Journal of Experimental Botany, Vol. 51, No. 347, pp. 1127-1133,
June 2000
© 2000 Oxford University Press
Antagonistic action of low-fluence and high-irradiance modes of response of phytochrome on germination and ß-mannanase activity in Datura ferox seeds
IFEVA, Facultad de Agronomía, Universidad de Buenos Aires, CONICET, Av. San Martín 4453, 1417, Buenos Aires, Argentina
Received 31 August 1999; Accepted 18 February 2000
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Abstract |
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Seed germination is often induced by a pulse of red light perceived by phytochrome and cancelled by a subsequent pulse of far-red light. When the pulse of red light is followed by several hours of darkness, a pulse of far-red light is no longer effective and prolonged far-red is necessary to block germination. The aim was to investigate whether the red light pulse and prolonged far-red light act on the same or different processes during germination of Datura ferox seeds. Forty-five hours after the inductive red light pulse, germination could not be blocked by one pulse or six hourly pulses of far-red light, but was significantly reduced by 6 h of continuous far-red light. The pulse of red light increased embryo growth potential and the activities of ß-mannanase and ß-mannosidase extracted from the micropylar region of the endosperm. Continuous far-red light had no effect on embryo growth potential or ß-mannosidase activity, but severely reduced the activity of ß-mannanase. The effect of far-red light had the features of a high-irradiance response of phytochrome. Both germination and ß-mannanase activity were restored by a pulse of red light given after the end of the continuous far-red treatment. It is concluded that the low-fluence response and the high-irradiance response modes of phytochrome have antagonistic effects on seed germination and that the control of ß-mannanase activity is one process where this antagonism is established.
Key words: Germination, phytochrome, high-irradiance response.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Light can influence seed germination in a large number of species (Taylorson and Hendricks, 1977
; Bewley and Black, 1982). This effect can be promotive or inhibitory depending on spectral composition, irradiance and timing of irradiation, temperature, water availability, and physiological status of the seeds (Frankland and Taylorson, 1983). The promotion of germination often shows a biphasic relationship with the fluence of a red light (R) pulse (Blaauw-Jansen and Blaauw, 1975; Cone et al., 1985). A first phase is saturated by very low fluences of R or by a pulse of far-red light (FR) and is called very-low-fluence response (VLFR). The second phase is observed with higher fluences of R and is called low-fluence response (LFR). A pulse of FR given immediately after a pulse of R in the LFR range is able to revert the promotion induced by R to the level induced by FR alone (Mancinelli, 1994; Casal and Sánchez, 1998). If a dark period is interposed between the R and the FR pulse, the extent of reversion of the promotion induced by R decreases gradually, as the effect of R escapes from reversibility. In Arabidopsis thaliana the VLFR is mediated by phytochrome A and the LFR is mediated by phytochrome B and other members of the phytochrome family different from phytochrome A (Botto et al., 1995, 1996; Shinomura et al., 1996; Poppe and Schäfer, 1997; Casal et al., 1998).
The control of germination in seeds with coat-imposed dormancy lies in the balance between the expansive force of the embryo and the resistance opposed by the surrounding tissues, mainly the endosperm (Bewley, 1997). Both parameters show a LFR. In lettuce seeds, the active form of phytochrome, Pfr, increases the growth potential of the embryo (Carpita et al., 1979) and modifies the internal structure of endosperm cells in the micropylar region (Psaras et al., 1981). Whether germination in lettuce includes modifications in the walls of endosperm cells is still a matter of debate (Halmer and Bewley, 1979; Dutta et al., 1994, 1997; Nonogaki and Morohashi, 1999). In tomato seeds, Pfr promotes endosperm softening, mannan-degrading enzyme activities and germination (Nomaguchi et al., 1995). In Datura ferox, the LFR of seed germination involves both an increase in the expansive force of the embryo and a decrease in the mechanical resistance of the micropylar portion of the endosperm (Sánchez et al., 1986; de Miguel and Sánchez, 1992). Endosperm softening involves degradation of the main cell-wall component (a ß,1–4 mannan) by endo-ß-mannanase and ß-mannosidase (Sánchez et al., 1990). The mobilization of reserve proteins and extensive ultrastructural modifications in other parts of the cells accompany the changes in the walls. This pre-radicle protrusion syndrome is almost completely confined to the micropylar portion of the endosperm (Sánchez et al., 1990; Mella et al., 1995; Sánchez and de Miguel, 1997).
