Introduction
There is undeniable evidence that the Earth's climate has changed in the last century, to an extent that cannot be attributed to normal climate cycles (Wheeler and von Braun, Reference Wheeler and von Braun2013). As a result of anthropogenic emissions, the current CO2 level (407 μmol mol–1) may surpass 700 μmol mol–1 by the end of the century (Stocker et al., Reference Stocker, Qin, Plattner, Tignor, Allen, Boschung, Nauels, Xia, Bex and Midgley2013). Additionally, warming of 0.65–1.06°C has already occurred between the period of 1880 to 2012, with the last 30 years being the warmest in nearly a millennium. From 2016 to 2035 alone, climate models predict a further temperature increase of 0.3–0.7°C, and by 2100 it is expected that temperatures will rise at least another 1.5°C, and as much as 6.4°C (Stocker et al., Reference Stocker, Qin, Plattner, Tignor, Allen, Boschung, Nauels, Xia, Bex and Midgley2013). Other effects of climate change include increased incidences of water stress and modifications to ecosystem suitability for native species (Stocker et al., Reference Stocker, Qin, Plattner, Tignor, Allen, Boschung, Nauels, Xia, Bex and Midgley2013). Climate change has been reported to have a large effect on plant recruitment, and especially on the regeneration process. Temperature and water status of soil do not only affect the distribution of plants, but they also control the initiation and breaking of seed dormancy and radical emergence during germination (Walck et al., Reference Walck, Hidayati, Dixon, Thompson and Poschlod2011). Moreover, the chemical properties of seeds and their ability to germinate have been reported to be affected by environmental factors either during their development on the mother plant, or after dispersal in the soil (Gutterman, Reference Gutterman2000a). In general, seed dormancy can be classified as physical, physiological or morphophysiological, in addition to other categories (Baskin and Baskin, Reference Baskin and Baskin1998). Physiological dormancy is the most common type of seed dormancy in plants, and it is controlled by the presence of internal germination inhibitors, such as phenolics (Baskin and Baskin, Reference Baskin and Baskin1998), which can also be present in the seed coat (Debeaujon et al., Reference Debeaujon, Léon-Kloosterziel and Koornneef2000). Seeds of mouse-ear cress (Arabidopsis thaliana L.) have a non-deep physiological dormancy, which means that an embryo released from surrounding structures grows normally and that dormancy is broken by stratification or after-ripening (Baskin and Baskin, Reference Baskin and Baskin2004). In Arabidopsis, seed dormancy has been investigated for many years, with more focus on the genetic and physiological levels (Holdsworth et al., Reference Holdsworth, Bentsink and Soppe2008). These studies have shown that the phytohormone abscisic acid (ABA) plays a central role in inducing seed dormancy in this species (Bewley and Black, Reference Bewley and Black1994). However, ABA-deficient and -insensitive mutants of Arabidopsis have reduced dormancy (Nambara et al., Reference Nambara, Keith, McCourt and Naito1994). Several environmental factors are known to affect seed germination. Among them, temperature is the main factor affecting seed germination either by altering the parent plant metabolism and/or changing seed chemical composition (Fitter and Hay, Reference Fitter and Hay2012). It has been shown that lower temperatures increase dormancy levels in mature seeds of Arabidopsis (Kendall et al., Reference Kendall, Hellwege, Marriot, Whalley, Graham and Penfield2011; Piskurewicz et al., Reference Piskurewicz, Iwasaki, Susaki, Megies, Kinoshita and Lopez-Molina2016). Higher temperature inhibits the ability of plants to accumulate chemicals, which have an important role in inducing seed dormancy (Dornbos and McDonald, Reference Dornbos and McDonald1986). However, earlier reports have shown that higher temperatures could enhance seed germination (Fenner, Reference Fenner1991; Baskin and