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Salinity affects production and salt tolerance of dimorphic seeds of Suaeda salsa
Accepted Manuscript Salinity affects production and salt tolerance of dimorphic seeds of Suaeda salsa Fengxia Wang, Yange Xu, Shuai Wang, Weiwei Shi, Ranran Liu, Gu Feng, Jie Song PII:
S0981-9428(15)30051-6
DOI:
10.1016/j.plaphy.2015.07.005
Reference:
PLAPHY 4223
To appear in:
Plant Physiology and Biochemistry
Received Date: 9 May 2015 Revised Date:
30 June 2015
Accepted Date: 1 July 2015
Please cite this article as: F. Wang, Y. Xu, S. Wang, W. Shi, R. Liu, G. Feng, J. Song, Salinity affects production and salt tolerance of dimorphic seeds of Suaeda salsa, Plant Physiology et Biochemistry (2015), doi: 10.1016/j.plaphy.2015.07.005. 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.
ACCEPTED MANUSCRIPT Title page
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Title
Salinity affects production and salt tolerance of dimorphic seeds of Suaeda salsa
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Author names and affiliations
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Fengxia Wanga, Yange Xua, Shuai Wanga, Weiwei Shia, Ranran Liua, Gu Fengb, Jie Songa
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a
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250014, PR China
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b
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PR China
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Key Laboratory of Plant Stress, College of Life Science, Shandong Normal University, Jinan
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College of Resource and Environmental Science, China Agricultural University, Beijing 100094,
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Corresponding author
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Jie Song
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Tel.: +86 531 86182568; Fax: +86 531 86180107.
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E-mail address: [email protected]
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Postal address: Wenhua East Road 88, Jinan China, 250014
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Abbreviations:
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IAA, indole-3-acetic acid; ZR, zeatin riboside; ABA, abscisic acid; GA, gibberellic acid.
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ABSTRACT: The effect of salinity on brown seeds/black seeds ratio, seed weight, endogenous
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hormone concentrations, and germination of brown and black seeds in the euhalophyte Suaeda
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salsa was investigated. The brown seeds/black seeds ratio, seed weight of brown and black seeds
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and the content of protein increased at a concentration of 500 mM NaCl compared to low salt
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conditions (1 mM NaCl). The germination percentage and germination index of brown seeds from
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plants cultured in 500 mM NaCl were higher than those cultured in 1 mM NaCl, but it was not true
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for black seeds. The concentrations of IAA (indole-3-acetic acid), ZR (free zeatin riboside) and
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ABA (abscisic acid) in brown seeds were much greater than those in black seeds, but there were no
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differences in the level of GAs (gibberellic acid including GA1 and GA3) regardless of the degree of
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salinity. Salinity during plant culture increased the concentration of GAs, but salinity had no effect
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on the concentrations of the other three endogenous hormones in brown seeds. Salinity had no
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effect on the concentration of IAA but increased the concentrations of the other three endogenous
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hormones in black seeds. Accumulation of endogenous hormones at different concentrations of
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NaCl during plant growth may be related to seed development and to salt tolerance of brown and
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black S. salsa seeds. These characteristics may help the species to ensure seedling establishment
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and population succession in variable saline environments.
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Key words: endogenous hormone; germination; salinity; seed development; seed dimorphism;
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Suaeda salsa.
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1. Introduction
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It is estimated that over 800 million hectares of land are salt-affected throughout the world
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(Munns, 2005). Meanwhile, global annual losses in agricultural production from salt-affected land
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are in excess of US$12 billion and rising (Shabala, 2013). Therefore, it has become imperative to
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identify plants with high economic value that can grow under high salt conditions. Suaeda salsa L.
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is a leaf-succulent halophytic herb with high salt tolerance during germination and seedling stages.
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Recently, it was suggested that S. salsa should be Suaeda maritima subsp. salsa (L.) Soó. It was
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also suggested that the species used in the present study may be Suaeda liaotungensis Kit. 2
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Therefore, the nomenclature and taxonomic position of the study species need to be identified
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(Suaeda salsa was still used in the present study). S. salsa seeds germinate in late April. The plants
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flower from July onwards, and seeds begin to mature in late September (Song and Wang, 2015).
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Nowadays, the species is economically important because its fresh branches have high value as a
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vegetable and its seed oil is edible and rich in unsaturated fatty acids. Moreover, because the plant
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is capable of removing salts and heavy metals from saline soils, S. salsa can also be used in the
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restoration of heavily-salinized land
Song and Wang, 2015).
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Seed dimorphism and polymorphism within a plant refers to there being different seeds whose
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morphological structure and physiological properties are different from each other. Seed
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dimorphism is a powerful germination strategy in unpredictable environments, such as deserts and
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high salt soil areas (Wang et al., 2010). Currently, studies of seed dimorphism have become
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important for understanding plant adaptability (Wei et al., 2007; Yao et al., 2010). Seed dimorphism
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and polymorphism exists in halophytes such as Suaeda moquinii (Khan et al., 2001) and Salsola
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affinis (Wei et al., 2007). S. salsa also produces dimorphic seeds, i.e., some seeds are brown and
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have a soft outer seed coat (big seed), and other seeds are black and have a hard and smooth outer
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seed coat (small seed)
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(IAA) and gibberellic acid (GA) are important hormones which play crucial roles in regulating the
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cell cycle, proliferation and differentiation of plant cells (Rijavec et al., 2009; Uchiumi and
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Okamoto, 2010). However, it is not clear whether these hormones are related to seed development
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in dimorphic seeds of S. salsa. Brown seeds have a higher salt tolerance than black seeds, for
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example the 50% inhibition of germination was achieved for black seeds between 300 and 400 mM
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NaCl and between 600 and 800 mM NaCl for brown seeds Li et al., 2005). It has been reported that
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certain hormones such as cytokinin (CTK) and auxin (i.e., IAA) can increase the salt tolerance of
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seeds in certain plant species (Park et al., 2011). It is also still unclear whether these hormones are
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related to seed salt tolerance in dimorphic seeds of S. salsa.
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Li et al., 2005; Song et al., 2008). Cytokinins (CTKs), indole-3-acetic acid
Germination is pivotal to plant establishment (Wang et al., 2015). A high salt concentration in 3
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the soil prevents germination, and seeds only germinate when temperature and edaphic conditions
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become favorable (Ungar, 1996; Khan et al., 1997; Tessier et al., 2000). Seeds of halophytes and
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nonhalophytes respond similarly to salinity stress. For instance, the initial germination process is
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delayed under salt stress (Almansouri et al., 2001; Khajeh-Hosseini et al., 2003). Besides salinity,
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plant growth regulators also affect seed germination and dormancy under high salt conditions.
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Former reports indicated that salt tolerance of brown seeds and young seedlings from brown
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seeds of S. salsa, cultivated under high salinity, were greater than when cultivated under low
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salinity because salinity during seed maturation may increase the contents of amino acids in the
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embryos of the maturing brown seeds and by changing the lipid composition of membranes in
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mature brown seeds (Zhou et al., 2014). However, there is no information describing how salinity
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during plant culture affects brown seeds/black seeds ratio, the salt tolerance of black seeds, and
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endogenous hormone concentrations in dimorphic seeds of S. salsa. The present study investigated
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these aspects in an attempt to further understand how S. salsa adapted to variable saline
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environments during both seed production and germination stages.
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2. Results
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2.1. Morphology of seeds
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Both seeds and embryos of mature brown and black seeds from plants cultured at 500 mM NaCl
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were better developed than those from plants cultured at 1 mM NaCl (Fig. 1).
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2.2. The brown seeds/black seeds ratio and seed weight
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In a previous experiment, we found that S. salsa as an euhalophyte can not grow well without
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NaCl. Therefore, normally 1 mM NaCl rather than 0 mM NaCl was used as control during plant
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culture of the species. The brown seeds/black seeds ratio was 1.8 times greater at 500 mM NaCl
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than that at 1 mM NaCl during plant culture (Fig. 2A). Brown seed weight was higher than black
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seed weight regardless of the salinity. Salinity increased seed weight regardless of seed type, e.g.,
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the seed weight of brown and black seeds at 500 mM NaCl was 1.6 and 1.9 times higher compared
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to 1 mM NaCl, respectively (Fig. 2B, Table 1). 4
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2.3. The protein content in brown and black seeds
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The protein content in brown seeds was 1.8 and 1.9 times of that in black seeds at 1 and 500
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mM NaCl during plant culture, respectively. Salinity had no significant effect on the protein
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content of brown and black seeds (Fig. 2C, Table 1). 2.4. Germination index, final germination and total germination
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In general, non-halophytes and halophytes respond to salinity in a similar way during the
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germination stage; the initial germination process is delayed under salt stress (Khan and Ungar,
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1997; Li et al., 2005), i.e., unlike plant culture, salinity was not necessary during germination.