In the seeds of several species germination is promoted by a LFR, and inhibited by several hours of exposure to continuous FR given even after a pulse of FR is no longer able to cancel the effect of R (Negbi and Koller, 1964; Hartmann, 1966; Bewley and Black, 1982). This effect of continuous FR is known as a high-irradiance response (HIR) and is most efficient between 710–720 nm (Frankland and Taylorson, 1983). Interestingly, during de-etiolation, VLFR, LFR and HIR all have effects in the same direction. In seeds, a LFR promotes (with few exceptions) whereas a HIR inhibits germination (Casal and Sánchez, 1998). However, the hallmark of the HIR is the failure of reciprocity between continuous FR and hourly pulses of FR (Casal et al., 1998) and this failure has generally not been tested for seed germination.
The mechanism of inhibition of seed germination by the HIR mode has received little attention. Continuous FR reduces the growth potential of the embryo in Raphanus sativus (Schopfer and Plachy, 1993) but there are no studies about possible effects on endosperm softening. The aim of this work is to study whether the inhibition of the germination of Datura ferox seeds shows a HIR and, if so, whether this HIR affects embryo growth potential and/or some of the enzymes involved in mannan degradation in the micropylar endosperm.
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Materials and methods |
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Source of seeds
Datura ferox seeds were collected from plants invading soybean fields in Junín, province of Buenos Aires, Argentina. After harvest, the seeds were stored in dark glass jars at room temperature.
Light sources
R light was provided by Philips 40/15 40 W fluorescent lamps (Philips, Eindhoven, Netherlands). FR light was provided by a set of 150 W incandescent internal reflector lamps in combination with two red acetate filters, six 2 mm thick blue acrylic filters (Paolini 2031, La Casa del Acetato, Bs. As., Argentina) and a 10 cm water filter. The calculated proportion of Pfr was 0.87 and 0.1 for the R and FR sources, respectively (Casal et al., 1991).
Incubation conditions and light treatments
Seeds were sown on cotton wool saturated with distilled water in clear plastic boxes, wrapped in black plastic sheets. The seeds were incubated in darkness for 24 h, 9 h at 30 °C and 15 h at 20 °C, decoated under white light, immediately given a saturating R pulse, and returned to darkness at 20–30 °C for approximately 45 h before exposure to the specific light treatments of each experiment. Imbibition is necessary before decoating because it is difficult to remove the coats of dry seeds without causing injury. Decoating is used to improve the uniformity and the definition of the time of radicle emergence through the endosperm.
Seed dissection and measurements
To investigate embryo growth potential the endosperm cap was excised under green light to remove the obstacle for embryo expansion and the capacity of the embryos to grow in conditions of limited water supply was tested (Carpita et al., 1979; de Miguel and Sánchez, 1992). Decapped seeds were incubated at 25 °C in water solutions containing polyethylene glycol 6000 (PEG) (Sigma, St Louis, USA) to provide a range of water potentials. The length of the embryo was measured after 24 h of incubation.
The procedures to dissect the micropylar portion of the endosperm and to extract the enzymes ß-mannanase and ß-mannosidase were carried out following the protocols described earlier (Sánchez and de Miguel, 1997). The activity of ß-mannosidase was assayed using p-nitrophenyl-ß-D-mannopyranoside as substrate and that of endo-ß-mannanase by changes in viscosity of locust bean galactomannan solutions.
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Results |
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Inhibition of germination by a HIR
The population of Datura ferox seeds used in this study showed a LFR and no VLFR. Germination was around 80% 72 h after exposure to a R pulse (Fig. 1A
). A pulse of FR did not induce germination (data not shown) and fully cancelled the LFR when given immediately after R (Fig. 1B). Germination increased when the FR pulse was delayed, particularly beyond 24 h. Thus, most seeds escaped from Pfr control at 45 h after R (Fig. 1B).
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Although, in most seeds a short FR pulse given 45 h after R was no longer able to cancel the induction of germination by R (Fig. 1B
, C), 6 h of continuous exposure to FR significantly reduced germination (Fig. 1C). This effect was not stronger under longer periods (24 h) of continuous FR (data not shown). Hourly pulses of FR providing the same total fluence than continuous FR, were only as effective as a single pulse of FR (Fig. 1C). Therefore, even after the end of escape time, germination can be blocked by exposure to continuous FR and this effect has the characteristics of a HIR (Casal and Sánchez, 1998).
