 and 24 h, respectively (data not shown).
CMCase was extracted and assayed for seeds imbibed for 0, 2, 4, 6, 8, 9, 10, and 12 h. Activity fluctuated during imbibition. Activity was present in dry seed as evidenced by the loss of viscosity of CMC and decreased to a minimum after 6 h of imbibition (
CMCase activity in seeds imbibed in water for 9 h showed a 20% decrease in viscosity of 0.75% CMC as in
then transferred to water, CMCase activity from 9 h seed was the same as that from water only imbibed seed and seeds germinated albeit delayed compared to water controls (
Hour of Imbibition | CMCase activity1 | Normal germination (atypical germination2) | |||
---|---|---|---|---|---|
0 - 5.5 | 5.5 - 9 | −%Δµ after 24 h | % after 48 h | % after 96 h | |
water | water | 20 A3 | 84 | 95 | |
CH4 | CH | 3 C | 0 | 0 | |
CH | water | 20 A | 0 | 73 | |
water | CH | 2 C | 0 | 0 | |
MP4 | MP | 21 A | 5 | 9 | |
MP5 | MP | 3 C | 0 | 0 | |
MP5 | water | 5 BC | 39 (6) | 55 (11) | |
water5 | MP | 9 B | 8 (3) | 9 (3) | |
0 - 4.5 | 4.5 - 7 | ||||
water3 | MP | water | 2 C | 0 | 0 |
MP3 | water | MP | 18 A | 49 (2) | 64 (4) |
1Carboxymethylcellulase activity expressed as the % decrease in viscosity of 0.75% carboxymethylcellulose after a 24 h reaction time based on extraction from 1 g of dry seeds imbibed. 2Atypical germination, i.e., protrusion from cotyledonary end, observed and presented in parenthesis as described in Pavlista and Haber [
was little germination (
Under the standard conditions of 20˚C and continuous white light, CMCase activity of 9 h water-imbibed seeds was that reported in
In seeds imbibed for 0 - 1 h, CMCase was present throughout the seed, the radicle and the cotyledon ends (
CMCase activity was present in dry lettuce seeds and decreased to a minimum
Temperature | Light exposure | CMCase activity1 | Endosperm cracking2 | Germination |
---|---|---|---|---|
˚C | −%Δµ at 24 h | % after 12 h | % after 24 h | |
20 | continuous white light3 | 20 A4 | 47 A | 75 A |
20 | continuous darkness | 10 B | 50 A | 48 B |
20 | darkness + 0.25 h white at 3.5 h | 21 A | 50 A | 74 A |
20 | darkness + 1 h far-red at 3 h | 2 C | 8 C | 15 C |
24 | continuous white light | 22 A | 51 A | 70 A |
24 | continuous darkness | 4 C | 9 C | 23 C |
35 | continuous white light | 11 B | 40 B | 0 D |
35 | continuous darkness | 1 C | 0 C | 0 D |
1Carboxymethylcellulase activity expressed as the % decrease in viscosity of 0.75% carboxymethylcellulose after a 24 h reaction time based on extraction from 1 g of dry seeds imbibed. 2Endosperm cracks at the radicle end as seen using scanning electron microscopy as described in Pavlista and Valdovinos [
CMCase activity1, −%Δµ after 2 h | |||
---|---|---|---|
Seed part | Imbibed 0 - 1 h | Imbibed 9 - 10 h | |
Radicle end | One-third of seed | 14 | 20 A2 |
Embryonic axis | 8 | 1 B | |
Endosperm | 14 | 21 A | |
Cotyledon end | Two-thirds of seed | 11 | 1 B |
1Carboxymethylcellulase activity expressed as the % decrease in viscosity of 0.75% carboxymethylcellulose after a 2 h reaction time based on extraction from 1 g of dry seeds imbibed. 2Numbers in each column followed by different letters are significantly different at the 95% confidence level using the t-test on three replicated tests.
by 6 h of imbibition followed by a sharp increase and a maximum at 9 and 10 h of imbibition, three hours prior to the first signs of radicle elongations [
RNA and protein are required for lettuce seed germination [
Light promotes lettuce seed germination while imbibing in darkness promotes a secondary dormancy (skotodormancy) [
To determine the molecular weight of CMCase at different times of imbibition, extracts at different times of imbibition were applied to Sephadex chromatography columns. Unexpectantly, three molecular-weight-based CMCase activity peaks, labeled E1, E2 and E3, were measured. E1 was the heaviest and E3 the lightest. E1 activity did not change during imbibition. However, E2 and especially the lightest E3 activity changed over time. E2 and E3 activities were greatly reduced at 6 and 12 h of imbibition while being present at 9 h to the same degree as in dry seeds. In view of the study on synthesis inhibitors, this indicated that E2 and E3 were degraded between 0 and 6 h, and after 10 h of imbibition, and synthesized de novo prior to 9 h.
For CMCase to play a major role in endosperm weakening at the radicle end, it is critical for the enzyme to be present in the endosperm there. This study observed that in dry seeds CMCase was present throughout the seed, both in the embryo as well as in the endosperm surrounding it. However, in seeds imbibed for 9 - 10 h, CMCase activity was measured only in the endosperm in the third of the seed surrounding the radicle. This showed that the enzyme was in the right place at the right time.
Other enzymes have also been studied for the role in lettuce seed germination. Since the principle carbohydrate component in the endosperm is mannose [
Endosperm weakening at the radicle end (micropylar region) is a pre-requisite for normal lettuce seed germination [
Pavlista, A.D. (2017) Carboxymethylcellulase Activity in Lettuce Seeds Prior to Germination. American Journal of Plant Sciences, 8, 706-719. https://doi.org/10.4236/ajps.2017.84049