Baskin, Reference Baskin and Baskin1998). Elevated CO2 has been reported to decrease (Farnsworth and Bazzaz, Reference Farnsworth and Bazzaz1995; Andalo et al., Reference Andalo, Godelle, Lefrance, Mousseau and Till-Bottraud1996), to increase (Edwards et al., Reference Edwards, Clark and Newton2001) or to have no effect (Way et al., Reference Way, Ladeau, McCarthy, Clark, Oren, Finzi and Jackson2010), on seed germination. The responses of plants to elevated CO2 are species dependent (Farnsworth and Bazzaz, Reference Farnsworth and Bazzaz1995) and variation among genotypes also exists (Andalo et al., Reference Andalo, Godelle, Lefrance, Mousseau and Till-Bottraud1996). In their meta-analytical study, considering 79 crop and wild species, Jablonski et al. (Reference Jablonski, Wang and Curtis2002) reported a 14% decrease in seed nitrogen content in response to elevated CO2, particularly in the non-leguminous species. This could lead to decreased viability of seeds (Andalo et al., Reference Andalo, Godelle, Lefrance, Mousseau and Till-Bottraud1996). It has been shown that higher nitrogen content in seeds increases germination rate (Hara and Toriyama, Reference Hara and Toriyama1998), but not germination percentage per se (Hampton et al., Reference Hampton, Boelt, Rolston and Chastain2013). However, it has also been found that higher content of nitrogen in seeds of some plant species can induce dormancy (Peterson and Bazzaz, Reference Peterson and Bazzaz1978; Goudey et al., Reference Goudey, Saini and Spencer1987, Reference Goudey, Saini and Spencer1988; Luzuriaga et al., Reference Luzuriaga, Escudero and Pérez-García2006). Water availability and temperature are the most important factors in determining seed germination (Baskin and Baskin, Reference Baskin and Baskin1998). Water stress has a negative impact on seed germination as well as on the early seedling growth stages (Toscano et al., Reference Toscano, Romano, Tribulato and Patanè2017). Arabidopsis seed germination was negatively affected by water stress (Auge et al., Reference Auge, Blair, Burghardt, Coughlan, Edwards, Leverett and Donohue2015). However, it has been reported that water stress during seed maturation on the mother plant causes the seeds to transit from the state of development to the state of germination (Kermode et al., Reference Kermode, Bewley, Dasgupta and Misra1986). This transition happens because of alterations in protein content (Lalonde and Bewley, Reference Lalonde and Bewley1986) and messenger RNA (Bewley et al., Reference Bewley, Kermode and Misra1989). Germination processes determine the distribution and composition of plant communities (Harper et al., Reference Harper, Freeman, Ostler and Klikoff1978); however, fewer studies have been conducted on this topic compared with that of plant growth and development in relation to elevated CO2. Although several studies have examined the interactive effects of two or more climate change main drivers on seed germination (Alexander and Wulff, Reference Alexander and Wulff1985; Qaderi and Reid, Reference Qaderi and Reid2008; Gurvich et al., Reference Gurvich, Pérez-Sánchez, Bauk, Jurado, Ferrero, Funes and Flores2017), there is still a need for many more multi-factor studies. The objectives of this study were: (1) to determine germination responses of wild-type (WT) and its relative abscisic acid-insensitive mutant (abi1-1) of A. thaliana seeds to temperature, CO2 and watering regime, as individual factors and in combination; (2) to examine the effects of these factors on the fate of seeds that are tested for germination; and (3) to evaluate changes in seed total phenolics from WT and abi1-1 plants and examine its relevance to seed germinability. It was hypothesized that exposure of Arabidopsis plants to higher temperature, elevated CO2 and water stress would decrease subsequent seed germination pattern and increase number of non-germinated (dormant) seeds through increased total phenolic content of seeds, and that seed germinability would be related to genotype.
