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Soil temperature and moisture regulate seed dormancy cycling of a dune annual in a temperate desert
Accepted Manuscript Title: Soil temperature and moisture regulate seed dormancy cycling of a dune annual in a temperate desert Authors: Ruiru Gao, Ruihua Zhao, Zhenying Huang, Xuejun Yang, Xiaoya Wei, Zhan He, Jeffrey L. Walck PII: DOI: Reference:
S0098-8472(18)31062-1 https://doi.org/10.1016/j.envexpbot.2018.08.010 EEB 3536
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
13-7-2018 10-8-2018 10-8-2018
Please cite this article as: Gao R, Zhao R, Huang Z, Yang X, Wei X, He Z, Walck JL, Soil temperature and moisture regulate seed dormancy cycling of a dune annual in a temperate desert, Environmental and Experimental Botany (2018), https://doi.org/10.1016/j.envexpbot.2018.08.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Original Articles
Soil temperature and moisture regulate seed dormancy cycling of a dune annual in a temperate desert
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Running head: Temperature and moisture regulating dormancy and germination
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Ruiru Gaoa,b, Ruihua Zhaob, Zhenying Huanga*, Xuejun Yanga, Xiaoya Weib, Zhan
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany,
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a
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Heb, Jeffrey L. Walckc
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Chinese Academy of Sciences, Beijing 100093, P.R. China The School of Life Sciences, Shanxi Normal University, Linfen, P.R.China
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Department of Biology, Middle Tennessee State University, Murfreesboro, TN
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b
37132, USA
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Hghlights
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* Corresponding author. E-mail: [email protected]
Soil seeds of Agriophyllum squarrosum have annual dormancy cycling
Freshly harvested seeds were in non-deep physiological (conditional) dormancy
Germination of soil seeds were promoted by wetting-drying cycles
Dormancy was induced by warm temperature and particularly low soil 1
moisture
Rainfall and temperature regulated seed dormancy/germination seasonal pattern
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Abstract
Plants have evolved diverse strategies to ensure their survival and regeneration in
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specific environments. Although temperature and soil moisture control seed dormancy, most studies have concentrated on temperature and little is known about the influence
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of moisture for species in the arid region. Responses of seed dormancy and germination
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of Agriophyllum squarrosum (Amaranthaceae), a pioneer and dominant species in Mu
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Us Sandland in northern China, to variations in soil moisture and temperature were
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examined. Our study showed that (1) freshly harvested seeds were in non-deep
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physiological (conditional) dormancy; (2) seeds in the soil exhibited dormancy cycling being non-dormant in spring and dormant from summer to autumn; (3) dry conditions
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at cold or warm temperatures alleviated dormancy; (4) germination was promoted by
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wetting-drying cycles; and (5) dormancy was induced by warm temperature (15/25°) and particularly low soil moisture less than 14.0%. The seasonal pattern of seed dormancy/germination was regulated by seasonal rainfall and soil temperature. At the
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same time, a range of conditions enable dormancy break and germination regardless of soil moisture conditions allowing the species to persist in an unpredictable environment.
Key words：Agriophyllum squarrosum; dormancy cycle; seed germination; soil 2
seed bank; soil temperature; soil moisture
1. Introduction Seeds are the most tolerant stage to environmental stresses in the life histories of plants
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(Gutterman,1993). Dormant seeds allow plants to persist during unfavorable seasons for seedling establishment and non-dormant seeds regenerate plant populations when
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favorable seasons occur (Mapes et al., 1989; Rees, 1996). Seed dormancy has been considered as a bet-hedging strategy of plants to cope with uncertain environments
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(Gremer and Venable, 2014). Plants have evolved diverse strategies to ensure
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successful germination at the right time in a suitable place (Linkies et al., 2010), and
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and Flores, 2005; Nonogaki, 2014).
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seed dormancy is important for plants to adapt to unpredictable environments (Jurado
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Seeds buried in the soil sense and integrate a range of environmental signals to continually adjust their degree of dormancy to co-ordinate germination with seedling
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emergence at a suitable space and time (Copete et al., 2015). In natural habitats,
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variation in germination timing for some species with physiological dormancy is controlled by seasonal climate cues resulting in dormancy cycling (Baskin and Baskin, 1985). This is an important strategy for a seed population to avoid sibling competition
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or extinction of all individuals due to natural disasters (Nonogaki, 2014). In arid and semi-arid regions, the temporal distributions of seed dormancy/germination might especially be an adaptation of plants to these dramatically fluctuating environments. Agriopyllum squarrosum (L.) Moq. (Amaranthaceae) is an annual plant with both
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aerial and soil seed banks (Liu et al., 2006; Gao et al., 2014). The seed dispersal of the species is extended over time, which forms the aerial seed bank (Gao et al., 2014). Differences between the soil and aerial seed banks occur: seeds from the soil seed bank germinate earlier than those from the aerial seed bank, which regulates the timing of
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seedling emergence through a bet-hedging strategy (Gao et al., 2014). Seed germination is suppressed by light and is promoted by darkness (Wang et al., 1998; Zheng et al.,
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2004; Tobe et al., 2005), and it is markedly low at constant temperatures (10°C) but high at alternating temperatures in darkness (Wang et al., 1998; Zheng et al., 2004). In
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addition, seeds germinate at low soil moisture (-0.8 MPa) (Cui et al., 2007), indicating
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that the species is strongly drought tolerant. When burial depth is beyond 5 cm, no seeds
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germinate (Tobe et al., 2005; Zheng et al., 2004) and a persistent soil seed bank develops.
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Liu et al. (2007) found that seeds remained viable in mobile dunes when buried deeper
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than 1.0 m, but the surface seed bank (<5 cm) was depleted in the growing season (Gao et al., 2014). Thus, the spatial and temporal patterns of the seed banks along with
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germination responses have important impacts on the seedling recruitment of A.
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squarrosum (Bai et al., 2004; Liu et al., 2007; Ma and Liu, 2008). Freshly matured seeds of A. squarrosum at our study site are mostly dispersed in
late autumn and winter, when the subsurface of the soil is frozen and the surface is
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unfrozen but often dry (Gao et al., pers. obs.). Anaysis of weather data for the past 50 years (1959-2009) at the study site showed that, in late winter and early spring, as snow and frozen soil melt and temperatures warm the subsurface of the soil remains moist but the surface may become dry. Thus, cold stratification may be severely limited from
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late autumn to early spring due to temperatures below freezing and to dry conditions. Discrete rainfall events start in mid-spring and continue until late summer as temperatures dramatically rise to 40°C. Soil moisture becomes very limited in the summer with high evaporation and sporadic rain. During the spring and summer, seeds
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are exposed to wetting-drying cycles and to a gradient of moisture on the surface as well as the subsurface of the soil. Although seeds of A. squarrosum are available from
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the aerial and soil seed banks at any time of the year, we have observed seedlings mostly
from April to May and rarely at other times of the year (Gao et al., 2014). We surmised
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that seeds of this species may undergo dormancy cycling in the soil, which would
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explain our seasonal observations of seedling emergence. Taken together, we surmised
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that dormancy and germination would be controlled by the complex interaction of
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temperature and soil moisture in this semi-arid region.
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Although temperature and soil moisture are important environmental cues that control seed dormancy loss/induction and germination, most studies have concentrated
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on temperature effects (Footitt et al., 2015; Postma et al., 2016) and little is known
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about the influence of moisture (Copete et al., 2015), especially for species in arid and semiarid regions. As such, although we understand the life history strategies of A. squarrosum to cope with unpredictable environments, there is little information on the
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dynamics of seasonal seed dormancy cycling of seeds in the soil seed bank and how dormancy loss/induction and germination are regulated by environmental factors. Moreover, a discrepancy on the degree of dormancy in freshly matured seeds of A. squarrosum exists. While some researchers have found that seeds were non-dormant
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and can rapidly germinate (Wang et al., 1998), other researchers have shown that seeds were dormant and dry storage was needed to overcome this dormancy (Liu et al., 2013). The germination responses of offspring seeds of this species were altered by maternal environments, and seeds collected from plants inhabiting moist habitats were more
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dormant than those from dry habitats (Gao et al., 2015). Therefore, the degree of dormancy may be strongly regulated by soil moisture.
