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AOBPreview originally published online on April 12, 2007
Annals of Botany 2007 99(5):915-924; doi:10.1093/aob/mcm029
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© The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Sensitivity of Polygonum aviculare Seeds to Light as Affected by Soil Moisture Conditions

Diego Batlla*, Marcelo Nicoletta and Roberto Benech-Arnold

I.F.E.V.A./Cátedra de Cerealicultura, C.O.N.I.C.E.T./Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417DSE-Buenos Aires, Argentina

* For correspondence. E-mail batlla{at}agro.uba.ar

Received: 15 November 2006    Returned for revision: 8 January 2007    Accepted: 15 January 2007    Published electronically: 12 April 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

Background and Aims: It has been hypothesized that soil moisture conditions could affect the dormancy status of buried weed seeds, and, consequently, their sensitivity to light stimuli. In this study, an investigation is made of the effect of different soil moisture conditions during cold-induced dormancy loss on changes in the sensitivity of Polygonum aviculare seeds to light.

Methods: Seeds buried in pots were stored under different constant and fluctuating soil moisture environments at dormancy-releasing temperatures. Seeds were exhumed at regular intervals during storage and were exposed to different light treatments. Changes in the germination response of seeds to light treatments during storage under the different moisture environments were compared in order to determine the effect of soil moisture on the sensitivity to light of P. aviculare seeds.

Key Results: Seed acquisition of low-fluence responses during dormancy release was not affected by either soil moisture fluctuations or different constant soil moisture contents. On the contrary, different soil moisture environments affected seed acquisition of very low fluence responses and the capacity of seeds to germinate in the dark.

Conclusions: The results indicate that under field conditions, the sensitivity to light of buried weed seeds could be affected by the soil moisture environment experienced during the dormancy release season, and this could affect their emergence pattern.

Key words: Dark germination, dormancy loss, light, low fluence response (LFR), phytochrome, Polygonum aviculare, soil moisture, soil moisture fluctuations, very low fluence response (VLFR), weed seeds


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
In many weed species, such as Polygonum aviculare, light perceived by phytochromes is required to terminate dormancy and allow the germination process to proceed (Baskin and Baskin, 1985; Batlla and Benech-Arnold, 2005). Phytochromes are a family of chromoproteins that exists in two photo-convertible forms, the inactive form (Pr) which presents maximum absorption at 660 nm (red light), and the active form for dormancy termination (Pfr) which presents maximum absorption at 730 nm (far-red light). Dormancy termination through light stimuli could be mediated by two action modes of phytochromes, the low fluence response (LFR) and the very low fluence response (VLFR). Termination of dormancy through the LFR generally requires light pulses that establish high Pfr levels in the seed, for example those established by a saturating pulse of red light (approx. 87 % Pfr). In the LFR mode, this promotive effect of red light could be cancelled if seeds are subsequently exposed to a saturating pulse of far-red light, because almost 87 % of Pfr is converted back to Pr (the inactive form of phytochrome) (Smith, 1982; Kronenberg and Kendrick, 1986). In contrast, VLFR is saturated with very low amounts of Pfr, for example those that result from exposing seeds to a saturating pulse of far-red light (approx. <3 % Pfr) or to very low fluences of red light (Scopel et al., 1991; Casal and Sánchez, 1998). Generally, seeds present the two type of responses (VLFR and LFR), giving rise to biphasic germination response curves according to the proportion of Pfr (Cone et al., 1985). It has been suggested that these phytochrome action modes mediate different seed responses under field conditions; while LFR is mainly involved in gap detection, VLFR mediates buried seed responses to the brief light pulse received during cultivation (Botto et al., 1998; Casal and Sánchez, 1998).

Seasonal variations in soil temperature often determine cyclic changes in the dormancy status of weed seed banks. For example, for the case of P. aviculare seeds, low winter temperatures alleviate dormancy, while, conversely, high summer temperatures reinforce dormancy, determining the existence of an annual cyclic dormancy pattern (Courtney, 1968; Batlla and Benech-Arnold, 2003). In many weed species, these cyclic changes in the dormancy level of the seed population are related to changes in the sensitivity of seeds to light stimuli (Froud-Williams et al., 1984; Benech-Arnold et al., 2000). Normally, seeds display an increase in light sensitivity as a result of dormancy loss and a decrease in light sensitivity as a result of dormancy induction (Derkx and Karssen, 1993, 1994; Batlla et al., 2004). Recently, Batlla and Benech-Arnold (2005) showed that storage of P. aviculare seeds at low temperatures under moist soil produced a gradual increase in the sensitivity of the seed population to light stimuli, resulting in a successive acquisition of LFR and VLFR, and, eventually, the loss of the light requirement for germination for a fraction of the population. The authors developed a predictive model based on a thermal time index which permits the prediction of changes in the sensitivity to light according to the temperature experienced by seeds during burial.