Materials and methods
Plant material and growth conditions
Seeds of the WT and abi1-1 mutant of A. thaliana ecotype Landsberg erecta were surface sterilized with 95% ethanol for 5 min and germinated in Petri dishes containing liquid Murashige and Skoog basal medium (MS) for 6 days. Then, from each genotype two seedlings were planted in each of 72 pots, containing a mixture of peat moss, Perlite and Vermiculite (1:1:1, v/v/v), with pellets of slow-release fertilizer (NPK, 13 14-14 plus micronutrients; Chisso-Asahi Fertilizer Co., Tokyo, Japan). Pots were transferred to a growth chamber (model ATC26, Conviron, Controlled Environments, Winnipeg, MB, Canada), set to a temperature regime of 22/18°C on a 16 h photoperiod. The photosynthetic photon flux density (PPFD) was 300 μmol photon m–2 s–1. Plants were left to acclimate for 2 days (which was sufficient time), and then 18 plants of each genotype were randomly assigned to one of eight experimental treatments: (1) lower temperatures (22/18°C, 16 h light/8 h dark), ambient CO2 (400 μmol mol–1) and watering to field capacity (well-watered) as control; (2) lower temperatures, ambient CO2 and watering at wilting point (water-stressed); (3) lower temperatures, elevated CO2 (700 μmol mol–1) and well-watered; (4) lower temperatures, elevated CO2 and water-stressed; (5) higher temperatures (28/24°C, 16 h light/8 h dark), ambient CO2 and well-watered; (6) higher temperatures, ambient CO2 and water-stressed; (7) higher temperatures, elevated CO2 and well-watered; (8) higher temperatures, elevated CO2 and water-stressed.
One growth chamber was used for each of the lower and higher temperature regimes. In each growth chamber, two equal size Plexiglas cabinets (60 × 65 × 50 cm, GE Polymershapes, Dartmouth, NS, Canada) were placed; one was supplied with ambient CO2 and the other with elevated CO2 (see above). Plants were grown inside these cabinets for 45 days. Half of the plants were watered to field capacity (well-watered) and the other half at wilting point (water-stressed). Midday soil water potential was about –0.4 and –1.3 MPa (megapascal) and leaf water potential was about –1.0 and –2.0 MPa for the well-watered and water-stressed plants, respectively. Water potential was measured with a WP4C Dew Point PotentiaMeter (Decagon Devices Inc., Pullman, WA, USA). Inside the cabinets, pots were rotated weekly to reduce positional effects. The experiment was conducted three times and each time the chambers and cabinets were reversed.
Germination tests
Seeds from the dry-brown siliques were used for germination experiments. From each treatment and genotype (three plants), 50 seeds were sown in triplicate on blue germination filter paper (Anchor Paper 37 Co., St Paul, MN, USA), initially moistened with 10 ml of distilled water in 100 × 15 mm Petri dishes. After that, 2–3 ml of distilled water were added as needed (usually every other day) to keep the germination paper moist. Seeds were germinated in a growth chamber under the control conditions essentially as described above. Germination was scored daily, and germinated seeds with 2 mm or longer radicle were counted and removed. Petri dishes were placed randomly in the growth chamber at the beginning of the experiment and replaced in different random patterns after each daily count. At the conclusion of the experiment (58 days), intact seeds were subjected to viability test according to Qaderi and Cavers (Reference Qaderi and Cavers2002).
Total phenolic content
Phenolic compounds were extracted according to Abdelhady et al. (Reference Abdelhady, Motaal and Beerhues2011) with some modifications. From each treatment, six samples of dry seeds (0.5 g) were collected and homogenized using a pestle and mortar in 2.5 ml 80% methanol. The homogenate was then incubated for 2 h at room temperature and centrifuged at 4000 g for 20 min at 4°C. The supernatant was collected and stored at 4°C for further analysis. Total phenolic content was estimated by the method of Waterhouse (Reference Waterhouse and Wrolstad2002) by using 2N Folin-Ciocalteu reagent and Gallic acid in methanol (concentration range: 0 to 500 mg l–1) as a standard. A 10 µl aliquot of each sample was mixed with 790 µl of water, 50 µl of the 2N Folin-Ciocalteu reagent and 150 µl of 20% (w/v) sodium carbonate and incubated at room temperature for 2 h in the dark. The absorbance was measured at 760 nm for the solution with a UV/visible spectrophotometer (model Ultraspec 3100 pro, Biochrom Ltd, Cambridge, UK). The concentration of phenolics was determined (mg l–1) from the standard curve; the total phenolic content in samples was then expressed in relations of Gallic acid equivalent (GAE) by the following equation: P = (C × V)/M, where P is total phenolic content (mg GAE g–1 FM), C is the concentration of Gallic acid calculated from the calibration curve (mg l–1), V is the volume of extracting medium (ml–1), and M is the leaf fresh mass (g–1).