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Therefore, 0 mM NaCl was used as control during seed germination in the present experiment. The
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germination index of brown seeds harvested from plants cultured at 1 or 500 mM NaCl was higher
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than that of black seeds at 0 or 600 mM NaCl during germination (Fig. 3A, Table 2). Salinity
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decreased the germination index of brown and black seeds but more significantly in black seeds.
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The germination index of brown seeds from plants cultured at 500 mM NaCl was higher than at 1
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mM NaCl, and even higher when germination conditions were 600 mM NaCl. The value for brown
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seeds from plants cultured at 500 mM NaCl was 1.5 and 2.8 times higher than plants cultured at 1
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mM NaCl at germination conditions of 0 and 600 mM NaCl, respectively. The opposite trend was
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observed in black seeds (Fig. 3A, Table 2). The final germination values followed the same trend as
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the germination index (Fig. 3B, Table 2).
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High salinity (600 mM NaCl) had no adverse effects on the total germination of brown seeds
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from plants cultured at both 1 and 500 mM NaCl after un-germinated brown seeds were pretreated
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with 600 mM NaCl and transferred to 0 mM NaCl, but a concentration of 600 mM NaCl decreased
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the total germination of black seeds regardless of the NaCl concentration during plant culture. The
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total germination of black seeds from plants cultured at 500 mM NaCl was lower than that at 1 mM
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NaCl regardless of NaCl concentrations during germination. For example, total germination of
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black seeds from plants cultured at 500 mM NaCl was 69.4% at 0 mM NaCl during germination 5
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and 76.9% at 600 mM NaCl during germination compared to plants cultured at 1 mM NaCl,
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respectively (Fig. 3C, Table 2).
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2.5. Endogenous hormone concentration The concentrations of IAA, ZR and ABA in brown seeds were much higher than those in black
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seeds, but there was no difference in GAs levels (Fig. 4, Table 2). Salinity during plant culture
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increased the concentration of GAs, but salinity had no effect on the concentrations of the other
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three endogenous hormones in brown seeds. Salinity had no effect on the concentration of IAA but
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increased the concentrations of the other three endogenous hormones in black seeds (Fig. 4, Table
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3. Discussion
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In the course of evolution, the ability of certain plant species to produce heteromorphic seeds
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has been mostly observed in species found in semi-arid, saline-rich and other unfavorable
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environments (Imbert, 2002). Seed heteromorphism is often considered a bet-hedging strategy to
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ensure seedling establishment and population succession for certain plant species in areas with high
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seasonal fluctuations. Chenopodium album plants that suffered higher salinity stress produced more
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salt-resistant seeds (brown seeds); brown seeds were larger and more salt tolerant than black seeds,
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and could germinate more rapidly in a wider range of environments (Yao et al., 2010). High levels
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of salinity did not change the seed morph ratio in Suaeda aralocaspica (Wang et al., 2012a). In the
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present study, salinity increased the brown seeds/black seeds ratio (Fig. 2A). The salt content in
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saline soils where S. salsa growth occurs is always high. S. salsa cannot be found in soils that
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contain less than 5 g of salt kg–1 dry soil, and the optimal salt concentration for its growth is
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between 15 and 20 g kg–1 dry soil. When the salt concentration in soil exceeds 20 g kg–1 dry soil in
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inland saline soil or in the intertidal zone, S. salsa forms a monospecific community (Gu, 1999).
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Therefore, the ability of S. salsa to produce more salinity-resistant seeds can help the species to
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ensure seedling establishment and population succession in variable saline environments.
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Morphological features of seeds usually indicate ecological adaptation to environments where 6
ACCEPTED MANUSCRIPT their mother plants naturally occur (Minuto et al., 2011). It has been suggested that heavy seeds of
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Primula spp. germinate in greater numbers and more quickly than light seeds, and seedlings of P.
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farinosa derived from heavy seeds produce larger rosettes, and more flowers and seeds than those
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from lighter seeds; this ensures that seedlings derived from heavy seeds are more fit than seedlings
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from lighter seeds (Tremayne and Richards, 2000). In dimorphic seeds, brown seeds are higher than
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black seeds in certain dimorphic species such as Suaeda maritima (Wetson et al., 2008), Suaeda
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acuminata (Wang et al., 2012b) and Chenopodium album (Yao et al., 2010). A pleiotropic gene
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expressed in early developing seeds of weedy red rice in activated a conserved network of eight
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genes for flavonoid biosynthesis to produce the pigments in the lower epidermal cells of the
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pericarp tissue and enhanced seed weight (Gu et al., 2011). The physiological and molecular
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mechanisms related to pigments and seed weight in dimorphic seeds of S. salsa are noteworthy to
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be investigated. In the present study, brown seeds were heavier than black seeds from plants
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cultured in both 1 and 500 mM NaCl. In a previous study, embryos of mature brown seeds,
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especially the cotyledons, were better developed than those of mature black seeds which were
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collected from S. salsa plants in saline soils (Song et al., 2008). In the present study, embryos of
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mature brown seeds were better developed than mature black seeds regardless of salinity during
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plant growth (Fig. 1). Correspondingly, salinity (600 mM NaCl during germination) had no adverse
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effect on the total germination of brown seeds, but decreased the total germination of black seeds
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regardless of the NaCl concentration used during plant culture (Fig. 3C). Therefore, heavy brown
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seeds were more salt tolerance than light black seeds regardless of the NaCl concentration during
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plant culture. Maternal environmental effects occur when the phenotype or growth environment of
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the mother plant affects the offspring phenotype beyond the direct effect of transmitted genes
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(Wang et al., 2012a). Seeds from high-salinity maternal plants had a higher germination percentage
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regardless of the level salinity during germination (Wang et al., 2012a). Therefore, producing more
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brown seeds with higher salinity-resistance at 500 mM NaCl compared to 1 mM NaCl during plant
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culture can help the species to adapt to saline environments. The fruit/seed development process is a complex interplay including cell division and
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differentiation (Uchiumi and Okamoto, 2010). Physiological regulation by plant hormones produces
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many types of effects, such as the enhancement of stress resistance, seed development, and
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maturation in plants (Kim et al., 2014). During early embryogenesis, CTK and IAA primarily affect
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seed development; during mid-embryogenesis, the embryo continuously grows by cell enlargement.
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GAs are primarily involved in regulating this process; during late embryogenesis, ABA acts as a
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key regulator as seeds lose water, desiccate, and mature during late embryogenesis (Kim et al.,
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2014). Fruit development in rice is associated with IAA synthesis in the ovary following
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pollination/fertilization and subsequent transport of IAA from the ovary to the rachilla–pedicel
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(Uchiumi and Okamoto, 2010). ZR is the main transportable form of cytokinins (CTKs) and travels
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from the root to other parts of the plant. CTKs are in relation to cell size, cell number and
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endoreduplication in developing maize caryopsis (Rijavec et al., 2009). Increased GA level can
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induce the formation of parthenocarpic fruits in tomato and parthenocarpic fruit development in
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cucumber. However, GA does not function as growth regulator during early fruit development in
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rice (Uchiumi and Okamoto, 2010). In Ginseng (Panax ginseng Meyer), contents of GAs and ABA
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changed during seed development. For example, the total concentration of GAs and ABA during
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different seed growth stages suggests antagonism between these hormones. Similarly, the embryo
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growth ratio during the seed collection period revealed a similar trend in which the total GA and
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ABA contents were inversely related (Kim et al., 2014). In the present study, ZR and IAA content
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was much higher in brown seeds than black seeds which indicated that ZR and IAA may take
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compared to black seeds. Whether GAs play important role in promoting the development of brown
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seeds need to be further investigated during the process of seed development. ABA plays a vital
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role in the processes of seed growth, development, and germination (Kim et al., 2014). ABA
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accumulates halfway through seed development and is synthesized in maternal organs to induce
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accumulation of storage proteins (Karssen et al., 1983). In the present study, the concentration ABA
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were higher in brown seeds than black seeds regardless of the NaCl concentrations utilized during
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plant culture (Fig. 4). Correspondingly, the protein content was higher and weight of brown seeds
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was heavier than black seeds (Fig. 2). Therefore, more ABA accumulated in brown seeds may relate
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to higher protein content which may contribute to heavier seed weight in brown seeds compared to
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black seeds. Seed of wheat (Triticum aestivum L.) that had a higher protein content and/or was
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larger could produce more vigorous seedlings and sometimes higher yields; regardless of genotype
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or environment, seedling vigor was consistently related to seed protein in wheat; when seed size
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was eliminated as a factor by using uniformly sized seed, the seed protein content and vigor
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relationships were significant (Ries and Everson, 1973). Therefore, higher protein content in brown
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seeds may relate to heavier seed size and higher salt resistance compare to black seeds in S. salsa.