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Recently, Fan et al. (2017) presented a conceptual model of the seed/seedling
dynamics of A. squarrosum. They stressed the need to understand better the dormancy
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and germination of this important dune-stabilizing species within an ecological context.
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To the best our knowledge, no studies on A. squarrosum have examined the roles that
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temperature and soil moisture play in regulating seed dormancy/induction and
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germination, i.e. considering the seasonal patterns of temperature and soil moisture in
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the semi-arid region. Thus, we (1) determined the degree of dormancy in fresh seeds from the population that we studied by testing the effects of dry storage and testa
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scarification in comparison to fresh (intact) seeds. In addition, we (2) examined the
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seasonal pattern of dormancy and germination of seeds buried in soil over time. Simultaneously, we tested the effects of (3) a moisture gradient at cold temperature, simulating conditions from late autumn to early spring and (4) wetting-drying cycles
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and (5) a moisture gradient at warm temperature, simulating conditions from midspring to summer. Using these combined methods of laboratory experiments and controlled field trials allowed us to explore how seed dormancy and germination are controlled in relation to seasonal soil temperature and moisture fluctuations. The results
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will allow us to further understand the adaptive strategies of annual dune plants in temperate semi-arid regions.
2. Material and methods
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2.1 Study species Agriophyllum squarrosum inhabits semi-arid and arid regions, and is widely distributed
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from central to eastern Asia (Kong, 1996; Naqinezhad, 2012; Qiao et al., 2012), where it is a pioneer species on mobile and semi-fixed sand dunes (Nemoto and Lu, 1992).
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The species could be used to stabilize mobile sand-dunes (Wang et al., 2009) and also
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is of nutrient value due to high content of protein and unsaturated fatty acids and linoleic
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acid in seeds (Korbanjhon, 2011); as such, it has the potential to be a human food crop
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(Chen et al., 2014). The vegetative parts of the plant are already used as forage for
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animals in winter, such as Camelus bactrianus (Zhao et al., 2006). Additional information on the species can be found in Kong (1996) and Gao et al. (2014, 2015).
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2.2 Plant material
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Fifty plants were randomly selected in a natural population of A. squarrosum at the Ordos Sandland Ecological Research Station of the Chinese Academy of Sciences (39°29′37.6″ N, 110°11′29.4″ E, 1296 m a.s.l.), located in the north-eastern Mu Us
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Sandland in Inner Mongolia, China, on 20 October 2012. The average annual precipitation in this region is 345.8 mm and is largely restricted to the period from June to August. The mean annual temperature is 6.8°C with a minimum of -16.9°C in January and maximum of 28.1°C in July (Gao et al., 2014, 2015). The landscape at the
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Station consists of mobile and semi-fixed sand dunes, on which A. squarrosum is common. The collected plants were placed in a paper bag and taken to a laboratory, where seeds were removed from each plant by rubbing the infructescences by hand. 2.3 General procedures for germination tests
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Germination tests were conducted at 5/15, 10/20, 15/25 and 20/30°C (12/12 h) in darkness due to the inhibitory effect of light on seed germination (Gao et al., 2014). The
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four temperature regimes represented mean minimum/maximum temperatures for late
April and October, May and September, June and August, and July, respectively. Four
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replicates of 25 seeds each were placed into 7-mm diameter Petri dishes on two layers
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of filter papers moistened with 3 ml distilled water. Germination was checked daily
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under a green safe light in a dark room. Germinated seeds (radicle emerged) were
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removed, and the test was completed after 20 d when no additional seeds germinated
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for 4 successive days, unless stated otherwise. Following the test, nongerminated seeds were pinched with forceps to determine whether the embryo was firm. If the embryo
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was firm, the seed was viable. Germination percentages were calculated based on
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number of viable seeds.
2.4 Determination of the degree of dormancy in fresh seeds Within 1 week after collection, fresh (intact) seeds were incubated at 5/15, 10/20, 15/25
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and 20/30°C in darkness. In addition, germination of seeds from scarification and dry storage treatments was also tested. The scarification treatment consisted of taking 400 fresh seeds within 1 week after collection, making a small hole through the testa with a dissecting needle and not damaging the embryo, and incubating them over the same
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conditions as the control. The dry storage treatment consisted of storing 500 fresh seeds dry in a paper bag in the laboratory (25.0 ± 2.0°C) for 1 month and then testing germination under the same conditions as the control seeds. 2.5 Seasonal patterns of dormancy and germination
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Fresh seeds were placed in 152 fine-mesh nylon bags (150 seeds per bag, 8 cm length x 2-3 cm width), which were buried in the natural habitat of A. squarrosum surrounded
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by a wire fence to prevent animal damage. Although 76 bags were needed for the
experiment, we buried an additional 76 bags in case of mice predation. Burial occurred
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on 1 December 2012 and exhumations were done at 1 month intervals between 1
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January 2013 and 30 July 2014. At the time of burial, the soil (consisting mostly of
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sand) was frozen and covered with 5-10 cm of snow; thus, the snow was carefully
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removed and a pickax used to dig five parallel trenches (ca. 1 m length x 8 cm width x
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6 cm depth) at 10 cm apart. The bags were randomly allocated to a trench, placed flat on the bottom of the trenches and equally spaced apart. Then, they were covered with
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unfrozen soil, which later froze, and snow (to same depth as before burial); the locations
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of each end of trenches were marked with a stick that protruded above the soil and snow. At each exhumation, snow, if present, was removed and four bags of seeds from a trench were removed; snow and soil were replaced after removal. The bags were taken back
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to the laboratory where seeds from all of them were mixed and tested for germination at 5/15, 10/20, 15/25, 20/30 and 25/35°C in darkness. The 25/35°C represented the extreme temperature that sometimes occurs in July. No germinated seeds were observed in the bags at any exhumation date. During the experimental period, data on daily
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rainfall and on soil temperatures [maximum (MaxT) and minimum (MinT) temperature] at a depth of 5 cm below the soil surface were recorded by the weather station at the Ordos Sandland Ecological Research Station which is close to the experimental site (37.3 m).
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2.6 Effects of a moisture gradient at cold temperature Eighteen hundred seeds were equally placed in nine fine-mesh nylon bags, with three
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bags each being buried 5 cm deep in three opaque plastic (stratification) boxes with lids (40 cm length × 30 cm width × 15 cm height) filled with 1.0 kg dry sand (at about 10
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cm depth), which was collected from natural habitats. The sand in two boxes was
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moistened with 90 and 180 mL of distilled water each making the water content 9.0%
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and 18.0%, respectively; no water was added to the third box (i.e. water content of dry
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sand was close to 0.0%). The 0.0, 9.0 and 18.0% represents the extreme minimum,
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mean and maximum soil moistures which were tested at the study site, respectively. The boxes were kept in a 5°C incubator for 2 months. During this period, the boxes
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were reweighed to keep the moisture content constant and water was replenished, if
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needed. During cold stratification, no seeds germinated. After 2 months at 5°C, bags of seeds were exhumed, the seeds mixed and divided among dishes, and incubated at 5/15, 10/20, 15/25 and 20/30°C in darkness.
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2.7 Effects of wetting-drying cycles at warm temperature Sixteen dishes (with 100 fresh seeds each) were equally divided into four treatments, subjected to one, two, three, or four cycles of wetting and drying; one dish served as a control [i.e. zero (no water addition) cycle]. A cycle consisted of a 12 h wet period
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followed by a 12 h dry period at 25°C. At the beginning of the wet period, the filter paper was moistened with 5 ml of distilled water and lids were placed on the dishes for 12 h; then, the lids were removed to facilitate drying for 12 h. During the dry period, a hair drier (held far enough away as to not to blow seeds out of dishes) was used to
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ensure that the seeds was dried for half an hour to a constant weight at 25°C. No germinated seeds were found during the wetting-drying cycles. Following the dry
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period, the next wet period started. After all cycles were completed, germination was
tested. Seeds from each dish in a treatment or in the control were mixed and transferred
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to each of eight dishes to test germination. Four dishes from a treatment or the control
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were incubated at 20°C and the other four dishes were incubated at 15/25°C; incubation
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occurred in darkness.