Data analysis
The effects of temperature, CO2, watering regime, genotype, and their interactions were determined on seed germination pattern, fate of seeds that were set for germination, and total phenolic content of the WT and its relative ABA-insensitive mutant (abi1-1) of A. thaliana using a four-way analysis of variance (ANOVA). Differences among treatments were determined by a one-way ANOVA, using Scheffé’s multiple comparison procedure at the 5% confidence level (SAS Institute, 2011). The following equation was used to calculate the coefficients of germination rate for each replicate: N/∑ n i d i, where N is final germination percentage, n i is the number of germinated seeds on the particular day on which a count was made, and d i is the number of days from the start of the experiment (Alm et al., Reference Alm, Stoller and Wax1993). All values of coefficients of germination rate are between 0 (no germination) and 1 (fastest germination rate), multiplied by 100 to facilitate interpretation. A multiple Pearson's correlation coefficient at the 5% confidence interval was used to determine relationships between parameters (Minitab, Inc., 2014).
Results
Germination percentage
Seeds that matured under higher temperatures had a higher percentage of germination than the seeds matured under lower temperatures (Table 1). Although not significant, seeds of the abi1-1 mutant had a higher germination percentage than those of the WT genotype (Table 1). Overall, subsequent seed germination was significantly affected by the plant growth temperature, the two-way interactions between temperature (T) × watering regime (W), T × genotype (G), and W × G, the three-way interaction among carbon dioxide (C) × W × G, and the four-way interaction (Table 2). Based on the four-way interaction, the WT seeds that matured on the water-stressed plants that were grown under higher temperatures at ambient CO2 had the highest germination percentage, whereas the WT seeds that matured on the water-stressed plants that were grown under lower temperatures at elevated CO2 had the lowest germination percentage (P < 0.05; Figs 1A,B and 2A,B).
Fig. 1. Effects of temperature, carbon dioxide and watering regime on germination pattern and total phenolic content of Arabidopsis thaliana seeds. Plants were grown under two temperature regimes (22/18°C and 28/24°C; 16 h light/8 h dark), two carbon dioxide concentrations (400 and 700 μmol mol–1) and two watering regimes (well-watered and water-stressed) in controlled-environment growth chambers. (A,C,E) WT, (B,D,F) abi1-1. (A,B) Germination percentage; (C,D) coefficients of germination rate; (E,F) total phenolic content. Dark grey bars represent well-watered plants and light grey bars water-stressed plants. Different letters above the bars (mean ± SE) denote significant differences (P < 0.05) within each parameter according to Scheffé’s multiple-comparison procedure. Lower case letters represent differences within genotypes, whereas upper case letters represent differences between genotypes.
Fig. 2. Effects of temperature, carbon dioxide and watering regime on the fate of non-germinated seeds of Arabidopsis thaliana. (A) WT, (B) abi1-1. ACO2, ambient CO2; ECO2, elevated CO2; WW, well-watered; WS, water-stressed. Other details are as in Fig. 1.
Table 1. Effects of temperature, carbon dioxide, watering regime and genotype on germinated and non-germinated seeds, and total phenolic content of Arabidopsis thaliana
Mature seeds were collected from A. thaliana plants (wild-type and abi1-1 mutant) that were grown under two temperature regimes (22/18°C and 28/24°C; 16 h light/8 h dark), two carbon dioxide concentrations (400 and 700 μmol mol–1) and two watering regimes (well-watered and water-stressed) in controlled-environment growth chambers for 45 days, after 8 days of initial growth under 22/18°C. Seeds were germinated under a temperature regime of 22/18°C (16 h light/8 h dark), light intensity of 300 μmol m–2 s–1, and relative humidity of ~65% for 58 days. Data are means ± SE of nine samples of 50 seeds from three trials for all measured parameters, except for total phenolic content (means ± SE of six samples from two trials). Means followed by different letters within each parameter and condition are significantly different (P < 0.05) according to Scheffé’s multiple-comparison procedure. For coefficients of germination rate, all means have been multiplied by 100 to facilitate data interpretation.