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GA is the original stimulus for germination and germination may occur even when the content of an
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inhibitor is relatively high (Kucera et al., 2005). The higher germination index of brown seeds from
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plants cultured in 500 mM NaCl compared to 1 mM NaCl may be due to higher concentration of
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GAs. However, this hypothesis can not explain why germination index in brown seeds was much
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higher than black seeds, but there was no difference in GAs concentration between brown seeds and
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black seeds.
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Under salt stress, plants always accumulate inorganic ions or lowmolecular-weight substances 9
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into the cells. Higher concentrations of inorganic ions such as Na+, K+, and Cl- (Song et al., 2014),
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and organic substances such as protein (Fig. 2) and amino acids (Li, 2012) in brown seeds than
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black seed may contribute higher salt tolerance of brown seeds compared to black seeds, and help
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brown seeds to germinate more rapidly than black seeds. In dimorphic seeds, seed permeability is
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related to density and thickness of the seed coat, both parameters being low in light-coloured seeds.
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The permeability of dark seed coats can be reduced by the number of layers making up the coat, by
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cell density, and by certain chemical reactions such as phenolic oxidationexclusive to dark seeds
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(Matilla et al., 2005). The different structure of seed coat may affect the germination rate in brown
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and black seeds, i.e., a soft outer seed coat of brown seed can help brown seeds take up water and
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germinate more rapidly than black seeds in S. salsa (Li et al., 2005). More inorganic and organic
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substance accumulated in brown seeds compared to black seeds, and the soft outer seed coat of
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brown seed may contribute higher germination index of brown seeds compared to black seeds.
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Endogenous hormones can affect salt tolerance of plants (Yang et al., 2014). For example,
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cytokinin (CTK) and auxin (i.e., IAA) can increase the salt tolerance of seeds in certain plant
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species (Park et al., 2011). In the present study, the concentrations of ZR and IAA in brown seeds
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were much higher than those in black seeds (Fig. 4). The higher salt tolerance of brown seeds may
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be attributable to higher ZR and IAA concentrations in brown seeds than black seeds. Salinity
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during seed maturation may also increase the salt tolerance of brown seeds by increasing oxygen
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production, and by changing the lipid composition of membranes in mature brown seeds (Zhou et
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al., 2014), rather than ZR and IAA as salinity had no effect on the concentrations of ZR and IAA in
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brown seeds (Fig. 4). Li et al. (2011) showed that euhalophytes require salts during the mature seed
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stage to maintain seed viability and to ensure seedling emergence and population establishment.
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used in developing saline agriculture. It has been suggested that t-zr (trans zeatin riboside) can
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counteract many processes induced by ABA (Polanska et al., 2006). Higher ABA concentration may
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counteract higher GAs or ZR levels in black seeds from plants cultured in 500 mM NaCl compared
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to 1 mM NaCl. The plants growing in saline regions exhibited higher ABA/t-zr ratio as compared to
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plants of non-saline soil, and it is inferred that the ratio of ABA/t-zr can serve as index for salt
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tolerance (Hassan and Bano, 2105). In the present study, the ABA/ZR ratio was 10.2 and 11.4,
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respectively in brown seeds, and 10.6 and 9.4, respectively in black seeds from plants cultured in 1
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and 500 mM NaCl. Correspondingly, salinity during plant growth increased salt tolerance of brown
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seeds, but decreased salt tolerance of black seeds. This indicated that in the present study ABA/ZR
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ratio may serve as index for salt tolerance of dimorphic seeds in S. salsa. Germination impairments
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at high salinity would not be caused solely by increased ABA synthesis under high salinity, but may
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also result from the toxic effect of salt and/or the delayed mobilization of seed reserves (Atia et al.,
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2009). Therefore, less salt tolerance of black seeds from plants cultured in 500 mM NaCl relative to
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1 mM NaCl may be related to the counteraction of ABA with GAs or ZR and the toxic effect of salt
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during plant culture and/or the delayed mobilization of seed reserves. The mechanism of how
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salinity during plant culture differentially affect salt tolerance of brown and black seeds needs to be
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further studied.
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In conclusion, salinity during plant growth can improve the brown seeds/black seeds ratio and
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the weight of brown and black seeds. Salinity during plant growth increased salt tolerance of brown
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seeds but this was not the case for black seeds. Interaction between endogenous hormones and their
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accumulation in dimorphic seeds during plant growth at different concentrations of NaCl may be
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related to seed development and salt tolerance of brown and black seeds. These characteristics may
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help the species to ensure seedling establishment and population succession in variable saline
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environments.
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4. Materials and Methods
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4.1. Plant material and seed collection Seeds of S. salsa in inland saline soils (N37o20ˊ; E118o36ˊ) were collected in the Yellow River
6
Delta in Shandong, the middle-east province of China in October, 2013. Dry seeds were stored in a
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refrigerator at 0oC before use.
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4.2. Plant culture and NaCl treatments
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Brown seeds of S. salsa were sown in round, 3-L plastic pots with drainage holes in April 2012.
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There were six seedlings and 3.0 kg of river sand in each pot. The plants were cultured in a
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glasshouse with natural light. The temperature in the glasshouse was 28 ± 5oC during the day and
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20 ± 3oC at night. A nutrient solution was supplied daily and the volume applied (300 mL per pot)
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was in excess of the volume required to saturate the sand. The nutrient solution contained 0.5 mM
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Ca(NO3)2, 3 mM CaCl2, 1 mM KNO3, 1 mM K2SO4, 1 mM MgSO4, 1 mM KH2PO4, 1 mM NaCl,
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45 µM Fe-EDTA, 23 µM H3BO3, 4.55 µM MnCl2, 0.16 µM CuSO4, 0.38 µM ZnSO4, and 0.28 µM
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Na2MoO4. The pH was adjusted to 6.2 ± 0.1 with NaOH and H2SO4.
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After pre-culture for 50 days, three seedlings were removed from each pot of the saline inland
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population (leaving three seedlings per pot). The pots were then treated with either 1 or 500 mM
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NaCl (six pots for each concentration of NaCl). The NaCl was dissolved in the solution described
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above. For the 500 mM treatment, the chance of osmotic shock was reduced by adding 50 mM
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NaCl on the first day and increasing the concentration by 50 mM on each subsequent day until the
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final concentration of 500 mM was reached on the tenth day (Li et al., 2011). One month after final
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salinity concentrations were reached, the plant began to flower. This main experiment was
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terminated 122 days after final salinity concentrations were reached. Then dry mature brown and
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black seeds were collect from plants and stored in a refrigerator at < 4oC for use of microscopy,
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determination of brown seeds/black seeds ratio, seed weight and protein content, as well as
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germination and endogenous hormone content.
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4.3. Microscopy
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Seed coats of mature brown and black seeds from plants cultured at 1 or 500 mM NaCl were
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removed carefully, and the embryos and seeds were observed and photographed under a light
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microscope (Olympus SZX-ILLD2-200, Olympus Corporation, Tokyo, Japan).
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4.4. Determination of brown seeds/black seeds ratio and seed weight
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Seeds of S. salsa were harvested from each of three replicate pots. Brown seeds and black seeds
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on the same branches of a plant (as one replicate) were counted, and the brown seeds/black seeds
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ratio was determined. One thousand grain brown seeds or black seeds from the same plant (as one
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replicate) were counted and the weight was measured. There are 3 replicates for each sample.