Although results obtained so far clearly evidence that soil temperature is an over-riding factor governing dormancy changes of weed seed banks, and consequently changes in the sensitivity of seed banks to light, it has been hypothesized that soil moisture could also affect the dormancy level of buried weed seeds (Karssen, 1980/81; Egley and Duke, 1985; Benech-Arnold et al., 2000; Batlla and Bench-Arnold, 2004). Buried seeds of summer annual weeds are certainly subjected to different soil moisture conditions during their dormancy release season (winter) according to the annual rainfall pattern and burial depth. Shallowly buried seeds are exposed to soil moisture fluctuations that could affect their dormancy status (Karssen, 1982; Downs and Cavers, 2000). Laboratory studies showed that desiccation and subsequent re-hydration of seeds could stimulate germination and modify seed light requirements (Lush and Groves, 1981; Karssen et al., 1988; Bouwmeester and Karssen, 1989, 1993a). For example, Bouwmeester and Karssen (1993b) showed that buried seeds of Sisymbrium officinale exposed to a desiccation treatment prior to incubation in water not only germinated under a wider range of temperatures, but a considerable fraction of the population lost the light requirement for germination. Similar results for P. aviculare seeds were recently reported by Batlla and Benech-Arnold (2006), who observed that seeds subjected to soil moisture fluctuations during cold-induced dormancy loss showed in situ germination values 4-fold greater than those observed for seeds subjected to a constantly moistened soil environment. Seeds buried in deeper layers of the soil would not be exposed to such fluctuations in soil moisture, but would be exposed to different soil moisture environments depending on weather and soil characteristics. The effects of interactions between temperature, and soil or seed moisture, on seed dormancy changes have been reported for several species (Foley, 1994; Martinez-Ghersa et al., 1997; Kruk and Benech-Arnold, 1998; Steadman et al., 2003). However, to the best of our knowledge, there is no experimental evidence showing how soil moisture conditions during dormancy loss could affect the sensitivity of buried seeds to light.

In this work, the effect of constant and fluctuating soil moisture conditions during cold-induced dormancy loss on the sensitivity of P. aviculare seeds to light was investigated. To determine the effect of soil moisture fluctuations on seed light sensitivity, data obtained in the present experiment were compared with those recently reported by Batlla and Benech-Arnold (2005) for P. aviculare seeds stored at similar temperatures under a constant moist soil environment. Comparisons between previously published results and results presented in the present paper are justified because experiments were carried out simultaneously and using the same seed lot.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Seed collection
Seeds of Polygonum aviculare L. were collected in a wheat field at Balcarce (latitude 37°45'S, longitude 58°15'W), Argentina, at the time of their natural dispersal (March 2000 and 2002 in experiment 1 and 2, respectively). After collection, the seeds were air-dried, winnowed with a seed blower (Burrows model 1836-3, Evanston, IL, USA) to eliminate light seeds, and stored at 10 % moisture content in glass jars at ambient temperature (approx. 20 °C) for 3 months in experiment 1, and 5 months in experiment 2, until the experiments commenced.

Experiment 1
Bags made of fine-mesh nylon gauze with four compartments containing seeds (50 seeds per compartment) were buried at 5 cm depth in 12 cm diameter black plastic pots filled with soil (Vertic Argiudoll according to the USDA taxonomy), previously oven-dried at 70 °C for 3 d. Pots were stored at three dormancy-releasing temperatures (1·6, 7 and 12 °C) (Batlla and Benech-Arnold, 2003), under three different soil moisture regimes:

  1. Dry soil regime (DS). Soil in the pots was maintained dry (2 % moisture content) during the entire storage period.
  2. Moist soil regime (MS). Pots were irrigated to saturation, sealed at the top with black nylon, and allowed to drain for 48 h. Then the nylon that sealed the pots was removed and pots were weighed to determine the weight corresponding to field capacity for each pot. At regular intervals during the storage period, pots were re-weighed, and water was added until they reached their original weight to maintain their initial field capacity status (36 % moisture content).
  3. Fluctuating soil moisture regime (FS). Pots were irrigated to saturation, sealed at the top with black nylon, and allowed to drain for 48 h. Then the nylon that sealed the pots was removed and pots were weighed to determine the weight corresponding to field capacity for each pot. At regular intervals of approx. 15 d during the storage period, nylon bags containing seeds were moved (in total darkness) from pots containing moist soil (field capacity status) to pots containing dry soil, and vice versa. During periods of storage under moist soil, pots were re-weighed every 5 d, and water was added until they attained their original weight to maintain their initial field capacity status.
At intervals of approx. 15 d throughout the storage period, three bags containing seeds (three replications) were exhumed from pots placed at each storage temperature under each soil moisture regime, and were exposed to the germination tests. For seeds subjected to FS, germination tests were conducted each time seeds were transferred. The entire experiment lasted 137 d. The moisture content of exhumed seeds was determined in sub-samples by weighing, before and after drying for 24 h in an oven at 100 °C.

Experiment 2
Bags made of fine-mesh nylon gauze with three compartments containing seeds (50 seeds per compartment) were buried at 2 cm depth in 8 cm diameter and 4 cm high open-bottom pots filled with a mixture of 50 % fine sand: 50 % soil (Vertic Argiudoll according to the USDA taxonomy) previously oven-dried at 70 °C for 3 d. The pots were fitted at their base with a fine-mesh of nylon gauze to allow proper water exchange. Pots were stored at three dormancy-releasing (stratification) temperatures (1·6, 7·7 and 13·4 °C) under four different constant soil moisture contents.

Different soil moisture contents were imposed by the method proposed by Fernandez and Reynolds (2000) based on the sub-irrigation system previously developed by Snow and Tingey (1985). Briefly, pots were placed at different heights (5·6, 21·2, 29 and 36·8 cm) over columns of a uniform porous medium obtained through piled up floral foam blocks (448 Secure-Firm density/Instant soaking, Floralife, Inc., Walterboro, SC, USA) that were placed in plastic containers with a constant water table (Fig. 1). This procedure ensures uniform and repeatable water content in the pots over time (Wookey et al., 1991; Saulescu et al., 1995; Fernández and Reynolds, 2000). The water table was maintained constant automatically through a Mariotte siphon system connected to the container by a transparent plastic hose of 0·5 cm diameter (Fig. 1). The Mariotte siphon system consisted of a 2 L polyethylene bottle equipped with a vertical inserted glass pipe, which allowed air to enter the bottle, so that the water in the plastic container should be constantly maintained at the height of the bottom end of the glass pipe, where the water pressure was equal to the atmospheric pressure (Araki et al., 1998). Prior to experiment set-up, the foam blocks were repeatedly rinsed with distilled water as recommended by the manufacturers. Soil in the pots was gently pressed with a weight in order to permit proper contact with the foam surface to improve water exchange.


Figure 1
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  		FIG. 1. Diagram of the device used to maintain pots of soil at different soil moisture contents. (1) Plastic pot containing soil; (2) hygroscopic foam block; (3) plastic container; (4) hose for siphoning water from the bottle to the container; (5) plastic bottle; (6) glass pipe; (7) rubber stopper; (8) water table in the bottle; (9) constant water table in the container.

 
After 31, 57 and 79 d of storage, three bags containing seeds (three replications) were exhumed from pots placed at each storage temperature under the different soil moisture contents, and were exposed to the germination tests. Throughout the experiment, soil and seed moisture content was monitored in additional control pots by weighing before and after drying for 24 h in an oven at 100 °C.

In both experiments, soil temperature in the pots was recorded hourly during the storage period using temperature sensors (LI-COR model 1015, Lincoln, NE, USA) connected to a DataLogger (LI-COR model 1000, Lincoln, NE, USA). At the beginning of the experiments, three replicates of 50 seeds, which had not been buried, were exposed to the germination tests to quantify the initial dormancy level of the seed population.

Germination test
Seeds were removed from the mesh bags and were rinsed with distilled water to remove adhering soil particles. Afterwards, replicates of 50 seeds were placed in 9 cm diameter plastic Petri dishes on two discs of Whatman No. 3 filter paper that had been moistened with 5 mL of distilled water. Dishes containing seeds were wrapped with aluminium foil to avoid exposing seeds to light, and were incubated at 15 °C for 18 h to allow seeds stored under different soil moisture conditions to achieve a similar moisture status. After this first period of incubation, the dishes were unwrapped (except treatment 4) and exposed to light treatments determining different phytochrome photoequilibria (Pfr/P) in the LFR and the VLFR range previously calculated by Batlla and Benech-Arnold (2005). In experiment 1, seeds were exposed to treatments 1–4, while in experiment 2 seeds were only exposed to treatments 1, 2 and 4.