Table 2. Analysis of variance (F value) for effects of temperature, carbon dioxide, watering regime and genotype on germination pattern, non-germinated seeds (viable, rotten and empty), and total phenolic content in seeds of Arabidopsis thaliana
Arabidopsis (wild-type and abi1-1 mutant) plants were grown under two temperature regimes (22/18°C and 28/24°C; 16 h light/8 h dark), two carbon dioxide concentrations (400 and 700 μmol mol–1) and two watering regimes (well-watered and water-stressed) in controlled-environment growth chambers for 45 days, after 8 days of initial growth under 22/18°C. Experiments were conducted three times. Mature seeds were collected and germinated under a temperature regime of 22/18°C (16 h light/8 h dark), light intensity of 300 μmol m–2 s–1, and relative humidity of ~65% for 58 days. Significance values: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Coefficient of germination rate
Seeds that matured under lower temperatures had a higher germination rate than seeds that matured under higher temperatures (Table 1). Also, seeds that matured on the abi1-1 mutant plants had a higher germination rate than the seeds that matured on the WT genotype plants. Germination rate was affected significantly by temperature, genotype and the two-way interactions between T × W, T × G and W × G (Table 2). The T × W interaction showed that seeds that matured on the well-watered plants grown under higher temperatures had the fastest rate, while seeds that matured on the well-watered plants grown under lower temperatures had the slowest rate. With regard to the T × G interaction, seeds that matured on the WT plants grown under higher temperatures had the fastest rate, and seeds that matured on the abi1-1 plants grown under lower temperatures had the slowest rate. The interaction between W and G revealed that seeds that matured on the water-stressed WT plants had the fastest rate, and seeds that matured on the water-stressed abi1-1 plants had the slowest rate (P < 0.05; Fig. 1C,D).
Fate of non-germinated seeds
A viability test at the end of this experiment showed that the percentage of non-germinated viable seeds was significantly higher for seeds that matured on plants that were grown under lower temperatures than for seeds that matured on plants that were grown under higher temperatures (Table 1). Similarly, seeds that matured on the well-watered plants had higher percentage of non-germinated viable seeds than seeds that matured on the water-stressed plants (Table 1). Overall, the percentage of non-germinated viable seeds was significantly affected by temperature, watering regime, the two-way interactions between T × W and T × G, and the four-way interaction (Table 2). Based on the four-way interaction, the abi1-1 seeds that matured on the well-watered plants grown under higher temperatures at elevated CO2 had the highest percentage of non-germinated viable seeds, whereas the WT seeds that matured on the water-stressed plants grown under higher temperatures at ambient CO2 had the lowest percentage of non-germinated viable seeds (P < 0.05; Fig. 2A,B).
The germination test revealed that seeds that matured under higher temperatures were less rotten than seeds that matured under lower temperatures (Table 1). The percentage of rotten seeds was significantly affected by temperature, the interactions between T × W, T × G and W × G, the three-way interaction among C × W × G, and the four-way interaction (Table 2). Based on these interactions, the WT seeds that matured on the water-stressed plants grown under lower temperatures at ambient CO2 had the highest percentage of rotten seeds, whereas the WT seeds that matured on the water-stressed plants grown under higher temperatures at ambient CO2 had the lowest percentage of rotten seeds (P < 0.05; Fig. 2A,B).