A LFR can abolish the inhibition imposed by the HIR
In seeds exposed to a R pulse followed by 45 h of darkness and 6 h of continuous FR, a second R pulse was able to increase germination compared with those seeds that did not receive this second R pulse (Fig. 2). These results indicate that while a HIR can block the promotion of germination induced by a R pulse (LFR), a new LFR can restore germination. In seed batches with low dormancy the promotive effect of the R pulse given after continuous FR was observed at 25 °C (Fig. 2), but alternating temperatures (20–30 °C) were needed for the re-induction of the germination when dormancy was deeper (data nor shown).
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Embryo growth potential is not involved in the inhibition of germination by HIR
In Datura ferox, seed germination depends on the balance between embryo growth potential and the mechanical resistance of the endosperm. To measure the embryo growth potential the seeds were de-tipped to remove the restriction to embryo growth imposed by the micropylar portion of the endosperm and the growth of the embryos was tested under limited supply of water. When the seeds were exposed to a R or FR pulse and then incubated for 51 h before de-tipping, the embryos of seeds treated with R showed a larger growth potential than those treated with FR (see R and FR controls in Fig. 3). To examine whether the HIR involves changes in embryo growth potential, seeds that had received a pulse of R followed by 45 h of darkness were exposed to either 6 h of continuous FR or a single FR pulse. The length of the embryo measured after 24 h in darkness was similar for seeds treated with either continuous FR or a single FR pulse (Fig. 3). The same results were obtained if the test of embryo growth potential was initiated 8 h after the end of the continuous FR treatment (data not shown) Therefore, whereas a LFR promotes germination and embryo growth potential a HIR inhibits germination but not embryo growth potential in Datura ferox seeds.
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). At this point, all the seeds were de-tipped under green safelight and transferred to polyethylene glycol solutions of the indicated water potential. The length of the embryos was measured after 24 h of further incubation in darkness (25 °C). Each datum point is the average of 10 embryos. The length of the embryo in seeds given only a R (
) or FR (
) pulse followed by 51 h of darkness is indicated.
HIR inhibits ß-activity
In Datura ferox, endosperm softening is related to the hydrolysis of a ß,1–4 mannan produced by a Pfr-dependent increase in both ß-mannanase and ß-mannosidase activities (Sánchez et al., 1990; Sánchez and de Miguel, 1997). The time required for the R effect on ß- mannanase activity to escape from the reversion by FR was approximately 45 h (Fig. 4A). To test the effects of HIR on ß-mannanase activity, seeds were given a R pulse, incubated for 45 h in darkness, exposed either to continuous FR or to a single FR pulse and then returned to darkness. The activity of ß-mannanase was significantly reduced by 6 h of continuous FR and continued to decrease after transfer from continuous FR to darkness. A pulse of FR had no effect on ß-mannanase and no measurements of activity were possible 9 h after FR (see 54 h in Fig. 4B) because the seeds started to germinate. Continuous FR reduced the activity of ß-mannanase but not the activity of ß-mannosidase (Fig. 4B).
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At equal total fluence, hourly FR pulses were not able to mimic the effect of continuous FR on ß-mannanase activity indicating that the effect was a HIR (Fig. 5
).