Treatments
Four treatments were applied.

  1. Pfr/P = 76 %: 60 min red light with a photon fluence rate of 28 mmol m–2 s–1 (LFR range).
  2. Pfr/P = 3 %: 20 min far-red light with a photon fluence rate of 32 mmol m–2 s–1 (VLFR range).
  3. Pfr/P = 7·6 x 10–4 %: 10 s far-red light with a photon fluence rate of 0·7 mmol m–2 s–1 (VLFR range).
  4. Darkness: three dishes (three replicates) were placed in the same environment as treatment 1 for 60 min but remained wrapped in aluminium foil throughout.
Red light was provided by 40 W fluorescent tubes (Philips 40/15, Germany) in combination with a water filter and red acetate sheet filters (La casa del acetato, Buenos Aires, Argentina). Far-red light was provided by a 150 W incandescent reflector lamp (Phillips R95, Buenos Aires, Argentina) in combination with a water filter and a RG9 filter (Schott, Mainz, Germany); in treatment 3 (7·6 x 10–4 Pfr/P), the fluence rate of the incandescent lamp was diminished by a rheostat to obtain a lower photon flux. Scans of the red and far-red light sources used to establish the different phytochrome photoequilibria are presented in Fig. 2.


Figure 2
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  		FIG. 2. Relative spectral photon distribution (400–800 nm) of the red light source (solid line) and the far-red light source (dashed line) used in the light treatments.

 
During exposure of seeds to the light sources, the temperature in the cabinets was maintained at 17 (±2) °C. Petri dishes containing seeds that had been unwrapped for light treatments were again wrapped in aluminium foil and incubated for 15 d at 15 °C. This incubation temperature was near the optimum germination temperature (16 °C) reported previously by Kruk and Benech-Arnold (1998) for this species. At the end of the incubation period, dishes were unwrapped and the percentage of germination was determined in each dish. The criterion for seed germination was visible radicle protrusion. Exhumation and manipulation of seeds were carried out in total darkness, except for seeds exposed to red light (treatment 1) which were exposed to dim green light during manipulation. Seeds that germinated in situ during storage, if any, were counted under dim green light in seed samples assigned to treatment 1 prior to exposure to red light. Seeds germinated in situ were discarded after counting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Effect of soil moisture fluctuations on seed responses to light
Seeds exhumed from pots containing dry soil (DS) stored at the different temperatures showed germination percentages lower than 5 % for any light treatment during the entire storage period, and did not reveal any changes in their dormancy status (data not shown). The moisture content of exhumed seeds that had been stored under dry soil conditions, either continuously in DS or for periods of approx. 15 d in the fluctuating soil moisture regime (FS), was 12 (±2·6) %, while seeds exhumed after storage under moist soil conditions, either continuously in the moist soil regime (MS) or for periods of approx. 15 d in FS, showed a value of 39·6 (±3·6) % moisture content. Differences of approx. 0·8–2 °C were registered between soil temperatures during storage under moist (field capacity status) and under dry soil conditions. Mean soil temperatures during dry storage periods were 2·4, 9 and 14 °C, while moist storage periods showed mean soil temperatures of 1·6, 7 and 12 °C. For convenience, mean soil temperatures recorded during moist storage periods (1·6, 7 and 12 °C) will be referred to as storage temperatures for seeds subjected to FS, recognizing that temperatures were somewhat higher during the dry storage period.

A significant increase in the percentage of seeds germinating in situ was observed for seeds stored at 1·6 and 7 °C under FS in comparison with that observed for seeds stored at similar temperatures under MS (Fig. 3). Seeds stored at 1·6 °C under FS showed approx. 40 % germination in situ at the end of the storage period, while seeds stored under MS at the same temperature for an equal time period showed values of germination in situ lower than 5%. Similar differences were observed between seeds stored under FS and MS at 7 °C. Finally, seeds stored at 12 °C under both soil moisture regimes did not germinate in situ during storage.


Figure 3
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  		FIG. 3. In situ germination of Polygonum aviculare seeds during storage at either 1·6 °C (circles) or 7 °C (triangles) and under a continuously moist (open symbols) or a fluctuating soil moisture regime (filled symbols). Verticals bars indicate s.e. if larger than symbols.