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4.5. Determination of protein content in brown and black seeds
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The protein content was measured using the method of Coomassie Brilliant Blue staining. In
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brief, each replicate of 0.5 g dry mature brown or black seeds were grinded with 1.5 mL ice-cold 50
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mM phosphate buffer (pH 7.0) and centrifuged at 15,000 g for 10 minutes at 2-4oC. After that, 10
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uL supernatant (sample solution) was mixed with 4 mL reaction reagent to get the test solution. The
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reaction reagent was prepared by dissolving 100 mg Coomassie Brilliant Blue G-250 in 50 mL 95%
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ethanol, and then diluted with 100 mL 85% (w/v) phosphoric acid and deionized water to get a
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volume of 1000 mL. The solution was stored in a refrigerator at < 4oC. The absorbance of test
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solution was read at 595 nm. The protein content was determined by the formula (a×v1)/(v0×m),
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where a is protein content calculated from the standard curve (using bovine serum albumin as the
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standard), v1 is the total volume of the test solution, v0 is the volume of sample which was measured,
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and m is the weight of seeds.
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4.6. Determination of germination
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In the germination experiments, 25 dry mature brown or black seeds obtained from plants
25
cultured in 1 or 500 mM NaCl were sown in Petri dishes (90 mm) on two layers of moistened filter
26
paper. These seeds were maintained at NaCl concentrations of 0 or 600 mM. NaCl was dissolved in 13
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a solution lacking NaCl, as described in the paragraph describing plant culture conditions. Every 2
2
days, after the solution in each Petri dish was removed, 10 mL of test solution was added and
3
removed as completely as possible, and 5 mL test solution was added (Song et al., 2008). Seeds
4
were considered to have germinated when the emerging radicle was at least 2 mm. The final
5
germination was determined by the formula a/b×100%, where a is the number of seeds that
6
germinated in each Petri dish, b is the total number of seeds provided in each Petri dish during the
7
initial 20 days. Germination index (Gi) in the initial 5 days was determined by the formula Gi=
8
∑(Gt/Dt), where Gt is the number of germinated seeds on t day, and Dt was the number of
9
corresponding days. Three replicates of each treatment were used.
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After 20 days, ungerminated seeds from each Petri dish were transferred to a nutrient solution
11
without NaCl for an additional 10 days, as described in the paragraph describing plant culture
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conditions. In this stage, the total germination was determined by the formula (a+c)/b×100%, where
13
c is the number of seeds that germinated in the nutrient solution without NaCl in each Petri dish
14
during the additional 10 days of incubation. Three replicates of each treatment were used.
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4.7. Determination of endogenous hormone concentration
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Dry mature black and brown seeds were stored in a refrigerator at 0oC for 60 days before being used for the determination of endogenous hormone content. Contents of indole-3-acetic acid (IAA)
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free zeatin riboside (ZR), abscisic acid (ABA) and gibberellic acids (GAs including GA1 and GA3)
19
were analyzed by an indirect ELISA technique. In brief, the samples (0.1g dry black or brown seeds)
20
were homogenised in liquid nitrogen and extracted in cold 80% (v/v) methanol with butylated
21
hydroxytoluene (1 mM) overnight at 4°C. The extracts were collected after centrifugation at 10000
22
×g (4°C) for 20 min, then the extracts were passed through a C18 Sep-Pak catridge (Waters, Milford,
23
MA) and dried in N2. After that the residues were dissolved in PBS (0.01 M, pH 7.4) in order to
24
determine the levels of IAA, GAs, ABA and ZR according to the method of Yang et al. (2001).
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Three replicates were used for each treatment.
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4.8. Statistical analyses
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Data for the brown seeds/black seeds ratio were subjected to one-way analyses of variance
2
(ANOVAs); the independent variable was the salt treatment applied to the source plants (1 vs. 500
3
mM NaCl). Data of endogenous hormones, seed weight and protein content of seeds were subjected
4
to two-way ANOVAs; the independent variables were the type of seeds (brown seeds or black seeds)
5
and the salt treatment applied to the source plants (1 vs. 500 mM NaCl). Data for final germination,
6
total germination and germination index were subjected to a three-way ANOVAs; the independent
7
variables were the type of seeds (brown seeds or black seeds), the salt treatment applied to the
8
source plants (1 vs. 500 mM NaCl) and the salt treatment applied to the seeds in Petri dishes (0 vs.
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600 mM NaCl). SAS™ software (SAS Institute Inc., 1989) was used for statistical analysis.
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Acknowledgments
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The financial support from National Natural Science Research Foundation of China
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(31370420), the State Basic Research Program of China (2014CB954202), and the Program for
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Scientific Research Innovation Team in Colleges and Universities of Shandong Province is greatly
14
appreciated.
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F-values from a two-way ANOVA describing how endogenous hormone (IAA and ZR), protein
4
content and seed weight in S. salsa seeds are affected by (A) seed type (brown seeds vs. black
5
seeds), (B) NaCl treatment during plant culture (1 vs. 500 mM), and their interactions.
Dependent variable 56.44*
IAA concentration
648.18***
GA concentration
4.07**
ABA concentration Protein content Seed weight
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10
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52.77***
92.86***
71.05***
0.28NS
219.29***
17.27**
0.94NS
108.84***
1.05NS
0.34NS
975.89***
508.45***
22.57**
Significant differences at P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 are indicated; NS indicates no significant difference.
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0.01NS
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2.02NS
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A×B
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F-values from a three-way ANOVA describing how final germination, total germination, and
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germination index in S. salsa seeds are affected by (A) seed type (brown seeds vs. black seeds),
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(B) NaCl treatment during plant culture (1 vs. 500 mM), (C) NaCl treatment during seeds
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germination (0 vs. 600 mM), and their interactions. B
C
A×B
Final germination
3.38NS
0.38NS
0.01NS
3.38NS
Total germination
1.81NS
0.52NS
0.23NS
Germination index
4.47*
0.37NS
0.51NS
B×C
A×B×C
0.1NS
3.93NS
8.32**
6.13*
1441NS
0.71NS
1.14NS
9.15**
1.34NS
6.08*
10.21**
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A×C
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Significant differences at *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 are indicated; NS indicates no significant
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difference.
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Fig. 1. Morphology of brown seeds of Suaeda salsa from plants cultured at 1 mM NaCl (A) and
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500 mM NaCl (B), and embryos of brown seeds at 1 mM NaCl (C) and 500 mM NaCl (D); black
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seeds at 1 mM NaCl (E) and 500 mM NaCl (F), and embryos of black seeds at 1 mM NaCl (G)
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and 500 mM NaCl (H). Bar in each graph indicates 250 µm.
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Fig. 2. The brown seeds/black seeds ratio (A), seed weight (B) and seed protein content (C) of S.
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salsa cultured in 1 or 500 mM NaCl during plant growth. Values are means + SD, n = 3. For each
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pair of bars, means with different letters are significantly different at P ≤ 0.05.
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Fig. 3. Germination index (A), final germination (B) and total germination (C) of brown and black
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seeds from plants cultured in 1 or 500 mM NaCl at 0 or 600 mM NaCl during germination. Values
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are means + SD, n = 3. For each pair of bars, means with different letters are significantly different
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at P ≤ 0.05.
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Fig. 4. Concentration of IAA (A), ZR (B), GA (C), and ABA (D) in brown and black seeds from
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plants cultured in 1 or 500 mM NaCl. Values are means + SD, n = 3. For each pair of bars, means
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with different letters are significantly different at P ≤ 0.05.
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Fig. 1.
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B
C
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Fig. 2.
b
2.00 1.00 0.00 1 mM 500 mM NaCl during plant growth
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0.7 0.6
B
1 mM NaCl
a
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0.5 0.4
b a
0.3 b
0.2 0.1 0
50 45 40 35 30 25 20 15 10 5 0
C a
a
a
Brown seeds
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Protein content (mg g -1 FW)
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Seed weight (g 1000 seed -1)
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Fig. 3.
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Germination index
A
a
0 mM NaCl 600 mM NaCl
a
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b 10 b
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a
a b
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120
a
a
100
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80 60
b
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a
a
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0
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120
C a a
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500 mM 1 mM 500 mM NaCl during plant growth Brown seeds Black seeds
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Fig. 4.
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a
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a
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ACCEPTED MANUSCRIPT Highlights: High salt during plant growth can improve the brown seeds/black seeds ratio and the weight of brown and black seeds in Suaeda salsa plants. Salinity during plant growth increased salt tolerance of brown seeds, but decreased salt tolerance of black seeds in Suaeda salsa.
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Endogenous hormone accumulation in dimorphic seeds of Suaeda salsa during plant growth may be related to seed development and to salt tolerance of dimorphic seeds.