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2.8 Effects of a moisture gradient at warm temperature
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Seeds stored dry for 1 month were sown in paper cups (height: 10.0 cm) filled with 250 g of sand with distilled water to make a moisture gradient (1.0, 2.0, 4.0, 6.0, 8.0, 10.0,
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12.0, 14.0, 16.0 and 18.0%); seeds were placed at a depth of 0.5 cm below sand surface.
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The moisture level of the sand was kept constant by reweighing the cups and replenishing water, if needed. Four replicates with 25 seeds each were arranged for each moisture regime and incubated at 25°C in darkness. Germination (cotyledon emergence
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from sand) was checked daily and seedlings, if present, were removed. The experiment was completed after 30 d. nongerminated seeds were removed from the soil, scarified (see above), and tested for germination at 15/25°C for 14 d in darkness. 2.9 Data analyses
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Statistical analyses were conducted with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). One-way analyses of variances (ANOVA) were used to assess the effects of soil moisture and of wetting-drying cycles on germination, and two-way ANOVAs to test the effects of condition (control, dry storage, scarification) across temperatures and of
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moisture regime during cold stratification across temperatures on germination. Germination percentages were transformed by normal logarithm to enhance normality
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and homogeneity of variance. If ANOVAs showed significant effects, a least significant
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difference (LSD) test was used to determine differences between treatments.
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3. Results
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3.1 Determination of the degree of dormancy in fresh seeds
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Compared with the control, dry storage and scarification enhanced germination but the
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response was highly dependent on the incubation temperature (factors and interaction, P < 0.05). Increased germination was most notable at 5/15°C and 20/30°C for treated
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seeds (Fig. 1). Following these two treatments, germination ranged from 88.0% to 100%
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across all temperature regimes.
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Fig. 1. The effects of dry storage for 1 month and scarification of the testa of fresh seeds
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on germination (mean ± SE) of Agriophyllum squarrosum seeds at four temperatures in
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darkness. Bars with different upper-case letters indicate significant differences among temperatures under the same treatment and those with different lower-case letters show
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significant differences among treatments at the same temperature (P < 0.05; LSD test).
3.2 Seasonal patterns of dormancy and germination
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In the study site, temperature and precipitation clearly showed seasonal fluctuations during the experiment (Fig. 2a, b). Seeds exhumed in April 2013 germinated to a higher
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percentage at 5/15-15/25°C (≥86%) compared to seeds exhumed in January 2013 (Fig. 2c). However, germination of exhumed seeds decreased dramatically during summer 2013 with only 0-5% of them germinated in all the temperature regimes in June 2013. Seeds exhumed from January to May 2014 exhibited an increase in germination with those exhumed in April and May 2014 germinating to the highest percentages at all 13
temperatures. Germination of seeds exhumed monthly from June to July 2014
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drastically decreased.
Fig. 2. Changes in daily maximum (MaxT) and minimum temperatures (MinT) at a soil depth of 5 cm (a) and daily rainfall (b) at the study site, and seasonal dynamics of 14
dormancy and germination (mean ± SE) for Agriophyllum squarrosum seeds buried in its natural habitat and tested at five temperatures in darkness (c).
3.3 Effects of a moisture gradient at cold temperature
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Moisture levels during cold stratification significantly influenced germination during incubation, but the response was highly dependent on the incubation temperature
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(factors and interaction, P < 0.05). Seed germination increased with increasing
incubation temperatures across the same moisture regime (Fig. 3). At 5/15, 10/20 and 15/25°C, germination significantly decreased at 9.0% and/or 18.0% moisture (P < 0.05);
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but at 20/30°C, germination was not significantly different among the moisture regimes
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(P > 0.05).
Fig. 3. The effects of a moisture gradient at 5°C for 2 months on germination (mean ± SE) of Agriophyllum squarrosum seeds at four temperatures in darkness. Bars with 15
different upper-case letters indicate significant differences among temperatures under the same moisture regime and those with different lower-case letters show significant differences among moisture regimes at the same temperature (P < 0.05; LSD test).
3.4 Effects of wetting-drying cycles at warm temperature
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Wetting and drying cycles significantly affected germination (P < 0.05). Germination
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percentages and rates increased with increasing number of wetting-drying cycles at 20°C and at 15/25°C, with germination being higher at 15/25°C than at 20°C (Fig. 4).
Final germination resulting from the cycles was 80-88% compared to the control of 72%
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at 15/25°C, and final germination from cycles was 49-73% compared to the control of
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22% at 20°C.
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Fig. 4. The effects of wetting-drying cycles at 25°C on germination (mean ± SE) of Agriophyllum squarrosum seeds at 20°C or 15/25°C. Bars with different lower-case
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letters show significant differences among treatments or control (P < 0.05; LSD test).
3.5 Effects of a moisture gradient at warm temperature Soil moisture significantly influenced germination (P < 0.05). Germination gradually
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increased from 8 to 40% when moisture increased from 1 to 14%, respectively; germination decreased to 11 and 7% at 16 and 18% moisture, respectively (Fig. 5). When nongerminated seeds were removed from the sand, scarified and transferred to filter paper moistened with distilled water, germination increased to 10-16% at moisture
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levels of 1-8% and to 39-53% at moisture levels of 10-18% (Fig. 5).
Fig. 5. The effects of a moisture gradient at 25°C on germination (mean ± SE) of
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Agriophyllum squarrosum seeds at 15/25°C. Seeds were buried at 5 cm depth, and germination scored as cotyledon emergence from sand. Following 30 d, nongerminated seeds were removed, scarified, and tested for germination at 15/25°C for 14 d. Bars
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with different upper-case and lower-case letters show significant differences among soil moisture regimes within soil emergence or within scarification treatment (P < 0.05; LSD test).
4. Discussion
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Freshly matured seeds of A. squarrosum germinated to high percentages at only relatively narrow range of temperatures (10/20 and 15/25 °C) (Fig. 1). Following dry storage or scarification, they germinated to high percentages across the entire range of temperatures. We conclude that seeds of this species are conditionally dormant at
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maturity and the class and level of dormancy would be non-deep physiological (Baskin and Baskin, 2007). In contrast, Wang et al. (1998) found that seeds were non-dormant,
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but this was after they had been stored dry at laboratory temperature for almost 1 year. We suggest that dormancy (or conditional dormancy) was broken during storage in
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Wang et al.’s study. Although Liu et al. (2013) tested fresh seeds and found them to be
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in primary dormancy, they did not determine the class and level of dormancy. Our
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results clarified the degree of dormancy in fresh seeds as well as the dormancy
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class/level.
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Gernerally, after-ripening is an effective way to break physiological dormancy (Rubio de Casas et al., 2012). Our results showed that seeds of A. squarrosum needed
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after-ripening to overcome dormancy, which was completed by dry storage for 1 month
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at ca. 25°C (Fig. 1). Similar results were also found in Nicotiana tabacum and Bromus tectorum (Leubner-Metzger, 2002; Bair et al., 2006). After-ripening weakens the endosperm and ruptures the testa (Leubner-Metzger, 2002) and alters the gene
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expression pattern of oxidases that enhance ethylene and gibberellin (IglesiasFernandez and Matilla, 2009). Scarification in seeds with physiological dormancy weakens the seed and/or fruit coat and allows the radicle to penetrate these coats more easily (Baskin and Baskin, 2007). Seeds of A. squarrosum had a water-permeable testa,
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but scarification along with dry storage readily broke dormancy and promoted germination (Fig.1). These responses to both treatments are typical for species with non-deep physiological dormancy (Baskin and Baskin, 2007). We found that seeds of A. squarrosum cycled in their degree of dormancy in
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relation to seasonal temperature and rainfall patterns while in the soil seed bank (Fig. 2). Fan et al. (2017) presented a conceptual model of the seed/seedling dynamics of A.