 
Seeds stored at the different temperatures exposed to treatment 1 (seeds germinated in situ + seeds germinated after exposure to red light establishing a Pfr/P of 76 %) did not evidence changes in their germination response in relation to the different soil moisture storage conditions (Figs 4A, 5A and 6A). In contrast, seeds with a calculated Pfr/P of 3 % or 7·6 x 10–4 %, and seeds incubated in the dark generally showed changes in their germination response in relation to the soil moisture regime under which they were stored (Figs 4B–D, 5B–D and 6B–D). The extent of these differences depended on storage temperature; seeds stored at lower temperatures (i.e. 1·6 °C; Fig. 4B–D) showed larger differences between seeds stored under both soil moisture regimes than that observed for seeds stored at higher temperatures (i.e. 7 and 12 °C; Figs 5B–D and 6B–D). For example, seeds stored at 1·6 °C in which a Pfr/P of 3 and 7·6 x 10–4 % was imposed showed a decrease in germination after periods of storage under dry soil conditions, and an increase in seed germination response to far-red light after periods of storage under moist soil conditions (Fig. 4B, C); these changes in the germination response of the seed population were steeper during initial phases of the storage period. In addition, seed storage at this temperature under dry soil conditions followed by a period of storage under moist soil conditions generally resulted in an increase of the fraction of the population that could germinate during the germination test in comparison with that observed for seeds stored under MS at the same temperature for identical time periods. For example, seeds exhumed after 80 d of storage at 1·6 °C under MS exposed to light treatment 2 (3 % Pfr/P) showed 40 % germination, while seeds stored under FS at the same temperature for the same time period and exposed to the same light treatment showed 63 % germination (Fig. 4B). However, germination percentages for seeds stored under both soil moisture regimes at the end of the storage period were not significantly different (P > 0·05).


Figure 4
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  		FIG. 4. The percentage of germination of Polygonum aviculare seeds in response to different Pfr/P photoequilibria: 76 % (A), 3 % (B) and 7·6 x 10–4 % (C), and for seeds kept in darkness (D), in relation to days of storage at 1·6 °C under a moist soil regime (open symbols) and a fluctuating soil moisture regime (filled symbols). The percentage of germination for 76 % Pfr/P was calculated by adding together the fraction of seeds germinated in situ at the corresponding soil moisture regime, if any (see Fig. 3), and the fraction of seeds germinated after exposure of seeds to treatment 1. Vertical bars represent s.e. if larger than symbols.

 


Figure 5
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  		FIG. 5. The percentage of germination of Polygonum aviculare seeds in response to different Pfr/P photoequilibria: 76 % (A), 3 % (B) and 7·6 x 10–4 % (C), and for seeds kept in darkness (D), in relation to days of storage at 7 °C under a moist soil regime (open symbols) and a fluctuating soil moisture regime (filled symbols). The percentage of germination for 76 % Pfr/P was calculated by adding together the fraction of seeds germinated in situ at the corresponding soil moisture regime, if any (see Fig. 3), and the fraction of seeds germinated after exposure of seeds to treatment 1. Vertical bars represent s.e. if larger than symbols.

 
Seeds with 3 and 7·6 x 10–4 % Pfr/P stored at 7 °C under FS also showed a decrease in germination after periods of storage under dry soil conditions and increases after periods of storage under moist soil conditions (Fig. 5B, C). However, these changes were not of the magnitude of those observed for seeds stored at 1·6 °C, and generally no significant differences were observed between the percentages of germination for seeds stored under both soil moisture regimes. Seeds stored at 12 °C with a Pfr of 3 and 7·6 x 10–4 % did not show marked differences between the percentages of germination for seeds stored under MS and FS during the whole storage period (Fig. 6B, C).


Figure 6
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  		FIG. 6. The percentage of germination of Polygonum aviculare seeds in response to different Pfr/P photoequilibria: 76 % (A), 3 % (B) and 7·6 x 10–4 % (C), and for seeds kept in darkness (D), in relation to days of storage at 12 °C under a moist soil regime (open symbols) and a fluctuating soil moisture regime (filled symbols). Vertical bars represent s.e. if larger than symbols.