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The work presented here was carried out in collaboration with all authors. Dr. Jie Song designed the experiments and revised the paper. Ms. Fengxia Wang performed the experiments, analyzed the data, and wrote the paper. Ms. Yange Xu and Mr. Shuai Wang assisted in analyzing the brown seeds/black seeds ratio, seed weight and endogenous hormone production. Ms.Weiwei Shi helped in measuring endogenous hormone concentrations. Ms. Ranran Liu helped finishing the determination of germination of brown and black seeds. Prof. Gu Feng assisted in writing the paper.
S0981-9428(15)30051-6
DOI:
10.1016/j.plaphy.2015.07.005
Reference:
PLAPHY 4223
To appear in:
Plant Physiology and Biochemistry
Received Date: 9 May 2015 Revised Date:
30 June 2015
Accepted Date: 1 July 2015
Please cite this article as: F. Wang, Y. Xu, S. Wang, W. Shi, R. Liu, G. Feng, J. Song, Salinity affects production and salt tolerance of dimorphic seeds of Suaeda salsa, Plant Physiology et Biochemistry (2015), doi: 10.1016/j.plaphy.2015.07.005. 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.
ACCEPTED MANUSCRIPT Title page
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Title
Salinity affects production and salt tolerance of dimorphic seeds of Suaeda salsa
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Author names and affiliations
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Fengxia Wanga, Yange Xua, Shuai Wanga, Weiwei Shia, Ranran Liua, Gu Fengb, Jie Songa
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a
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250014, PR China
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b
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PR China
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Key Laboratory of Plant Stress, College of Life Science, Shandong Normal University, Jinan
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College of Resource and Environmental Science, China Agricultural University, Beijing 100094,
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Corresponding author
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Jie Song
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Tel.: +86 531 86182568; Fax: +86 531 86180107.
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E-mail address: [email protected]
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Postal address: Wenhua East Road 88, Jinan China, 250014
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Abbreviations:
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IAA, indole-3-acetic acid; ZR, zeatin riboside; ABA, abscisic acid; GA, gibberellic acid.
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ABSTRACT: The effect of salinity on brown seeds/black seeds ratio, seed weight, endogenous
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hormone concentrations, and germination of brown and black seeds in the euhalophyte Suaeda
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salsa was investigated. The brown seeds/black seeds ratio, seed weight of brown and black seeds
4
and the content of protein increased at a concentration of 500 mM NaCl compared to low salt
5
conditions (1 mM NaCl). The germination percentage and germination index of brown seeds from
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plants cultured in 500 mM NaCl were higher than those cultured in 1 mM NaCl, but it was not true
7
for black seeds. The concentrations of IAA (indole-3-acetic acid), ZR (free zeatin riboside) and
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ABA (abscisic acid) in brown seeds were much greater than those in black seeds, but there were no
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differences in the level of GAs (gibberellic acid including GA1 and GA3) regardless of the degree of
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salinity. Salinity during plant culture increased the concentration of GAs, but salinity had no effect
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on the concentrations of the other three endogenous hormones in brown seeds. Salinity had no
12
effect on the concentration of IAA but increased the concentrations of the other three endogenous
13
hormones in black seeds. Accumulation of endogenous hormones at different concentrations of
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NaCl during plant growth may be related to seed development and to salt tolerance of brown and
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black S. salsa seeds. These characteristics may help the species to ensure seedling establishment
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and population succession in variable saline environments.
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Key words: endogenous hormone; germination; salinity; seed development; seed dimorphism;
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Suaeda salsa.
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1. Introduction
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It is estimated that over 800 million hectares of land are salt-affected throughout the world
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(Munns, 2005). Meanwhile, global annual losses in agricultural production from salt-affected land
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are in excess of US$12 billion and rising (Shabala, 2013). Therefore, it has become imperative to
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identify plants with high economic value that can grow under high salt conditions. Suaeda salsa L.
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is a leaf-succulent halophytic herb with high salt tolerance during germination and seedling stages.
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Recently, it was suggested that S. salsa should be Suaeda maritima subsp. salsa (L.) Soó. It was
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also suggested that the species used in the present study may be Suaeda liaotungensis Kit. 2
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Therefore, the nomenclature and taxonomic position of the study species need to be identified
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(Suaeda salsa was still used in the present study). S. salsa seeds germinate in late April. The plants
3
flower from July onwards, and seeds begin to mature in late September (Song and Wang, 2015).
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Nowadays, the species is economically important because its fresh branches have high value as a
5
vegetable and its seed oil is edible and rich in unsaturated fatty acids. Moreover, because the plant
6
is capable of removing salts and heavy metals from saline soils, S. salsa can also be used in the
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restoration of heavily-salinized land
Song and Wang, 2015).
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Seed dimorphism and polymorphism within a plant refers to there being different seeds whose
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morphological structure and physiological properties are different from each other. Seed
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dimorphism is a powerful germination strategy in unpredictable environments, such as deserts and
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high salt soil areas (Wang et al., 2010). Currently, studies of seed dimorphism have become
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important for understanding plant adaptability (Wei et al., 2007; Yao et al., 2010). Seed dimorphism
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and polymorphism exists in halophytes such as Suaeda moquinii (Khan et al., 2001) and Salsola
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affinis (Wei et al., 2007). S. salsa also produces dimorphic seeds, i.e., some seeds are brown and
15
have a soft outer seed coat (big seed), and other seeds are black and have a hard and smooth outer
16
seed coat (small seed)
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(IAA) and gibberellic acid (GA) are important hormones which play crucial roles in regulating the
18
cell cycle, proliferation and differentiation of plant cells (Rijavec et al., 2009; Uchiumi and
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Okamoto, 2010). However, it is not clear whether these hormones are related to seed development
20
in dimorphic seeds of S. salsa. Brown seeds have a higher salt tolerance than black seeds, for
21
example the 50% inhibition of germination was achieved for black seeds between 300 and 400 mM
22
NaCl and between 600 and 800 mM NaCl for brown seeds Li et al., 2005). It has been reported that
23
certain hormones such as cytokinin (CTK) and auxin (i.e., IAA) can increase the salt tolerance of
24
seeds in certain plant species (Park et al., 2011). It is also still unclear whether these hormones are
25
related to seed salt tolerance in dimorphic seeds of S. salsa.
26
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Li et al., 2005; Song et al., 2008). Cytokinins (CTKs), indole-3-acetic acid
Germination is pivotal to plant establishment (Wang et al., 2015). A high salt concentration in 3
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the soil prevents germination, and seeds only germinate when temperature and edaphic conditions
2
become favorable (Ungar, 1996; Khan et al., 1997; Tessier et al., 2000). Seeds of halophytes and
3
nonhalophytes respond similarly to salinity stress. For instance, the initial germination process is
4
delayed under salt stress (Almansouri et al., 2001; Khajeh-Hosseini et al., 2003). Besides salinity,
5
plant growth regulators also affect seed germination and dormancy under high salt conditions.
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Former reports indicated that salt tolerance of brown seeds and young seedlings from brown
7
seeds of S. salsa, cultivated under high salinity, were greater than when cultivated under low
8
salinity because salinity during seed maturation may increase the contents of amino acids in the
9
embryos of the maturing brown seeds and by changing the lipid composition of membranes in
10
mature brown seeds (Zhou et al., 2014). However, there is no information describing how salinity
11
during plant culture affects brown seeds/black seeds ratio, the salt tolerance of black seeds, and
12
endogenous hormone concentrations in dimorphic seeds of S. salsa. The present study investigated
13
these aspects in an attempt to further understand how S. salsa adapted to variable saline
14
environments during both seed production and germination stages.
15
2. Results
16
2.1. Morphology of seeds
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Both seeds and embryos of mature brown and black seeds from plants cultured at 500 mM NaCl
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were better developed than those from plants cultured at 1 mM NaCl (Fig. 1).