Seeds that matured on the abi1-1 plants were emptier than seeds that matured on the WT plants (Table 1). Genotype and the interactions between T × C, T × W, T × G and W × G significantly affected the percentage of empty seeds. With respect to the interaction between T × C, seeds that matured on plants grown under lower temperatures at ambient CO2 had highest number of empty seeds, while seeds that matured on plants grown under higher temperatures at ambient CO2 had lowest number of empty seeds. The T × W interaction showed that the water-stressed plants grown under lower temperatures produced the highest number of empty seeds, whereas the water-stressed plants grown under higher temperatures produced the lowest number of empty seeds. The interaction between T and G revealed that the abi1-1 seeds that matured on plants grown under higher temperatures had the highest percentage of empty seeds, whereas the WT seeds that matured on plants grown under the same temperatures had the lowest percentage of empty seeds. As per the W × G interaction, seeds that matured on the water-stressed abi1-1 plants had the highest number of empty seeds, whereas seeds that matured on the well-watered WT plants had the lowest number of empty seeds (P < 0.05; Fig. 2A,B).
Total phenolic content
Seeds that matured under lower temperatures had higher total phenolic content than seeds that matured under higher temperatures (Table 1). Elevated CO2 increased total phenolic content, which was higher in the abi1-1 seeds than in the WT seeds (Table 1). Total phenolic content was significantly affected by temperature, CO2, genotype, the two-way interaction between T × W, T × G and W × G, the three-way interaction among C × W × G, and the four-way interaction (Table 2). The highest total phenolic content occurred in seeds that matured on the water-stressed WT plants that were grown under lower temperatures at elevated CO2, but the lowest total phenolic content occurred in seeds that matured on the water-stressed WT plants that were grown under higher temperatures at ambient CO2 (P < 0.05; Fig. 1E,F).
Relationship between seed categories
Pearson's correlation analysis showed numerous significant and interesting relationships between seed germination pattern, fate of non-germinated seeds and total phenolic content (Table 3). For example, germination percentage was negatively correlated with non-germinated seeds (viable, rotten and empty); similarly, germination percentage was negatively correlated with total phenolic content. Germination rate had a significant positive relationship with germination percentage, but a negative relationship with rotten seeds. The percentage of non-germinated viable seeds was positively correlated with the percentage of rotten and empty seeds and total phenolic content (Table 3).
Table 3. Pearson's correlation coefficients between germination percentage, coefficients of germination rate, non-germinated seeds (viable, rotten and empty) and total phenolic content in seeds of Arabidopsis thaliana
Arabidopsis plants (wild-type and abi1-1 mutant) were grown under two temperature regimes (22/18°C and 28/24°C; 16 h light/8 h dark), two carbon dioxide concentrations (400 and 700 μmol mol–1) and two watering regimes (well-watered and water-stressed) in controlled-environment growth chambers for 45 days, after 8 days of initial growth under 22/18°C. Experiments were conducted three times (n = 9), except for the total phenolics (two times, n = 6). Seeds were collected and germinated under a temperature regime of 22/18°C (16 h light/8 h dark), light intensity of 300 μmol m–2 s–1, and relative humidity of ~65% for 58 days. Significance values: *P < 0.05; ***P < 0.001.
Discussion
Effects of temperature
This study showed that maturation temperatures had the greatest effect on subsequent seed germinability and total phenolic content of the progeny. In the current study, higher maturation temperatures were followed by more complete and faster germination of Arabidopsis seeds (Table 1; Fig. 1A–D). The positive effect of higher temperatures on germination percentage and rate agrees with the results of earlier studies on seeds from other plant species (Qaderi et al., Reference Qaderi, Cavers, Hamill, Downs and Bernards2006; Pérez-Sánchez et al., Reference Pérez-Sánchez, Jurado, Chapa-Vargas and Flores2011). Higher temperatures increased the germination percentage of Scotch thistle (Onopordum acanthium L.) cypselas either by making the cypsela coats thinner (Qaderi et al., Reference Qaderi, Cavers and Bernards2003), or by lowering phenolic compounds and surface wax in cypselas (Qaderi et al., Reference Qaderi, Cavers, Hamill, Downs and Bernards2006). In the current study, it is likely that higher temperatures have led to increased germination percentage by decreasing the germination-inhibiting factor – the phenolic compounds (Tables 1 and 3; Fig. 1E,F).