 
A significant increase in dark germination capacity (treatment 4) was observed for seeds stored at any temperature under FS in comparison with that observed for seeds stored under MS (Figs 4D, 5D and 6D). Seeds stored at 1·6 °C under FS showed 65 % of the population with capacity to germinate in the dark at the end of the storage period, while seeds stored at the same temperature under MS only presented 18 % of germination in darkness (Fig. 4D). In addition, seeds stored under FS at this temperature did not show significant differences (P > 0·05) between germination percentages obtained under the different light treatments after 114 d of storage, providing evidence for a loss of the light requirement for germination of the seed population. A similar response pattern, though with less marked differences between germination in darkness for seeds stored under both soil moisture regimes, was observed for seeds stored at 7 °C (Fig. 5D) and 12 °C (Fig. 6D). Significant differences (P < 0·05) between soil moisture regimes were established after 80 d of storage at 7 °C, showing values of 41 % (FS) and 18 % (MS) germination at the end of the storage period (Fig. 5D), and after 94 d of storage for seeds stored at 12 °C, showing values of 20 % (FS) and 4 % (MS) germination at the end of the storage period (Fig. 6D).

Effect of soil moisture content on seed responses to light
Placing pots at different heights over the porous medium produced significantly different soil moisture contents during the experiment (F = 62·75, P < 0·0001; Table 1). Moisture contents established in the pots were maintained fairly constant during the experimental period as denoted by the low s.e. values. However, a comparatively higher variability in soil moisture content during the experiment was recorded for pots placed at the highest position (s.e. 0·8). Indeed pots disposed at 36·8 cm over the water table showed a maximum and minimum value of 16 and 6 % moisture content, respectively.


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  		TABLE 1. Different soil and seed moisture contents imposed by the experimental sub-irrigation method in relation to the distance between the bottom of the pots and the water table

 
Although pots containing seeds were stored in cabinets at constant temperatures, slight differences in soil temperature (approx. 0·5–1·5 °C) were registered between the different storage soil moisture conditions (data not shown). These slight differences were mainly due to differences in soil moisture and arrangement of the treatments in the cabinets.

The different soil moisture contents established by the sub-irrigation method significantly affected the moisture content of buried seeds (F = 40·99, P < 0·0001; Table 1), although no significant differences in seed moisture content were observed between seeds stored at soil water contents of 19·6 and 17·2%. Seed moisture content was also unexpectedly affected by storage temperature (F = 30·11, P < 0·0001), seeds stored at the different temperatures showing significant differences in moisture content (Table 2). However, soil water content accounted for >50 % of the total variation in seed moisture content, while storage temperature only accounted for 24 % of this variation. A non-significant interaction between soil moisture content and storage temperature was detected (F = 1·44, P = 0·21).


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  		TABLE 2. Effect of storage temperature on seed water content

 
During storage under the different experimental conditions, no germination in situ and almost no germination (<5 %) for seeds incubated in darkness (treatment 4) was recorded (data not shown). To compare changes in the germination response of seeds to light treatments 1 and 2 during storage under the different soil moisture contents, germination data obtained for seeds exhumed during the storage period at the different dormancy-releasing temperatures were plotted on a common thermal time scale using the stratification thermal time index (Stt) previously developed by Batlla and Benech-Arnold (2003, 2005) (Fig. 7A, B). This index permits quantification of the effects of stratification temperature and time of storage over changes in seed responses to light in the LFR and the VLFR range using the following equation (Batlla and Benech-Arnold, 2005):


Formula 029M1

(1)
where Stt is stratification thermal time units (°Cd), t is the time in days, Tc is the dormancy release ‘ceiling’ temperature (°C) (the temperature at, or over, which dormancy release does not occur) and Ts is the daily mean storage temperature (°C). The optimal ‘ceiling’ temperature for dormancy release in P. aviculare seeds was determined to be 17 °C (Batlla and Benech-Arnold, 2003, 2005).


Figure 7
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  		FIG. 7. The percentage of germination of Polygonum aviculare seeds in relation to stratification thermal time (Stt) for seeds stored under different soil moisture contents in response to light treatment 1, Pfr/P 76 % (A). and light treatment 2, Pfr/P 3 % (B). The dotted line in (A) corresponds to the adjustment of the dose–response model y = 68·83/[1 + 10(log 468·7–x)x0·003854]; R2 = 0·91. Dotted and solid lines in (B) correspond to the adjustment of dose–response models to germination data obtained for seed stored at 19·6, 17·2 and 13·3 %, y = 26·58/[1 + 10(log 706·6–x)x0·002225]; R2 = 0·9, and for seeds stored at 22·8 % soil moisture content y = 40·6/[1 + 10(log 570–x)x0·0041]; R2 = 0·94, respectively. Vertical bars represent s.e. if larger than symbols.