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2.2. The brown seeds/black seeds ratio and seed weight
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In a previous experiment, we found that S. salsa as an euhalophyte can not grow well without
21
NaCl. Therefore, normally 1 mM NaCl rather than 0 mM NaCl was used as control during plant
22
culture of the species. The brown seeds/black seeds ratio was 1.8 times greater at 500 mM NaCl
23
than that at 1 mM NaCl during plant culture (Fig. 2A). Brown seed weight was higher than black
24
seed weight regardless of the salinity. Salinity increased seed weight regardless of seed type, e.g.,
25
the seed weight of brown and black seeds at 500 mM NaCl was 1.6 and 1.9 times higher compared
26
to 1 mM NaCl, respectively (Fig. 2B, Table 1). 4
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2.3. The protein content in brown and black seeds
2
The protein content in brown seeds was 1.8 and 1.9 times of that in black seeds at 1 and 500
3
mM NaCl during plant culture, respectively. Salinity had no significant effect on the protein
4
content of brown and black seeds (Fig. 2C, Table 1). 2.4. Germination index, final germination and total germination
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In general, non-halophytes and halophytes respond to salinity in a similar way during the
7
germination stage; the initial germination process is delayed under salt stress (Khan and Ungar,
8
1997; Li et al., 2005), i.e., unlike plant culture, salinity was not necessary during germination.
9
Therefore, 0 mM NaCl was used as control during seed germination in the present experiment. The
10
germination index of brown seeds harvested from plants cultured at 1 or 500 mM NaCl was higher
11
than that of black seeds at 0 or 600 mM NaCl during germination (Fig. 3A, Table 2). Salinity
12
decreased the germination index of brown and black seeds but more significantly in black seeds.
13
The germination index of brown seeds from plants cultured at 500 mM NaCl was higher than at 1
14
mM NaCl, and even higher when germination conditions were 600 mM NaCl. The value for brown
15
seeds from plants cultured at 500 mM NaCl was 1.5 and 2.8 times higher than plants cultured at 1
16
mM NaCl at germination conditions of 0 and 600 mM NaCl, respectively. The opposite trend was
17
observed in black seeds (Fig. 3A, Table 2). The final germination values followed the same trend as
18
the germination index (Fig. 3B, Table 2).
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High salinity (600 mM NaCl) had no adverse effects on the total germination of brown seeds
20
from plants cultured at both 1 and 500 mM NaCl after un-germinated brown seeds were pretreated
21
with 600 mM NaCl and transferred to 0 mM NaCl, but a concentration of 600 mM NaCl decreased
22
the total germination of black seeds regardless of the NaCl concentration during plant culture. The
23
total germination of black seeds from plants cultured at 500 mM NaCl was lower than that at 1 mM
24
NaCl regardless of NaCl concentrations during germination. For example, total germination of
25
black seeds from plants cultured at 500 mM NaCl was 69.4% at 0 mM NaCl during germination 5
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1
and 76.9% at 600 mM NaCl during germination compared to plants cultured at 1 mM NaCl,
2
respectively (Fig. 3C, Table 2).
3
2.5. Endogenous hormone concentration The concentrations of IAA, ZR and ABA in brown seeds were much higher than those in black
5
seeds, but there was no difference in GAs levels (Fig. 4, Table 2). Salinity during plant culture
6
increased the concentration of GAs, but salinity had no effect on the concentrations of the other
7
three endogenous hormones in brown seeds. Salinity had no effect on the concentration of IAA but
8
increased the concentrations of the other three endogenous hormones in black seeds (Fig. 4, Table
9
1).
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3. Discussion
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In the course of evolution, the ability of certain plant species to produce heteromorphic seeds
12
has been mostly observed in species found in semi-arid, saline-rich and other unfavorable
13
environments (Imbert, 2002). Seed heteromorphism is often considered a bet-hedging strategy to
14
ensure seedling establishment and population succession for certain plant species in areas with high
15
seasonal fluctuations. Chenopodium album plants that suffered higher salinity stress produced more
16
salt-resistant seeds (brown seeds); brown seeds were larger and more salt tolerant than black seeds,
17
and could germinate more rapidly in a wider range of environments (Yao et al., 2010). High levels
18
of salinity did not change the seed morph ratio in Suaeda aralocaspica (Wang et al., 2012a). In the
19
present study, salinity increased the brown seeds/black seeds ratio (Fig. 2A). The salt content in
20
saline soils where S. salsa growth occurs is always high. S. salsa cannot be found in soils that
21
contain less than 5 g of salt kg–1 dry soil, and the optimal salt concentration for its growth is
22
between 15 and 20 g kg–1 dry soil. When the salt concentration in soil exceeds 20 g kg–1 dry soil in
23
inland saline soil or in the intertidal zone, S. salsa forms a monospecific community (Gu, 1999).
24
Therefore, the ability of S. salsa to produce more salinity-resistant seeds can help the species to
25
ensure seedling establishment and population succession in variable saline environments.
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Morphological features of seeds usually indicate ecological adaptation to environments where 6
ACCEPTED MANUSCRIPT their mother plants naturally occur (Minuto et al., 2011). It has been suggested that heavy seeds of
2
Primula spp. germinate in greater numbers and more quickly than light seeds, and seedlings of P.
3
farinosa derived from heavy seeds produce larger rosettes, and more flowers and seeds than those
4
from lighter seeds; this ensures that seedlings derived from heavy seeds are more fit than seedlings
5
from lighter seeds (Tremayne and Richards, 2000). In dimorphic seeds, brown seeds are higher than
6
black seeds in certain dimorphic species such as Suaeda maritima (Wetson et al., 2008), Suaeda
7
acuminata (Wang et al., 2012b) and Chenopodium album (Yao et al., 2010). A pleiotropic gene
8
expressed in early developing seeds of weedy red rice in activated a conserved network of eight
9
genes for flavonoid biosynthesis to produce the pigments in the lower epidermal cells of the
10
pericarp tissue and enhanced seed weight (Gu et al., 2011). The physiological and molecular
11
mechanisms related to pigments and seed weight in dimorphic seeds of S. salsa are noteworthy to
12
be investigated. In the present study, brown seeds were heavier than black seeds from plants
13
cultured in both 1 and 500 mM NaCl. In a previous study, embryos of mature brown seeds,
14
especially the cotyledons, were better developed than those of mature black seeds which were
15
collected from S. salsa plants in saline soils (Song et al., 2008). In the present study, embryos of
16
mature brown seeds were better developed than mature black seeds regardless of salinity during
17
plant growth (Fig. 1). Correspondingly, salinity (600 mM NaCl during germination) had no adverse
18
effect on the total germination of brown seeds, but decreased the total germination of black seeds
19
regardless of the NaCl concentration used during plant culture (Fig. 3C). Therefore, heavy brown
20
seeds were more salt tolerance than light black seeds regardless of the NaCl concentration during
21
plant culture. Maternal environmental effects occur when the phenotype or growth environment of
22
the mother plant affects the offspring phenotype beyond the direct effect of transmitted genes
23
(Wang et al., 2012a). Seeds from high-salinity maternal plants had a higher germination percentage
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regardless of the level salinity during germination (Wang et al., 2012a). Therefore, producing more
2
brown seeds with higher salinity-resistance at 500 mM NaCl compared to 1 mM NaCl during plant
3
culture can help the species to adapt to saline environments. The fruit/seed development process is a complex interplay including cell division and
5
differentiation (Uchiumi and Okamoto, 2010). Physiological regulation by plant hormones produces
6
many types of effects, such as the enhancement of stress resistance, seed development, and
7
maturation in plants (Kim et al., 2014). During early embryogenesis, CTK and IAA primarily affect
8
seed development; during mid-embryogenesis, the embryo continuously grows by cell enlargement.