A significant reduction in the percentage of non-germinated viable seeds was found in seeds that matured under higher temperatures (Table 1). Also, negative correlation between germinated seeds and non-germinated viable seeds revealed that the germinability of seeds was increased by higher temperatures, as expected (Table 3). The thinner outer covering of seeds that usually forms under higher temperatures may also enhance the leaching of germination inhibitors from the embryo (Porter and Wareing, Reference Porter and Wareing1974) and, thus, decreases the number of non-germinated viable seeds that matured under higher temperatures. In the current study, seeds that matured under higher temperatures had decreased number of rotten seeds at the end of the germination test (Table 1). Because seeds that matured under lower temperatures took longer to germinate, possibly prolonged soaking of seeds increased the number of rotten seeds, which was followed by seed mortality. The negative relationship between germinated and rotten seeds may also explain the lower germination percentage of seeds that matured under lower temperatures, as more rotten seeds were found from this temperature regime upon viability test (Tables 1 and 3).
Effects of carbon dioxide
Elevated CO2 during seed maturation significantly increased total phenolic content (Table 1), but did not affect the germination pattern. Way et al. (Reference Way, Ladeau, McCarthy, Clark, Oren, Finzi and Jackson2010) also found that neither germination percentage nor germination rate of loblolly pine (Pinus taeda L.) seeds were affected by CO2 treatment. In the current study, the increased total phenolic content of seeds that matured at elevated CO2 (Table 1) is in agreement with the result of Karowe and Grubb (Reference Karowe and Grubb2011) who found higher concentrations of phenolic compounds in oilseed rape (Brassica rapa L.) plants grown at elevated CO2.
Effects of watering regime
Seeds that matured on the water-stressed plants had a decreased percentage of non-germinated viable seeds (Table 1). Drought condition of the mother plant interrupts the development of seeds that results in light and shrivelled seeds (DeLouche, Reference Delouche1980), which can have altered germinability. Hawkes (Reference Hawkes2004) reported that drought increased seed dormancy and decreased seed germination and/or viability of four herbs. Auge et al. (Reference Auge, Blair, Burghardt, Coughlan, Edwards, Leverett and Donohue2015) have also shown that water stress increased secondary dormancy in Arabidopsis. However, other studies have shown that water stress during seed maturation increases subsequent seed germination (see Gutterman, Reference Gutterman and Fenner2000b). For example, water stress decreased seed dormancy of wild oats (Avena fatua L.; Sawhney and Naylor, Reference Sawhney and Naylor1982), Johnson grass (Sorghum halepense (L.) Pers.; Benech Arnold et al., Reference Benech Arnold, Fenner and Edwards1992), and African mustard (Brassica tournefortii Gouan.; Gorecki et al., Reference Gorecki, Long, Flematti and Stevens2012). The absence of a consistent pattern of seed germination among plant species could be related to variation in the properties of maternal tissue of seeds that are affected by water stress during seed maturation (Gutterman, Reference Gutterman and Fenner2000b).
Effects of genotype
The current study showed that seeds that matured on the WT plants had a lower germination rate, lower percentage of empty seeds, and decreased total phenolic content than the seeds that matured on the abi1-1 mutant plants (Table 1). Non-significant difference in seed germination percentage between WT and abi1-1 is unusual. The Arabidopsis abi1-1 phosphatase mutation, which reduced abscisic acid-induced dormancy in seeds (Koornneef et al., Reference Koornneef, Reuling and Karssen1984) made the abi1-1 mutant seeds quicker and more germinable and therefore their germination rate was higher (Fig. 1C,D). However, the higher percentage of empty seeds and phenolic content of the abi1-1 mutant seeds (Table 1; Figs 1E,F and 2A,B), caused them to have similar germination percentage to that of WT seeds. The use of ABA-insensitive mutant seeds showed that under climate change, the ABA content of mature embryo will not be the only factor that can reduce or prevent seed germination. Also, increased empty seed number produced by the abi1-1 plants is related to the stronger effect of stress factors on the abi1-1 mutant than on the WT genotype. This is mainly because of the ABA signalling impairment in the guard cells of the abi1-1 mutant (Pei et al., Reference Pei, Kuchitsu, Ward, Schwarz and Schroeder1997), causing their stomata to remain open and, consequently, increasing transpiration and decreasing photosynthesis. Decreased accumulation of phenolics in the WT seeds could mean that these seeds were less affected by temperature and water stress than the abi1-1 seeds, as stressed plants usually accumulate more phenolic compounds (Harborne and Williams, Reference Harborne and Williams2000).