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squarrosum and postulated that there might be dormancy cycling in the species. We
confirmed that dormancy cycling existed in this species. If dispersed in late
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autumn/early winter, seeds of this species are prevented from germinating since (1) soil
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is frozen and (2) the minimum temperature required for germination (10/20°C) is higher
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than that in the field. With increasing temperature and soil moisture in the late winter
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into spring, seeds gradually germinated over a wide range of temperatures, including
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the lowest temperature (5/15°C) that prevented germination in the previous autumn (Fig. 2c). Indeed, seedlings of this species are mostly observed in the field in April (Gao et
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al., 2014) that corresponds to the time when seeds are non-dormant and germinate to
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their highest percentages. Between summer and late autumn, seeds are mostly dormant and germination is prevented in nature. Physiological dormancy is often broken for species dispersed in autumn in
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temperate regions by cold, moist (stratification) conditions (Walck et al., 1997a, Baskin and Baskin, 2014), such as in Suaeda corniculata subsp. mongolica (Cao et al., 2012). However, cold stratification may not overcome dormancy in seeds of some temperate plants, such as Stipa gobica (Katrin et al., 2008) and Suaeda acuminata (Wang et al.,
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2012). For A. squarrosum, treatment at 5°C was more effective at 0% moisture than at 9 or 18% moisture when seeds were incubated at 5/15 or 10/20°C (Fig. 3). This result is in contrast with other studies on cold stratification in which seeds stored dry at 5°C do not overcome dormancy (e.g. Walck et al., 1997b). As compared to fresh seeds (Fig.
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1), our treatment at 5°C and 0% moisture increased germination over the range of incubation temperatures especially at 5/15 and 20/30°C (Fig. 3). At temperatures that
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are most relevant for seedling emergence in nature in spring (5/15 and 10/20°C) seeds can readily germinate to high percentages at 10/20°C (without any treatment) but cold,
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dry conditions overcame dormancy in some seeds of the population to allow an increase
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in germination at 5/15°C, because seeds in the aerial seed bank are dry but cold during
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winter, and those in the soil may actually be in frozen soil, which would be cold but dry.
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Seed germination of some plants is promoted by wetting-drying cycles that result
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from the discrete rainfall events in water-limited ecosystems, such as the annuals Cyperus inflexus (Baskin and Baskin, 1982) and Plantago minuta (Zhang et al., 2014)
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or the perennials Cirsium vulgare (Downs and Cavers 2000), Agropyron spicatum
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(Kastner et al., 1981) and Mammillaria hernandezii (Santini and Martorell, 2013). For A. squarrosum, our results showed that its germination was enhanced by wetting-drying cycles (Fig. 4). However, germination from these cycles was highly dependent on
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constant vs. alternating temperature. Once temperatures warm and sporadic rain begins in mid-spring, wetting-drying cycles are an important environment cue that triggers germination in the natural habitat for A. squarrosum. Seeds of A. squarrosum stored dry for 1 month germinated to ca. 80% at 15/25°C
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(Fig. 1), whereas those stored dry for 1 month and then subjected to a moisture gradient for an additional month germinated to ca. 10-40% (Fig. 5). On one hand, seed germination of this species is highly drought tolerance: with one study reporting that germination was depressed by little soil moisture (beyond -0.94 MPa) (Tobe et al., 2005)
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and another study showing optimal germination occurred at median soil moisture (-0.2 MPa) (Cui et al., 2007). On the other hand, when the remaining nongerminated seeds
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in our experiment were removed, scarified, and placed at optimum conditions, germination was low when seeds were exposed to ≤8% moisture for 1 month and
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moderate to high when exposed to 10-18% moisture (Fig. 5). In comparison, fresh seeds
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that were scarified germinated to ca. 85% at the same temperature/moisture conditions
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(Fig. 1). We found that, although most of the rain occurs in summer in our study area,
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most of rain events were less than 10 mm, even there were several heavy rain events,
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but long interval occurred among these events (Fig. 2b). Thus, moisture is low at a soil depth of 5 cm resulting from strong transpiration. We suggest that soil moisture,
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especially at low moisture that would simulate conditions in nature during summer,
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played a role in inducing secondary dormancy in seeds of this species. Moreover, we suggest that the degree of secondary dormancy was deeper than that of primary dormancy. This latter point might explain the discrepancy in germination percentages
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of seeds exhumed in January - March 2013 compared to those exhumed in January March 2014 (Fig. 2). Seeds exhumed in January, February, and March 2013 germinated up to ca. 60, 90, and 90%, respectively, whereas those exhumed in 2014 did so up to ca. 30, 40, and 40%, respectively.
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Though many studies have focused on the regulation of dormancy cycling by seasonal change of temperatures, such as Arabidopsis (Baskin and Baskin, 1985; Footitt et al., 2014, 2015) or Chenopodium album (Bouwmeester and Karssen, 1993), our results suggest that temperature and soil moisture regulate dormancy cycling of A.
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squarrosum. Soil moisture induced dormancy, with deeper secondary dormancy occurring with low moisture as compared to high moisture evidenced by dormancy
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being overcome more easily (i.e. germination increased) in scarified seeds (Fig. 5). For Polygonum aviculare, however, dormancy was deeper under low moisture condition
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than under high or fluctuating moisture conditions (Batlla and Benech-Arnold, 2006).
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In the study area, surface soil stays droughty for most of the time in a year due to rare
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rainfall events and strong evaporation in summer. Seeds in the shallow seed bank may
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be subject to drought and enter secondary dormancy in summer and autumn, which are
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unfavorable for seedling survival. Although seeds that are deeply buried by mobile soil (Liu et al., 2007) may experience relatively moist soil, they do not germinate. Thus, a
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persistent soil seed bank may develop regardless of changes in soil moisture. Seed
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germination from the soil seed bank may be triggered by cold or warm temperatures in combination with dry conditions or wetting-drying cycles. This versatility in overcoming dormancy and stimulating germination over a range of temperature and
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moisture conditions during the favorable season for seedling establishment as well as preventing germination during the unfavorable season allows A. squarrosum to germinate in a highly unpredictable environment.
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5. Conclusion Freshly havested seeds of A. squarrosum had non-deep physiological (conditional) dormancy, and seeds in the soil demonstrated dormancy cycling associated with seasonal changes in temperature and moisture. Following autumn to winter dispersal,
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some dormancy break occurs at cold, particularly dry conditions at low temperatures, and along with the high germination at 10/20°C (without treatment) allows seedling
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emergence to occur in nature. As temperatures warm, after-ripening at dry conditions along with wetting-drying cycles corresponding to the onset of sporadic rain in spring,
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seedling emergence continues to occur. From summer to autumn, warm temperatures
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combined with dry soil conditions cause seeds to re-enter secondary dormancy. Seeds
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of A. squarrosum are highly versatile in their mechanisms to overcome dormancy and
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to germinate: cold or warm temperatures with dry conditions or wetting-drying cycles.
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However, germination is mostly restricted to mid-spring when seeds are non-dormant. They become dormant during the summer and autumn with warm temperatures and low
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soil moisture. Therefore, seasonal changes in soil temperature and moisture regulate
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seed dormancy and germination of A. squarrosum.
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Declarations of interest: none
Acknowledgments This work was supported by the National Key Technology R&D Program (2016YFC050080502) and the National Natural Science Foundation (31570416,
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31370705) of P.R. China, and the Strategy of CAS Biological Resources Service
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Network Planning Project [grant number ZSSD-014].
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Authors’ contribution Rui Ru Gao and Zhen Ying Huang formulated the conception and design of the study; Rui Ru Gao, Rui Hua Zhao, Xiao Ya Wei, and Zhan He conducted the
Rui Ru Gao analyzed the data and wrote the manuscript;
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experiments and acquired the data;
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Zhen Ying Huang, Xue Jun Yang and Jeffrey L. Walck revised the
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manuscript .