 
Seeds stored at the three dormancy-releasing temperatures under the different soil moisture contents exposed to a saturating pulse of red light (treatment 1) showed a similar germination response pattern in relation to Stt accumulation during storage (Fig. 7A). A dose–response model was fitted to germination data obtained for each soil moisture condition, and the non-linear extra sum of squares method (Motulsky and Ransnas, 1987; Bates and Watts, 1988) was used to evaluate whether the different soil moisture conditions affected the germination thermal time course. Results indicated that the germination dose–response pattern could be adequately described adjusting a single dose–response model [F = 0·968 (9,27), P = 0·48], showing that changes in seed responses to the light treatment were not affected by the different soil moisture conditions experienced during storage. The same test applied to dose–response curves fitted to germination data of seeds exposed to a saturating pulse of far-red light (treatment 2) showed that, in this case, the soil moisture content experienced by seeds during burial significantly affected the dose–response relationship [F = 7·63 (9,27), P < 0·0001]. Further comparisons between dose–response models adjusted to germination data for seeds stored under each soil moisture regime showed that the pattern of change in sensitivity to the light treatment for seeds stored at the highest soil moisture level (22·8 %) was significantly different from that displayed by seeds stored at soils with <20 % moisture content (Table 3; Fig. 7B). Moreover, changes in the germination response of seeds stored at soils with <20 % moisture content in relation to Stt accumulation could be adequately described adjusting a single dose–response model.


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  		TABLE 3. F-test for differences in variance explained by fitting single or separate models to germination data obtained for seeds stored under different soil moisture content regimes

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Results presented herein clearly show that the soil moisture environment experienced by seeds during cold-induced dormancy loss can affect seed responses to light stimuli. However, while different soil moisture conditions generally affected seed responses in the VLFR range (Figs 4B, C, 5B, C and 7B) and the percentage of the population that could germinate in darkness (Figs 4D, 5D and 6D), no effect was observed on seed responses to light in the LFR range (Figs 4A, 5A, 6A and 7A). These results are in agreement with those reported by Bouwmeester (1990) who observed that desiccation and subsequent re-hydration of Polygonum lapathifolium subsp. lapathifolium seeds increased the percentage of germination of seeds incubated in darkness, but did not affect the germination response of seeds exposed to red light (i.e. the LFR range).

Soil moisture fluctuations (FS) affected germination responses in the VLFR range (i.e. far-red light treatments) for seeds stored at low temperature (1·6 °C) (Fig. 4B, C), while little or no effect of FS was observed in that range for seeds stored at higher temperatures (7 and 12 °C) (Figs 5B, C, and 6B, C) or in the LFR range for seeds stored at any temperature (Figs 4A, 5A and 6A). However, seeds stored under a fluctuating soil moisture environment displayed germination percentages in darkness significantly higher than those recorded for seeds stored under a constantly moist soil environment, regardless of the temperature under which they had been stored (Figs 4D, 5D and 6D). This loss of the light requirement for germination due to exposure of seeds to soil moisture fluctuations resulted in higher values of germination in situ for seeds stored at 1·6 and 7 °C subjected to FS than displayed by seeds stored at similar temperatures under MS (Fig. 3). Almost identical results were recently reported by Batlla and Benech-Arnold (2006) for germination in situ of P. aviculare seeds stored under a fluctuating soil moisture environment.

Storage of seeds under different constant soil moisture contents modified the dynamics of acquisition of seed germination responses in the VLFR range in relation to stratification thermal time (Stt) accumulation (Fig. 7B). While changes in sensitivity to far-red light in relation to Stt accumulation for seeds stored under soil moisture contents of 19·6, 17·2 and 13·3 % could be described by adjusting a single dose–response model, changes in sensitivity to the same light treatment for seeds stored under a soil water content of 22·8 % required a different set of dose–response model parameters to be adequately described (Table 3). These results indicate that, in contrast to the lack of effect observed for seeds exposed to light in the LFR range (Fig. 7A), soil moisture content during storage under dormancy-releasing temperatures affected seed acquisition of VLFR.