Interactive effects of temperature, carbon dioxide, watering regime and genotype
Germination percentage was highest for seeds that matured on the water-stressed plants under higher temperatures, but lowest for seeds that matured on the water-stressed plants under lower temperatures. This result did not concur with the study of Gurvich et al. (Reference Gurvich, Pérez-Sánchez, Bauk, Jurado, Ferrero, Funes and Flores2017), who found that low water potentials and high temperatures negatively affected seed germination in cactus (Echinopsis candicans L.). Seeds that matured on the water-stressed WT plants grown under higher temperatures at ambient CO2 had 1.3 times higher germination percentage than the seeds that matured on the well-watered WT plants grown under lower temperatures at elevated CO2 (Figs 1A,B and 2A,B). Higher maturation temperatures and water stress, individually and together, increased germination percentage, but seeds that matured at elevated CO2 had decreased germination percentage. Germination percentage has been shown to decrease in Arabidopsis at elevated CO2 (Andalo et al., Reference Andalo, Godelle, Lefrance, Mousseau and Till-Bottraud1996). Elevated CO2 has been shown to increase the C/N ratio and that can cause a reduction in seed protein content, which can be used to provide amino acids necessary for embryo growth during germination (Andalo et al., Reference Andalo, Godelle, Lefrance, Mousseau and Till-Bottraud1996). Seeds that matured on the well-watered abi1-1 plants grown under higher temperatures at elevated CO2 had 10 times higher non-germinated viable seeds than the seeds that matured on the water-stressed WT plants grown under higher temperatures at ambient CO2 (P < 0.05; Fig. 2A,B). It is not surprising to see that seeds matured on the abi1-1 plants were not more germinable than those of WT plants; the outcome may be due to the accumulation of more phenolic compounds in those seeds. Also, the non-significant positive effect of elevated CO2 on seed viability is in agreement with the study of Prasad et al. (Reference Prasad, Boote, Allen and Thomas2003) who showed that elevated CO2 did not counteract the negative effects of high temperatures on seed viability. Earlier studies have shown that abi1-1 mutation lead to a total (Webb and Hetherington, Reference Webb and Hetherington1997) or 50% (Leymarie et al., Reference Leymarie, Vavasseur and Lascève1998) suppression of CO2, sensing in stomata of the abi1-1 mutant. The seeds from the water-stressed WT plants that were grown under lower temperatures at elevated CO2 had the highest total phenolic content; this could explain the lowest germination percentage and the highest germination rate for seeds that matured under this condition in comparison with other conditions (Fig. 1A–F).
In conclusion, among the main climate change components discussed in the current study, maturation temperature has more effects on subsequent seed germination, germination rate, rotten seeds and total phenolic content than CO2 and watering regime. Seeds that matured under higher temperatures could have less dormancy and exhibit more germiniability than those that matured under lower temperatures. The negative effects of higher temperatures on the non-germinated viable seeds that matured on the abi1-1 plants was not mitigated by the elevated CO2. It is, therefore, most likely that higher temperature and water stress will have a strong impact on plant performance, especially on the sensitive plant cultivars, and this could have implications for the survival of sensitive plants under future climates. This study showed that the climate change-related factors will have an effect on the fate of seeds after dispersal. Based on the results of this study, climate change might have little effect on the germinability of WT seeds, but could decrease seeds of ABA-insensitive mutants (e.g. abi1-1) through changing their phenolics and other such compounds. This study indicates that there are factors other than ABA that are involved in controlling seed germination in response to climatic factors. Further studies are required to fully understand seed germination patterns under multiple climate change factors.
Author ORCIDs
Mirwais M. Qaderi, 0000-0001-7050-4050
Financial support
Funding for this research was provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada through a Discovery grant to M.M.Q., and supported by a graduate scholarship from Yarmouk University (Irbid, Jordan) to M.I.A.