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S0098-8472(18)31062-1 https://doi.org/10.1016/j.envexpbot.2018.08.010 EEB 3536
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
13-7-2018 10-8-2018 10-8-2018
Please cite this article as: Gao R, Zhao R, Huang Z, Yang X, Wei X, He Z, Walck JL, Soil temperature and moisture regulate seed dormancy cycling of a dune annual in a temperate desert, Environmental and Experimental Botany (2018), https://doi.org/10.1016/j.envexpbot.2018.08.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Original Articles
Soil temperature and moisture regulate seed dormancy cycling of a dune annual in a temperate desert
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Running head: Temperature and moisture regulating dormancy and germination
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Ruiru Gaoa,b, Ruihua Zhaob, Zhenying Huanga*, Xuejun Yanga, Xiaoya Weib, Zhan
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany,
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a
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Heb, Jeffrey L. Walckc
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Chinese Academy of Sciences, Beijing 100093, P.R. China The School of Life Sciences, Shanxi Normal University, Linfen, P.R.China
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Department of Biology, Middle Tennessee State University, Murfreesboro, TN
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b
37132, USA
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Hghlights
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* Corresponding author. E-mail: [email protected]
Soil seeds of Agriophyllum squarrosum have annual dormancy cycling
Freshly harvested seeds were in non-deep physiological (conditional) dormancy
Germination of soil seeds were promoted by wetting-drying cycles
Dormancy was induced by warm temperature and particularly low soil 1
moisture
Rainfall and temperature regulated seed dormancy/germination seasonal pattern
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Abstract
Plants have evolved diverse strategies to ensure their survival and regeneration in
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specific environments. Although temperature and soil moisture control seed dormancy, most studies have concentrated on temperature and little is known about the influence
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of moisture for species in the arid region. Responses of seed dormancy and germination
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of Agriophyllum squarrosum (Amaranthaceae), a pioneer and dominant species in Mu
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Us Sandland in northern China, to variations in soil moisture and temperature were
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examined. Our study showed that (1) freshly harvested seeds were in non-deep
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physiological (conditional) dormancy; (2) seeds in the soil exhibited dormancy cycling being non-dormant in spring and dormant from summer to autumn; (3) dry conditions
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at cold or warm temperatures alleviated dormancy; (4) germination was promoted by
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wetting-drying cycles; and (5) dormancy was induced by warm temperature (15/25°) and particularly low soil moisture less than 14.0%. The seasonal pattern of seed dormancy/germination was regulated by seasonal rainfall and soil temperature. At the
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same time, a range of conditions enable dormancy break and germination regardless of soil moisture conditions allowing the species to persist in an unpredictable environment.
Key words：Agriophyllum squarrosum; dormancy cycle; seed germination; soil 2
seed bank; soil temperature; soil moisture
1. Introduction Seeds are the most tolerant stage to environmental stresses in the life histories of plants
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(Gutterman,1993). Dormant seeds allow plants to persist during unfavorable seasons for seedling establishment and non-dormant seeds regenerate plant populations when
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favorable seasons occur (Mapes et al., 1989; Rees, 1996). Seed dormancy has been considered as a bet-hedging strategy of plants to cope with uncertain environments
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(Gremer and Venable, 2014). Plants have evolved diverse strategies to ensure
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successful germination at the right time in a suitable place (Linkies et al., 2010), and
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and Flores, 2005; Nonogaki, 2014).
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seed dormancy is important for plants to adapt to unpredictable environments (Jurado
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Seeds buried in the soil sense and integrate a range of environmental signals to continually adjust their degree of dormancy to co-ordinate germination with seedling
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emergence at a suitable space and time (Copete et al., 2015). In natural habitats,
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variation in germination timing for some species with physiological dormancy is controlled by seasonal climate cues resulting in dormancy cycling (Baskin and Baskin, 1985). This is an important strategy for a seed population to avoid sibling competition
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or extinction of all individuals due to natural disasters (Nonogaki, 2014). In arid and semi-arid regions, the temporal distributions of seed dormancy/germination might especially be an adaptation of plants to these dramatically fluctuating environments. Agriopyllum squarrosum (L.) Moq. (Amaranthaceae) is an annual plant with both
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aerial and soil seed banks (Liu et al., 2006; Gao et al., 2014). The seed dispersal of the species is extended over time, which forms the aerial seed bank (Gao et al., 2014). Differences between the soil and aerial seed banks occur: seeds from the soil seed bank germinate earlier than those from the aerial seed bank, which regulates the timing of
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seedling emergence through a bet-hedging strategy (Gao et al., 2014). Seed germination is suppressed by light and is promoted by darkness (Wang et al., 1998; Zheng et al.,
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2004; Tobe et al., 2005), and it is markedly low at constant temperatures (10°C) but high at alternating temperatures in darkness (Wang et al., 1998; Zheng et al., 2004). In
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addition, seeds germinate at low soil moisture (-0.8 MPa) (Cui et al., 2007), indicating
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that the species is strongly drought tolerant. When burial depth is beyond 5 cm, no seeds
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germinate (Tobe et al., 2005; Zheng et al., 2004) and a persistent soil seed bank develops.
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Liu et al. (2007) found that seeds remained viable in mobile dunes when buried deeper
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than 1.0 m, but the surface seed bank (<5 cm) was depleted in the growing season (Gao et al., 2014). Thus, the spatial and temporal patterns of the seed banks along with
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germination responses have important impacts on the seedling recruitment of A.
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squarrosum (Bai et al., 2004; Liu et al., 2007; Ma and Liu, 2008). Freshly matured seeds of A. squarrosum at our study site are mostly dispersed in
late autumn and winter, when the subsurface of the soil is frozen and the surface is
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unfrozen but often dry (Gao et al., pers. obs.). Anaysis of weather data for the past 50 years (1959-2009) at the study site showed that, in late winter and early spring, as snow and frozen soil melt and temperatures warm the subsurface of the soil remains moist but the surface may become dry. Thus, cold stratification may be severely limited from
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late autumn to early spring due to temperatures below freezing and to dry conditions. Discrete rainfall events start in mid-spring and continue until late summer as temperatures dramatically rise to 40°C. Soil moisture becomes very limited in the summer with high evaporation and sporadic rain. During the spring and summer, seeds
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are exposed to wetting-drying cycles and to a gradient of moisture on the surface as well as the subsurface of the soil. Although seeds of A. squarrosum are available from
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the aerial and soil seed banks at any time of the year, we have observed seedlings mostly
from April to May and rarely at other times of the year (Gao et al., 2014). We surmised
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that seeds of this species may undergo dormancy cycling in the soil, which would
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explain our seasonal observations of seedling emergence. Taken together, we surmised
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that dormancy and germination would be controlled by the complex interaction of
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temperature and soil moisture in this semi-arid region.
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Although temperature and soil moisture are important environmental cues that control seed dormancy loss/induction and germination, most studies have concentrated
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on temperature effects (Footitt et al., 2015; Postma et al., 2016) and little is known
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about the influence of moisture (Copete et al., 2015), especially for species in arid and semiarid regions. As such, although we understand the life history strategies of A. squarrosum to cope with unpredictable environments, there is little information on the
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dynamics of seasonal seed dormancy cycling of seeds in the soil seed bank and how dormancy loss/induction and germination are regulated by environmental factors. Moreover, a discrepancy on the degree of dormancy in freshly matured seeds of A. squarrosum exists. While some researchers have found that seeds were non-dormant
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and can rapidly germinate (Wang et al., 1998), other researchers have shown that seeds were dormant and dry storage was needed to overcome this dormancy (Liu et al., 2013). The germination responses of offspring seeds of this species were altered by maternal environments, and seeds collected from plants inhabiting moist habitats were more
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dormant than those from dry habitats (Gao et al., 2015). Therefore, the degree of dormancy may be strongly regulated by soil moisture.