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A R pulse restores ß-mannanase activity after continuous FR
Germination of Datura ferox seeds is induced by a LFR, cancelled by a HIR and restored by a second LFR. The activity of ß-mannanase is also induced by a LFR and cancelled by a HIR. To investigate if a second LFR can restore ß-mannanase activity, the seeds were given a pulse of R, 45 h of darkness, 8 h of continuous FR, 14 h of darkness (to allow full expression of the inhibitory activity of the HIR), and a terminal R pulse followed by 16 h of darkness. The terminal R pulse restored ß-mannanase activity compared to control seeds that received no R after exposure to continuous FR (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Formation of Pfr sets in motion several processes that end in seed germination. In seeds with coat-imposed dormancy some of these processes take place in the embryo, while others are located in the micropylar portion of the endosperm. The time required for Pfr to complete its intervention is different for each one. In Datura ferox the escape time for promotion of embryo growth potential is approximately 24 h (de Miguel and Sánchez, 1992
) whereas for endosperm softening and germination it is longer than 45 h (de Miguel and Sánchez, 1992, Fig. 1B). Even after the apparent completion of Pfr action, prolonged FR (6 h) can inhibit germination (Fig. 1C). This effect of FR, which goes beyond converting existing Pfr into Pr, is known as a HIR and has been observed in the seeds of several species. Among these there are seeds with coat-imposed dormancy (i.e. lettuce), where continuous FR can act late in the germination process (Frankland and Taylorson, 1983). In this paper it is shown that in Datura ferox continuous FR is effective as late as 45 h after an inductive R pulse. At this time extensive physiological and structural changes have already taken place. The activity of mannan hydrolases has been high for more than 15 h, the mobilization of the protein bodies and the formation of vacuoles in the endosperm cap are well underway and endosperm softening is clearly perceptible (Mella et al., 1995; Sánchez and de Miguel, 1997). Despite these changes, 6 h of continuous FR blocked germination and reimposed the need for a high level of Pfr to restart the processes (Fig. 2). A likely target for this last-minute action was embryo growth potential. However, continuous FR did not affect embryo growth potential (Fig. 3). This observations is at odds with the reduction in embryo growth potential by continuous FR in Rapahnus sativus (Schopfer and Plachy, 1993). These divergent results might suggest distinct HIR action in different species (there is no endosperm effect in Raphanus). However, in R. sativus the effect of continuous FR was not compared with that of a FR pulse or hourly FR pulses. If a pulse of FR were effective in Rapahnus sativus the effect of continuous FR would be a LFR and not a HIR.
Continuous FR produced a significant decrease in ß-mannanase activity suggesting that it could interfere with mannan mobilization and endosperm softening. The observation that germination and ß-mannanase activity are restored by a R pulse given after continuous FR, gives support to this suggestion. Continuous FR is effective even after the micropylar endosperm has undergone considerable softening (de Miguel and Sánchez, 1992; Sánchez and de Miguel, 1997), but before softening reaches its maximum, i.e. when mannan content is still 30–40% of its original amount (de Miguel et al., 1999). Radicle protrusion is likely to require extensive cell-wall mannan degradation to reduce the mechanical resistance of the endosperm sufficiently. An interruption of this degradation process even at an advanced stage, could block radicle protrusion.
A HIR of ß-mannosidase was not found (Fig. 4B). Since endo-ß-mannanase action precedes that of the ß-mannosidase, the lack of HIR of the later enzyme could bear no consequence for the inhibition of mannan mobilization. Clearly, there are independent controls for each enzyme. These independent controls suggest that modulation of Pfr action on germination by different factors may not operate through a single master reaction controlling all Pfr-dependent processes. In fact, ABA blocks the promotion of seed germination by Pfr by inhibiting the enhancement of embryo growth potential without interfering with endosperm softening (de Miguel et al., 1999). Endosperm softening is inhibited at water potentials that do not decrease (or even enhance) embryo growth potential (de Miguel and Sánchez, 1992). Low water potential inhibits endosperm softening, decreases mannan mobilization and mannosidase activity, but does not affect ß-mannanase activity (Sánchez et al., 1989). Thus, Pfr appears to activate a number of processes required for germination, but the final result depends on the interaction with other internal or environmental signals resolved at different points of the complex web of reactions. In this context it seems that looking for a ‘germination enzyme’ or a ‘germination gene’ is a complicated endeavour. Probably in different scenarios the key enzymes or genes serving as germination predictors could be distinct.
A HIR of phytochrome that opposes a LFR action has been described. The seeds remain sensitive to both actions to a point very close to germination and ß-mannanase activity is one process where the antagonism is evident. This seems peculiar to seed germination. During de-etiolation of Arabidopsis thaliana seedlings, phytochrome B mediates the LFR and phytochrome A mediates the HIR of processes like hypocotyl growth inhibition, cotyledon unfolding, and the expression of some photosynthetic genes (Casal et al., 1998). In the later case, LFR and HIR not only go in the same direction, but also operate synergisticaly (Casal, 1995; Cerdán et al., 1999; Hennig et al., 1999).
Work in progress in this laboratory shows that the LFR promotes the transcription of a ß-mannanase gene; it is currently being investigated whether the HIR inhibits this promotion or is acting at a post-transcriptional stage, to identify the molecular level at which the antagonism takes place.
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Acknowledgments |
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This work was financially supported by CONICET (6682), FONCYT (2088) and VBA (TG36).
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Notes |
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1 To whom correspondence should be addressed. Fax: +54 11 4514 8730. E-mailsanchez{at}ifeva.edu.ar
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References |
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