9
GAs are primarily involved in regulating this process; during late embryogenesis, ABA acts as a
10
key regulator as seeds lose water, desiccate, and mature during late embryogenesis (Kim et al.,
11
2014). Fruit development in rice is associated with IAA synthesis in the ovary following
12
pollination/fertilization and subsequent transport of IAA from the ovary to the rachilla–pedicel
13
(Uchiumi and Okamoto, 2010). ZR is the main transportable form of cytokinins (CTKs) and travels
14
from the root to other parts of the plant. CTKs are in relation to cell size, cell number and
15
endoreduplication in developing maize caryopsis (Rijavec et al., 2009). Increased GA level can
16
induce the formation of parthenocarpic fruits in tomato and parthenocarpic fruit development in
17
cucumber. However, GA does not function as growth regulator during early fruit development in
18
rice (Uchiumi and Okamoto, 2010). In Ginseng (Panax ginseng Meyer), contents of GAs and ABA
19
changed during seed development. For example, the total concentration of GAs and ABA during
20
different seed growth stages suggests antagonism between these hormones. Similarly, the embryo
21
growth ratio during the seed collection period revealed a similar trend in which the total GA and
22
ABA contents were inversely related (Kim et al., 2014). In the present study, ZR and IAA content
23
was much higher in brown seeds than black seeds which indicated that ZR and IAA may take
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ACCEPTED MANUSCRIPT important functions as growth regulators which may promote the development of brown seeds
2
compared to black seeds. Whether GAs play important role in promoting the development of brown
3
seeds need to be further investigated during the process of seed development. ABA plays a vital
4
role in the processes of seed growth, development, and germination (Kim et al., 2014). ABA
5
accumulates halfway through seed development and is synthesized in maternal organs to induce
6
accumulation of storage proteins (Karssen et al., 1983). In the present study, the concentration ABA
7
were higher in brown seeds than black seeds regardless of the NaCl concentrations utilized during
8
plant culture (Fig. 4). Correspondingly, the protein content was higher and weight of brown seeds
9
was heavier than black seeds (Fig. 2). Therefore, more ABA accumulated in brown seeds may relate
10
to higher protein content which may contribute to heavier seed weight in brown seeds compared to
11
black seeds. Seed of wheat (Triticum aestivum L.) that had a higher protein content and/or was
12
larger could produce more vigorous seedlings and sometimes higher yields; regardless of genotype
13
or environment, seedling vigor was consistently related to seed protein in wheat; when seed size
14
was eliminated as a factor by using uniformly sized seed, the seed protein content and vigor
15
relationships were significant (Ries and Everson, 1973). Therefore, higher protein content in brown
16
seeds may relate to heavier seed size and higher salt resistance compare to black seeds in S. salsa.
17
GA is the original stimulus for germination and germination may occur even when the content of an
18
inhibitor is relatively high (Kucera et al., 2005). The higher germination index of brown seeds from
19
plants cultured in 500 mM NaCl compared to 1 mM NaCl may be due to higher concentration of
20
GAs. However, this hypothesis can not explain why germination index in brown seeds was much
21
higher than black seeds, but there was no difference in GAs concentration between brown seeds and
22
black seeds.
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Under salt stress, plants always accumulate inorganic ions or lowmolecular-weight substances 9
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2
into the cells. Higher concentrations of inorganic ions such as Na+, K+, and Cl- (Song et al., 2014),
3
and organic substances such as protein (Fig. 2) and amino acids (Li, 2012) in brown seeds than
4
black seed may contribute higher salt tolerance of brown seeds compared to black seeds, and help
5
brown seeds to germinate more rapidly than black seeds. In dimorphic seeds, seed permeability is
6
related to density and thickness of the seed coat, both parameters being low in light-coloured seeds.
7
The permeability of dark seed coats can be reduced by the number of layers making up the coat, by
8
cell density, and by certain chemical reactions such as phenolic oxidationexclusive to dark seeds
9
(Matilla et al., 2005). The different structure of seed coat may affect the germination rate in brown
10
and black seeds, i.e., a soft outer seed coat of brown seed can help brown seeds take up water and
11
germinate more rapidly than black seeds in S. salsa (Li et al., 2005). More inorganic and organic
12
substance accumulated in brown seeds compared to black seeds, and the soft outer seed coat of
13
brown seed may contribute higher germination index of brown seeds compared to black seeds.
14
Endogenous hormones can affect salt tolerance of plants (Yang et al., 2014). For example,
15
cytokinin (CTK) and auxin (i.e., IAA) can increase the salt tolerance of seeds in certain plant
16
species (Park et al., 2011). In the present study, the concentrations of ZR and IAA in brown seeds
17
were much higher than those in black seeds (Fig. 4). The higher salt tolerance of brown seeds may
18
be attributable to higher ZR and IAA concentrations in brown seeds than black seeds. Salinity
19
during seed maturation may also increase the salt tolerance of brown seeds by increasing oxygen
20
production, and by changing the lipid composition of membranes in mature brown seeds (Zhou et
21
al., 2014), rather than ZR and IAA as salinity had no effect on the concentrations of ZR and IAA in
22
brown seeds (Fig. 4). Li et al. (2011) showed that euhalophytes require salts during the mature seed
23
stage to maintain seed viability and to ensure seedling emergence and population establishment.
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2
used in developing saline agriculture. It has been suggested that t-zr (trans zeatin riboside) can
3
counteract many processes induced by ABA (Polanska et al., 2006). Higher ABA concentration may
4
counteract higher GAs or ZR levels in black seeds from plants cultured in 500 mM NaCl compared
5
to 1 mM NaCl. The plants growing in saline regions exhibited higher ABA/t-zr ratio as compared to
6
plants of non-saline soil, and it is inferred that the ratio of ABA/t-zr can serve as index for salt
7
tolerance (Hassan and Bano, 2105). In the present study, the ABA/ZR ratio was 10.2 and 11.4,
8
respectively in brown seeds, and 10.6 and 9.4, respectively in black seeds from plants cultured in 1
9
and 500 mM NaCl. Correspondingly, salinity during plant growth increased salt tolerance of brown
10
seeds, but decreased salt tolerance of black seeds. This indicated that in the present study ABA/ZR
11
ratio may serve as index for salt tolerance of dimorphic seeds in S. salsa. Germination impairments
12
at high salinity would not be caused solely by increased ABA synthesis under high salinity, but may
13
also result from the toxic effect of salt and/or the delayed mobilization of seed reserves (Atia et al.,
14
2009). Therefore, less salt tolerance of black seeds from plants cultured in 500 mM NaCl relative to
15
1 mM NaCl may be related to the counteraction of ABA with GAs or ZR and the toxic effect of salt
16
during plant culture and/or the delayed mobilization of seed reserves. The mechanism of how
17
salinity during plant culture differentially affect salt tolerance of brown and black seeds needs to be
18
further studied.
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In conclusion, salinity during plant growth can improve the brown seeds/black seeds ratio and
20
the weight of brown and black seeds. Salinity during plant growth increased salt tolerance of brown
21
seeds but this was not the case for black seeds. Interaction between endogenous hormones and their
22
accumulation in dimorphic seeds during plant growth at different concentrations of NaCl may be
23
related to seed development and salt tolerance of brown and black seeds. These characteristics may
11
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1
help the species to ensure seedling establishment and population succession in variable saline
2
environments.
3
4. Materials and Methods
4
4.1. Plant material and seed collection Seeds of S. salsa in inland saline soils (N37o20ˊ; E118o36ˊ) were collected in the Yellow River
6
Delta in Shandong, the middle-east province of China in October, 2013. Dry seeds were stored in a
7
refrigerator at 0oC before use.
8
4.2. Plant culture and NaCl treatments
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Brown seeds of S. salsa were sown in round, 3-L plastic pots with drainage holes in April 2012.
10
There were six seedlings and 3.0 kg of river sand in each pot. The plants were cultured in a
11
glasshouse with natural light. The temperature in the glasshouse was 28 ± 5oC during the day and
12
20 ± 3oC at night. A nutrient solution was supplied daily and the volume applied (300 mL per pot)
13
was in excess of the volume required to saturate the sand. The nutrient solution contained 0.5 mM
14
Ca(NO3)2, 3 mM CaCl2, 1 mM KNO3, 1 mM K2SO4, 1 mM MgSO4, 1 mM KH2PO4, 1 mM NaCl,
15
45 µM Fe-EDTA, 23 µM H3BO3, 4.55 µM MnCl2, 0.16 µM CuSO4, 0.38 µM ZnSO4, and 0.28 µM
16
Na2MoO4. The pH was adjusted to 6.2 ± 0.1 with NaOH and H2SO4.
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After pre-culture for 50 days, three seedlings were removed from each pot of the saline inland
18
population (leaving three seedlings per pot). The pots were then treated with either 1 or 500 mM
19
NaCl (six pots for each concentration of NaCl). The NaCl was dissolved in the solution described
20
above. For the 500 mM treatment, the chance of osmotic shock was reduced by adding 50 mM
21
NaCl on the first day and increasing the concentration by 50 mM on each subsequent day until the
22
final concentration of 500 mM was reached on the tenth day (Li et al., 2011). One month after final
23
salinity concentrations were reached, the plant began to flower. This main experiment was
24
terminated 122 days after final salinity concentrations were reached. Then dry mature brown and
25
black seeds were collect from plants and stored in a refrigerator at < 4oC for use of microscopy,
26
determination of brown seeds/black seeds ratio, seed weight and protein content, as well as
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germination and endogenous hormone content.