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Recently, Fan et al. (2017) presented a conceptual model of the seed/seedling
dynamics of A. squarrosum. They stressed the need to understand better the dormancy
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and germination of this important dune-stabilizing species within an ecological context.
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To the best our knowledge, no studies on A. squarrosum have examined the roles that
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temperature and soil moisture play in regulating seed dormancy/induction and
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germination, i.e. considering the seasonal patterns of temperature and soil moisture in
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the semi-arid region. Thus, we (1) determined the degree of dormancy in fresh seeds from the population that we studied by testing the effects of dry storage and testa
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scarification in comparison to fresh (intact) seeds. In addition, we (2) examined the
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seasonal pattern of dormancy and germination of seeds buried in soil over time. Simultaneously, we tested the effects of (3) a moisture gradient at cold temperature, simulating conditions from late autumn to early spring and (4) wetting-drying cycles
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and (5) a moisture gradient at warm temperature, simulating conditions from midspring to summer. Using these combined methods of laboratory experiments and controlled field trials allowed us to explore how seed dormancy and germination are controlled in relation to seasonal soil temperature and moisture fluctuations. The results
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will allow us to further understand the adaptive strategies of annual dune plants in temperate semi-arid regions.
2. Material and methods
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2.1 Study species Agriophyllum squarrosum inhabits semi-arid and arid regions, and is widely distributed
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from central to eastern Asia (Kong, 1996; Naqinezhad, 2012; Qiao et al., 2012), where it is a pioneer species on mobile and semi-fixed sand dunes (Nemoto and Lu, 1992).
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The species could be used to stabilize mobile sand-dunes (Wang et al., 2009) and also
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is of nutrient value due to high content of protein and unsaturated fatty acids and linoleic
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acid in seeds (Korbanjhon, 2011); as such, it has the potential to be a human food crop
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(Chen et al., 2014). The vegetative parts of the plant are already used as forage for
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animals in winter, such as Camelus bactrianus (Zhao et al., 2006). Additional information on the species can be found in Kong (1996) and Gao et al. (2014, 2015).
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2.2 Plant material
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Fifty plants were randomly selected in a natural population of A. squarrosum at the Ordos Sandland Ecological Research Station of the Chinese Academy of Sciences (39°29′37.6″ N, 110°11′29.4″ E, 1296 m a.s.l.), located in the north-eastern Mu Us
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Sandland in Inner Mongolia, China, on 20 October 2012. The average annual precipitation in this region is 345.8 mm and is largely restricted to the period from June to August. The mean annual temperature is 6.8°C with a minimum of -16.9°C in January and maximum of 28.1°C in July (Gao et al., 2014, 2015). The landscape at the
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Station consists of mobile and semi-fixed sand dunes, on which A. squarrosum is common. The collected plants were placed in a paper bag and taken to a laboratory, where seeds were removed from each plant by rubbing the infructescences by hand. 2.3 General procedures for germination tests
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Germination tests were conducted at 5/15, 10/20, 15/25 and 20/30°C (12/12 h) in darkness due to the inhibitory effect of light on seed germination (Gao et al., 2014). The
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four temperature regimes represented mean minimum/maximum temperatures for late
April and October, May and September, June and August, and July, respectively. Four
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replicates of 25 seeds each were placed into 7-mm diameter Petri dishes on two layers
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of filter papers moistened with 3 ml distilled water. Germination was checked daily
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under a green safe light in a dark room. Germinated seeds (radicle emerged) were
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removed, and the test was completed after 20 d when no additional seeds germinated
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for 4 successive days, unless stated otherwise. Following the test, nongerminated seeds were pinched with forceps to determine whether the embryo was firm. If the embryo
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was firm, the seed was viable. Germination percentages were calculated based on
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number of viable seeds.
2.4 Determination of the degree of dormancy in fresh seeds Within 1 week after collection, fresh (intact) seeds were incubated at 5/15, 10/20, 15/25
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and 20/30°C in darkness. In addition, germination of seeds from scarification and dry storage treatments was also tested. The scarification treatment consisted of taking 400 fresh seeds within 1 week after collection, making a small hole through the testa with a dissecting needle and not damaging the embryo, and incubating them over the same
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conditions as the control. The dry storage treatment consisted of storing 500 fresh seeds dry in a paper bag in the laboratory (25.0 ± 2.0°C) for 1 month and then testing germination under the same conditions as the control seeds. 2.5 Seasonal patterns of dormancy and germination
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Fresh seeds were placed in 152 fine-mesh nylon bags (150 seeds per bag, 8 cm length x 2-3 cm width), which were buried in the natural habitat of A. squarrosum surrounded
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by a wire fence to prevent animal damage. Although 76 bags were needed for the
experiment, we buried an additional 76 bags in case of mice predation. Burial occurred
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on 1 December 2012 and exhumations were done at 1 month intervals between 1
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January 2013 and 30 July 2014. At the time of burial, the soil (consisting mostly of
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sand) was frozen and covered with 5-10 cm of snow; thus, the snow was carefully
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removed and a pickax used to dig five parallel trenches (ca. 1 m length x 8 cm width x
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6 cm depth) at 10 cm apart. The bags were randomly allocated to a trench, placed flat on the bottom of the trenches and equally spaced apart. Then, they were covered with
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unfrozen soil, which later froze, and snow (to same depth as before burial); the locations
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of each end of trenches were marked with a stick that protruded above the soil and snow. At each exhumation, snow, if present, was removed and four bags of seeds from a trench were removed; snow and soil were replaced after removal. The bags were taken back
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to the laboratory where seeds from all of them were mixed and tested for germination at 5/15, 10/20, 15/25, 20/30 and 25/35°C in darkness. The 25/35°C represented the extreme temperature that sometimes occurs in July. No germinated seeds were observed in the bags at any exhumation date. During the experimental period, data on daily
9
rainfall and on soil temperatures [maximum (MaxT) and minimum (MinT) temperature] at a depth of 5 cm below the soil surface were recorded by the weather station at the Ordos Sandland Ecological Research Station which is close to the experimental site (37.3 m).
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2.6 Effects of a moisture gradient at cold temperature Eighteen hundred seeds were equally placed in nine fine-mesh nylon bags, with three
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bags each being buried 5 cm deep in three opaque plastic (stratification) boxes with lids (40 cm length × 30 cm width × 15 cm height) filled with 1.0 kg dry sand (at about 10
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cm depth), which was collected from natural habitats. The sand in two boxes was
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moistened with 90 and 180 mL of distilled water each making the water content 9.0%
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and 18.0%, respectively; no water was added to the third box (i.e. water content of dry
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sand was close to 0.0%). The 0.0, 9.0 and 18.0% represents the extreme minimum,
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mean and maximum soil moistures which were tested at the study site, respectively. The boxes were kept in a 5°C incubator for 2 months. During this period, the boxes
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were reweighed to keep the moisture content constant and water was replenished, if
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needed. During cold stratification, no seeds germinated. After 2 months at 5°C, bags of seeds were exhumed, the seeds mixed and divided among dishes, and incubated at 5/15, 10/20, 15/25 and 20/30°C in darkness.
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2.7 Effects of wetting-drying cycles at warm temperature Sixteen dishes (with 100 fresh seeds each) were equally divided into four treatments, subjected to one, two, three, or four cycles of wetting and drying; one dish served as a control [i.e. zero (no water addition) cycle]. A cycle consisted of a 12 h wet period
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followed by a 12 h dry period at 25°C. At the beginning of the wet period, the filter paper was moistened with 5 ml of distilled water and lids were placed on the dishes for 12 h; then, the lids were removed to facilitate drying for 12 h. During the dry period, a hair drier (held far enough away as to not to blow seeds out of dishes) was used to
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ensure that the seeds was dried for half an hour to a constant weight at 25°C. No germinated seeds were found during the wetting-drying cycles. Following the dry
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period, the next wet period started. After all cycles were completed, germination was
tested. Seeds from each dish in a treatment or in the control were mixed and transferred
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to each of eight dishes to test germination. Four dishes from a treatment or the control
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were incubated at 20°C and the other four dishes were incubated at 15/25°C; incubation
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occurred in darkness.