To evaluate the effect of soil moisture fluctuations on the sensitivity of P. aviculare seeds to light stimuli, the germination response to light treatments between seeds subjected to a constant moist soil regime (MS) and a fluctuating soil moisture regime (FS) was compared. Due to the fact that variations in the soil water content in FS also involved changes in the storage temperature to which seeds were exposed, it is possible that these fluctuations in the thermal environment could have had an effect on the dormancy level of the seed population, and probably on its light sensitivity, beyond the effect of fluctuations in soil water content. However, results obtained by Batlla et al. (2003) showed that the thermal amplitude of fluctuating temperature regimes should be at least 6 °C or greater to affect the dormancy of P. aviculare seeds. Thus, the low amplitude variations in temperature registered during the storage period (0·8–2 °C in 15 d cycles) were unlikely to have affected the dormancy level of the seeds. On the other hand, to calculate the fraction of the seed population germinated in response to 76 % Pfr/P for seeds stored under FS and MS at 1·6 and 7 °C, it was assumed that seeds germinated in situ would have inevitably germinated in response to treatment 1. Indeed seeds germinated in situ, if any, were added to the fraction of seeds germinated after the light treatment to calculate percentage germination at 76 % Pfr/P. This decision was based on the fact that treatment 1, in which seeds were exposed to a saturating pulse of red light and were incubated at a temperature (15 °C) near the optimum temperature for P. aviculare germination (16 °C), was considered to be an ‘optimum’ environment for seed germination.

Overall, the results presented here show that, at least for the present experimental conditions, seed acquisition of LFR responses during dormancy release is not affected by either soil moisture fluctuations or different constant soil moisture contents. In contrast, different soil moisture storage conditions affected seed acquisition of VLFR and the capacity of seeds to germinate in the dark. These results suggest that VLFR and LFR responses may be mediated by different phytochromes in P. aviculare seeds, as demonstrated for Arabidopsis thaliana seeds by Botto et al. (1995, 1996) and Shinomura et al. (1996). In the latter species, phytochrome B and other phytochrome species different from phytochrome A are the photoreceptors involved in the LFR, while phytochrome A are the photoreceptors involved in the VLFR. Soil moisture content during burial might affect either the abundance of phytochrome A or the status of its transduction chain, but not phytochrome B.

Because ‘pre-existing’ phytochrome in the active form (Pfr) presumably declines during burial due to dark reversion, the possibility of its contribution to determine germination of seeds in the absence of light after some period of burial is unlikely. Recent experiments indicate that exposure of imbibed seeds to low temperatures in the dark activates gibberellin biosynthesis and response pathways in a phytochrome-independent way (Gallagher and Cardina, 1998; Yamauchi et al., 2004), suggesting that cold-induced dark germination could be not under phytochrome control. Due to the fact that soil moisture fluctuations dramatically raise germination of seeds incubated in the dark, while showing no or a less marked effect on the seeds' LFR and VLFR, our results also suggest that in the present experiment dark germination seemed not to be mediated by the action of phytochrome. Based on previous investigations, which showed the effect of seed hydration–dehydration cycles on cell membranes (Hegarty, 1978; Simon, 1984), it can be speculated that exposure of seeds to hydration–dehydration cycles in the present experiment could have exposed membrane-located receptors to active gibberellins biosynthesized during cold-induced dormancy loss, activating germination. Bouwmeester (1990) arrived at a similar hypothesis based on desiccation experiments carried out with seeds of different weed species. The facts that the effect of fluctuations in soil water content over dark germination levels showed an inverse relationship with storage temperature, and that the effect was more pronounced with time of storage (i.e. seeds were exposed to an increasing number of hydration–dehydration cycles), support the proposed hypothesis.

From an ecological point of view, the present results suggest that different soil moisture environments could certainly affect seed bank germination dynamics and, consequently, emergence patterns under field conditions. For example, many experiments showed that the extent to which seeds acquire VLFR would determine the fraction of the seed bank that would emerge in response to tillage operations. The present results suggest that soil moisture conditions experienced by seeds during the dormancy release season, which would mainly depend on prevailing weather conditions and burial depth, would affect seed acquisitions of VLFR, and thus the fraction of the seed bank that would germinate in response to tillage operations.

In addition, the present results provide evidence that in the case of seeds that require light to terminate dormancy and germinate, soil moisture fluctuations could mean that a certain fraction of the seed population would bypass this requirement. The marked changes in soil moisture to which seeds were exposed during storage in the present experiments could only take place in the upper centimetres of the soil profile where fluctuation of environmental variables, such as water and temperature, are larger. From an ecological viewpoint, this loss of the light requirement for germination as a result of exposing seeds to soil moisture fluctuations could represent a depth detection mechanism (in addition to those previously reported related to the requirement for light and fluctuating temperatures) (Bouwmeester and Karssen, 1993b).


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Dr R. Fernández for his guidance on experimental design and Dr R. Sánchez for useful comments on the manuscript. This research was financially supported by Fundación Antorchas (project 13870-1) and a grant from the Agencia Nacional de Promoción Científica (PICT/99 08-06651).


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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