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2.8 Effects of a moisture gradient at warm temperature
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Seeds stored dry for 1 month were sown in paper cups (height: 10.0 cm) filled with 250 g of sand with distilled water to make a moisture gradient (1.0, 2.0, 4.0, 6.0, 8.0, 10.0,
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12.0, 14.0, 16.0 and 18.0%); seeds were placed at a depth of 0.5 cm below sand surface.
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The moisture level of the sand was kept constant by reweighing the cups and replenishing water, if needed. Four replicates with 25 seeds each were arranged for each moisture regime and incubated at 25°C in darkness. Germination (cotyledon emergence
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from sand) was checked daily and seedlings, if present, were removed. The experiment was completed after 30 d. nongerminated seeds were removed from the soil, scarified (see above), and tested for germination at 15/25°C for 14 d in darkness. 2.9 Data analyses
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Statistical analyses were conducted with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). One-way analyses of variances (ANOVA) were used to assess the effects of soil moisture and of wetting-drying cycles on germination, and two-way ANOVAs to test the effects of condition (control, dry storage, scarification) across temperatures and of
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moisture regime during cold stratification across temperatures on germination. Germination percentages were transformed by normal logarithm to enhance normality
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and homogeneity of variance. If ANOVAs showed significant effects, a least significant
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difference (LSD) test was used to determine differences between treatments.
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3. Results
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3.1 Determination of the degree of dormancy in fresh seeds
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Compared with the control, dry storage and scarification enhanced germination but the
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response was highly dependent on the incubation temperature (factors and interaction, P < 0.05). Increased germination was most notable at 5/15°C and 20/30°C for treated
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seeds (Fig. 1). Following these two treatments, germination ranged from 88.0% to 100%
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across all temperature regimes.
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Fig. 1. The effects of dry storage for 1 month and scarification of the testa of fresh seeds
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on germination (mean ± SE) of Agriophyllum squarrosum seeds at four temperatures in
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darkness. Bars with different upper-case letters indicate significant differences among temperatures under the same treatment and those with different lower-case letters show
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significant differences among treatments at the same temperature (P < 0.05; LSD test).
3.2 Seasonal patterns of dormancy and germination
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In the study site, temperature and precipitation clearly showed seasonal fluctuations during the experiment (Fig. 2a, b). Seeds exhumed in April 2013 germinated to a higher
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percentage at 5/15-15/25°C (≥86%) compared to seeds exhumed in January 2013 (Fig. 2c). However, germination of exhumed seeds decreased dramatically during summer 2013 with only 0-5% of them germinated in all the temperature regimes in June 2013. Seeds exhumed from January to May 2014 exhibited an increase in germination with those exhumed in April and May 2014 germinating to the highest percentages at all 13
temperatures. Germination of seeds exhumed monthly from June to July 2014
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drastically decreased.
Fig. 2. Changes in daily maximum (MaxT) and minimum temperatures (MinT) at a soil depth of 5 cm (a) and daily rainfall (b) at the study site, and seasonal dynamics of 14
dormancy and germination (mean ± SE) for Agriophyllum squarrosum seeds buried in its natural habitat and tested at five temperatures in darkness (c).
3.3 Effects of a moisture gradient at cold temperature
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Moisture levels during cold stratification significantly influenced germination during incubation, but the response was highly dependent on the incubation temperature
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(factors and interaction, P < 0.05). Seed germination increased with increasing
incubation temperatures across the same moisture regime (Fig. 3). At 5/15, 10/20 and 15/25°C, germination significantly decreased at 9.0% and/or 18.0% moisture (P < 0.05);
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but at 20/30°C, germination was not significantly different among the moisture regimes
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(P > 0.05).
Fig. 3. The effects of a moisture gradient at 5°C for 2 months on germination (mean ± SE) of Agriophyllum squarrosum seeds at four temperatures in darkness. Bars with 15
different upper-case letters indicate significant differences among temperatures under the same moisture regime and those with different lower-case letters show significant differences among moisture regimes at the same temperature (P < 0.05; LSD test).
3.4 Effects of wetting-drying cycles at warm temperature
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Wetting and drying cycles significantly affected germination (P < 0.05). Germination
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percentages and rates increased with increasing number of wetting-drying cycles at 20°C and at 15/25°C, with germination being higher at 15/25°C than at 20°C (Fig. 4).
Final germination resulting from the cycles was 80-88% compared to the control of 72%
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at 15/25°C, and final germination from cycles was 49-73% compared to the control of
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22% at 20°C.
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Fig. 4. The effects of wetting-drying cycles at 25°C on germination (mean ± SE) of Agriophyllum squarrosum seeds at 20°C or 15/25°C. Bars with different lower-case
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letters show significant differences among treatments or control (P < 0.05; LSD test).
3.5 Effects of a moisture gradient at warm temperature Soil moisture significantly influenced germination (P < 0.05). Germination gradually
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increased from 8 to 40% when moisture increased from 1 to 14%, respectively; germination decreased to 11 and 7% at 16 and 18% moisture, respectively (Fig. 5). When nongerminated seeds were removed from the sand, scarified and transferred to filter paper moistened with distilled water, germination increased to 10-16% at moisture
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levels of 1-8% and to 39-53% at moisture levels of 10-18% (Fig. 5).
Fig. 5. The effects of a moisture gradient at 25°C on germination (mean ± SE) of
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Agriophyllum squarrosum seeds at 15/25°C. Seeds were buried at 5 cm depth, and germination scored as cotyledon emergence from sand. Following 30 d, nongerminated seeds were removed, scarified, and tested for germination at 15/25°C for 14 d. Bars
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with different upper-case and lower-case letters show significant differences among soil moisture regimes within soil emergence or within scarification treatment (P < 0.05; LSD test).
4. Discussion
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Freshly matured seeds of A. squarrosum germinated to high percentages at only relatively narrow range of temperatures (10/20 and 15/25 °C) (Fig. 1). Following dry storage or scarification, they germinated to high percentages across the entire range of temperatures. We conclude that seeds of this species are conditionally dormant at
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maturity and the class and level of dormancy would be non-deep physiological (Baskin and Baskin, 2007). In contrast, Wang et al. (1998) found that seeds were non-dormant,
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but this was after they had been stored dry at laboratory temperature for almost 1 year. We suggest that dormancy (or conditional dormancy) was broken during storage in
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Wang et al.’s study. Although Liu et al. (2013) tested fresh seeds and found them to be
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in primary dormancy, they did not determine the class and level of dormancy. Our
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results clarified the degree of dormancy in fresh seeds as well as the dormancy
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class/level.
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Gernerally, after-ripening is an effective way to break physiological dormancy (Rubio de Casas et al., 2012). Our results showed that seeds of A. squarrosum needed
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after-ripening to overcome dormancy, which was completed by dry storage for 1 month
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at ca. 25°C (Fig. 1). Similar results were also found in Nicotiana tabacum and Bromus tectorum (Leubner-Metzger, 2002; Bair et al., 2006). After-ripening weakens the endosperm and ruptures the testa (Leubner-Metzger, 2002) and alters the gene
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expression pattern of oxidases that enhance ethylene and gibberellin (IglesiasFernandez and Matilla, 2009). Scarification in seeds with physiological dormancy weakens the seed and/or fruit coat and allows the radicle to penetrate these coats more easily (Baskin and Baskin, 2007). Seeds of A. squarrosum had a water-permeable testa,
19
but scarification along with dry storage readily broke dormancy and promoted germination (Fig.1). These responses to both treatments are typical for species with non-deep physiological dormancy (Baskin and Baskin, 2007). We found that seeds of A. squarrosum cycled in their degree of dormancy in
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relation to seasonal temperature and rainfall patterns while in the soil seed bank (Fig. 2). Fan et al. (2017) presented a conceptual model of the seed/seedling dynamics of A.
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squarrosum and postulated that there might be dormancy cycling in the species. We
confirmed that dormancy cycling existed in this species. If dispersed in late
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autumn/early winter, seeds of this species are prevented from germinating since (1) soil
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is frozen and (2) the minimum temperature required for germination (10/20°C) is higher
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than that in the field. With increasing temperature and soil moisture in the late winter
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into spring, seeds gradually germinated over a wide range of temperatures, including
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the lowest temperature (5/15°C) that prevented germination in the previous autumn (Fig. 2c). Indeed, seedlings of this species are mostly observed in the field in April (Gao et
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al., 2014) that corresponds to the time when seeds are non-dormant and germinate to
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their highest percentages. Between summer and late autumn, seeds are mostly dormant and germination is prevented in nature. Physiological dormancy is often broken for species dispersed in autumn in
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temperate regions by cold, moist (stratification) conditions (Walck et al., 1997a, Baskin and Baskin, 2014), such as in Suaeda corniculata subsp. mongolica (Cao et al., 2012). However, cold stratification may not overcome dormancy in seeds of some temperate plants, such as Stipa gobica (Katrin et al., 2008) and Suaeda acuminata (Wang et al.,
20
2012). For A. squarrosum, treatment at 5°C was more effective at 0% moisture than at 9 or 18% moisture when seeds were incubated at 5/15 or 10/20°C (Fig. 3). This result is in contrast with other studies on cold stratification in which seeds stored dry at 5°C do not overcome dormancy (e.g. Walck et al., 1997b). As compared to fresh seeds (Fig.
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1), our treatment at 5°C and 0% moisture increased germination over the range of incubation temperatures especially at 5/15 and 20/30°C (Fig. 3). At temperatures that
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are most relevant for seedling emergence in nature in spring (5/15 and 10/20°C) seeds can readily germinate to high percentages at 10/20°C (without any treatment) but cold,
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dry conditions overcame dormancy in some seeds of the population to allow an increase
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in germination at 5/15°C, because seeds in the aerial seed bank are dry but cold during
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winter, and those in the soil may actually be in frozen soil, which would be cold but dry.
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Seed germination of some plants is promoted by wetting-drying cycles that result
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from the discrete rainfall events in water-limited ecosystems, such as the annuals Cyperus inflexus (Baskin and Baskin, 1982) and Plantago minuta (Zhang et al., 2014)
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or the perennials Cirsium vulgare (Downs and Cavers 2000), Agropyron spicatum
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(Kastner et al., 1981) and Mammillaria hernandezii (Santini and Martorell, 2013). For A. squarrosum, our results showed that its germination was enhanced by wetting-drying cycles (Fig. 4). However, germination from these cycles was highly dependent on
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constant vs. alternating temperature. Once temperatures warm and sporadic rain begins in mid-spring, wetting-drying cycles are an important environment cue that triggers germination in the natural habitat for A. squarrosum. Seeds of A. squarrosum stored dry for 1 month germinated to ca. 80% at 15/25°C
21
(Fig. 1), whereas those stored dry for 1 month and then subjected to a moisture gradient for an additional month germinated to ca. 10-40% (Fig. 5). On one hand, seed germination of this species is highly drought tolerance: with one study reporting that germination was depressed by little soil moisture (beyond -0.94 MPa) (Tobe et al., 2005)
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and another study showing optimal germination occurred at median soil moisture (-0.2 MPa) (Cui et al., 2007). On the other hand, when the remaining nongerminated seeds
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in our experiment were removed, scarified, and placed at optimum conditions, germination was low when seeds were exposed to ≤8% moisture for 1 month and
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moderate to high when exposed to 10-18% moisture (Fig. 5). In comparison, fresh seeds
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that were scarified germinated to ca. 85% at the same temperature/moisture conditions
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(Fig. 1). We found that, although most of the rain occurs in summer in our study area,
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most of rain events were less than 10 mm, even there were several heavy rain events,
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but long interval occurred among these events (Fig. 2b). Thus, moisture is low at a soil depth of 5 cm resulting from strong transpiration. We suggest that soil moisture,
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especially at low moisture that would simulate conditions in nature during summer,
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played a role in inducing secondary dormancy in seeds of this species. Moreover, we suggest that the degree of secondary dormancy was deeper than that of primary dormancy. This latter point might explain the discrepancy in germination percentages
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of seeds exhumed in January - March 2013 compared to those exhumed in January March 2014 (Fig. 2). Seeds exhumed in January, February, and March 2013 germinated up to ca. 60, 90, and 90%, respectively, whereas those exhumed in 2014 did so up to ca. 30, 40, and 40%, respectively.
22
Though many studies have focused on the regulation of dormancy cycling by seasonal change of temperatures, such as Arabidopsis (Baskin and Baskin, 1985; Footitt et al., 2014, 2015) or Chenopodium album (Bouwmeester and Karssen, 1993), our results suggest that temperature and soil moisture regulate dormancy cycling of A.
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squarrosum. Soil moisture induced dormancy, with deeper secondary dormancy occurring with low moisture as compared to high moisture evidenced by dormancy
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being overcome more easily (i.e. germination increased) in scarified seeds (Fig. 5). For Polygonum aviculare, however, dormancy was deeper under low moisture condition
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than under high or fluctuating moisture conditions (Batlla and Benech-Arnold, 2006).
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In the study area, surface soil stays droughty for most of the time in a year due to rare
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rainfall events and strong evaporation in summer. Seeds in the shallow seed bank may
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be subject to drought and enter secondary dormancy in summer and autumn, which are
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unfavorable for seedling survival. Although seeds that are deeply buried by mobile soil (Liu et al., 2007) may experience relatively moist soil, they do not germinate. Thus, a
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persistent soil seed bank may develop regardless of changes in soil moisture. Seed
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germination from the soil seed bank may be triggered by cold or warm temperatures in combination with dry conditions or wetting-drying cycles. This versatility in overcoming dormancy and stimulating germination over a range of temperature and
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moisture conditions during the favorable season for seedling establishment as well as preventing germination during the unfavorable season allows A. squarrosum to germinate in a highly unpredictable environment.
23
5. Conclusion Freshly havested seeds of A. squarrosum had non-deep physiological (conditional) dormancy, and seeds in the soil demonstrated dormancy cycling associated with seasonal changes in temperature and moisture. Following autumn to winter dispersal,
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some dormancy break occurs at cold, particularly dry conditions at low temperatures, and along with the high germination at 10/20°C (without treatment) allows seedling
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emergence to occur in nature. As temperatures warm, after-ripening at dry conditions along with wetting-drying cycles corresponding to the onset of sporadic rain in spring,
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seedling emergence continues to occur. From summer to autumn, warm temperatures
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combined with dry soil conditions cause seeds to re-enter secondary dormancy. Seeds
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of A. squarrosum are highly versatile in their mechanisms to overcome dormancy and
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to germinate: cold or warm temperatures with dry conditions or wetting-drying cycles.
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However, germination is mostly restricted to mid-spring when seeds are non-dormant. They become dormant during the summer and autumn with warm temperatures and low
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soil moisture. Therefore, seasonal changes in soil temperature and moisture regulate
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seed dormancy and germination of A. squarrosum.
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Declarations of interest: none
Acknowledgments This work was supported by the National Key Technology R&D Program (2016YFC050080502) and the National Natural Science Foundation (31570416,
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31370705) of P.R. China, and the Strategy of CAS Biological Resources Service
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A
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Network Planning Project [grant number ZSSD-014].
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Authors’ contribution Rui Ru Gao and Zhen Ying Huang formulated the conception and design of the study; Rui Ru Gao, Rui Hua Zhao, Xiao Ya Wei, and Zhan He conducted the
Rui Ru Gao analyzed the data and wrote the manuscript;
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experiments and acquired the data;
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Zhen Ying Huang, Xue Jun Yang and Jeffrey L. Walck revised the
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manuscript .
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