2
4.3. Microscopy
3
Seed coats of mature brown and black seeds from plants cultured at 1 or 500 mM NaCl were
4
removed carefully, and the embryos and seeds were observed and photographed under a light
5
microscope (Olympus SZX-ILLD2-200, Olympus Corporation, Tokyo, Japan).
6
4.4. Determination of brown seeds/black seeds ratio and seed weight
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Seeds of S. salsa were harvested from each of three replicate pots. Brown seeds and black seeds
8
on the same branches of a plant (as one replicate) were counted, and the brown seeds/black seeds
9
ratio was determined. One thousand grain brown seeds or black seeds from the same plant (as one
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7
replicate) were counted and the weight was measured. There are 3 replicates for each sample.
11
4.5. Determination of protein content in brown and black seeds
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The protein content was measured using the method of Coomassie Brilliant Blue staining. In
13
brief, each replicate of 0.5 g dry mature brown or black seeds were grinded with 1.5 mL ice-cold 50
14
mM phosphate buffer (pH 7.0) and centrifuged at 15,000 g for 10 minutes at 2-4oC. After that, 10
15
uL supernatant (sample solution) was mixed with 4 mL reaction reagent to get the test solution. The
16
reaction reagent was prepared by dissolving 100 mg Coomassie Brilliant Blue G-250 in 50 mL 95%
17
ethanol, and then diluted with 100 mL 85% (w/v) phosphoric acid and deionized water to get a
18
volume of 1000 mL. The solution was stored in a refrigerator at < 4oC. The absorbance of test
19
solution was read at 595 nm. The protein content was determined by the formula (a×v1)/(v0×m),
20
where a is protein content calculated from the standard curve (using bovine serum albumin as the
21
standard), v1 is the total volume of the test solution, v0 is the volume of sample which was measured,
22
and m is the weight of seeds.
23
4.6. Determination of germination
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In the germination experiments, 25 dry mature brown or black seeds obtained from plants
25
cultured in 1 or 500 mM NaCl were sown in Petri dishes (90 mm) on two layers of moistened filter
26
paper. These seeds were maintained at NaCl concentrations of 0 or 600 mM. NaCl was dissolved in 13
ACCEPTED MANUSCRIPT
a solution lacking NaCl, as described in the paragraph describing plant culture conditions. Every 2
2
days, after the solution in each Petri dish was removed, 10 mL of test solution was added and
3
removed as completely as possible, and 5 mL test solution was added (Song et al., 2008). Seeds
4
were considered to have germinated when the emerging radicle was at least 2 mm. The final
5
germination was determined by the formula a/b×100%, where a is the number of seeds that
6
germinated in each Petri dish, b is the total number of seeds provided in each Petri dish during the
7
initial 20 days. Germination index (Gi) in the initial 5 days was determined by the formula Gi=
8
∑(Gt/Dt), where Gt is the number of germinated seeds on t day, and Dt was the number of
9
corresponding days. Three replicates of each treatment were used.
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After 20 days, ungerminated seeds from each Petri dish were transferred to a nutrient solution
11
without NaCl for an additional 10 days, as described in the paragraph describing plant culture
12
conditions. In this stage, the total germination was determined by the formula (a+c)/b×100%, where
13
c is the number of seeds that germinated in the nutrient solution without NaCl in each Petri dish
14
during the additional 10 days of incubation. Three replicates of each treatment were used.
15
4.7. Determination of endogenous hormone concentration
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Dry mature black and brown seeds were stored in a refrigerator at 0oC for 60 days before being used for the determination of endogenous hormone content. Contents of indole-3-acetic acid (IAA)
18
free zeatin riboside (ZR), abscisic acid (ABA) and gibberellic acids (GAs including GA1 and GA3)
19
were analyzed by an indirect ELISA technique. In brief, the samples (0.1g dry black or brown seeds)
20
were homogenised in liquid nitrogen and extracted in cold 80% (v/v) methanol with butylated
21
hydroxytoluene (1 mM) overnight at 4°C. The extracts were collected after centrifugation at 10000
22
×g (4°C) for 20 min, then the extracts were passed through a C18 Sep-Pak catridge (Waters, Milford,
23
MA) and dried in N2. After that the residues were dissolved in PBS (0.01 M, pH 7.4) in order to
24
determine the levels of IAA, GAs, ABA and ZR according to the method of Yang et al. (2001).
25
Three replicates were used for each treatment.
26
4.8. Statistical analyses
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Data for the brown seeds/black seeds ratio were subjected to one-way analyses of variance
2
(ANOVAs); the independent variable was the salt treatment applied to the source plants (1 vs. 500
3
mM NaCl). Data of endogenous hormones, seed weight and protein content of seeds were subjected
4
to two-way ANOVAs; the independent variables were the type of seeds (brown seeds or black seeds)
5
and the salt treatment applied to the source plants (1 vs. 500 mM NaCl). Data for final germination,
6
total germination and germination index were subjected to a three-way ANOVAs; the independent
7
variables were the type of seeds (brown seeds or black seeds), the salt treatment applied to the
8
source plants (1 vs. 500 mM NaCl) and the salt treatment applied to the seeds in Petri dishes (0 vs.
9
600 mM NaCl). SAS™ software (SAS Institute Inc., 1989) was used for statistical analysis.
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Acknowledgments
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The financial support from National Natural Science Research Foundation of China
12
(31370420), the State Basic Research Program of China (2014CB954202), and the Program for
13
Scientific Research Innovation Team in Colleges and Universities of Shandong Province is greatly
14
appreciated.
15
References
16
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Atia, A., Debez, A., Barhoumi, Z., Smaoui, A., Abdelly, C., 2009. ABA, GA3, and nitrate may
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Gu, F.T., 1999. Research in exploiting the green series of edibles-Suaeda Salsa. J. Binzhou Edueation College 5 (3), 43-48 [in Chinese]. Gu, X.Y., Foley, M.E., Horvath, D.P., Anderson, J.V., Feng, J.H., Zhang, L.H., Mowry, C.R., Ye,
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H., Suttle, J.C., Kadowaki, K.I., Chen, Z.X., 2011.
Association between seed dormancy and
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pericarp color is controlled by a pleiotropic gene that regulates abscisic acid and flavonoid
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synthesis in weedy red rice. Genet189, 1515-1524. 15
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Hassan, T.U., Bano, A., 2015. Physiological characterization of halophytic weeds growing in saline and non-saline regions of Pakistan. J. Anim. Plant Sci. 25, 463-471. Imbert, E., 2002. Ecological consequences and ontogeny of seed heteromorphism. Perspect. Plant Ecol. 5, 13-36. Karssen, C.M., Brinkhorst-van der Swan, D.L.C., Breekland, A.E., Koornneef, M., 1983.
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Khajeh-Hosseini, M., Powell, A.A., Bingham, I.J., 2003. The interaction between salinity stress and seed vigour during germination of soybean seeds. Seed Sci. Technol. 31, 715-725. Khan, M.A., Gul, B., Weber D.J., 2001. Germination of dimorphic seeds of Suaeda moquinii under
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F-values from a two-way ANOVA describing how endogenous hormone (IAA and ZR), protein
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content and seed weight in S. salsa seeds are affected by (A) seed type (brown seeds vs. black
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seeds), (B) NaCl treatment during plant culture (1 vs. 500 mM), and their interactions.
Dependent variable 56.44*
IAA concentration
648.18***
GA concentration
4.07**
ABA concentration Protein content Seed weight
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52.77***
92.86***
71.05***
0.28NS
219.29***
17.27**
0.94NS
108.84***
1.05NS
0.34NS
975.89***
508.45***
22.57**
Significant differences at P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 are indicated; NS indicates no significant difference.
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0.01NS
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A×B
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F-values from a three-way ANOVA describing how final germination, total germination, and
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germination index in S. salsa seeds are affected by (A) seed type (brown seeds vs. black seeds),
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(B) NaCl treatment during plant culture (1 vs. 500 mM), (C) NaCl treatment during seeds
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germination (0 vs. 600 mM), and their interactions. B
C
A×B
Final germination
3.38NS
0.38NS
0.01NS
3.38NS
Total germination
1.81NS
0.52NS
0.23NS
Germination index
4.47*
0.37NS
0.51NS
B×C
A×B×C
0.1NS
3.93NS
8.32**
6.13*
1441NS
0.71NS
1.14NS
9.15**
1.34NS
6.08*
10.21**
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A×C
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Significant differences at *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 are indicated; NS indicates no significant
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difference.
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Fig. 1. Morphology of brown seeds of Suaeda salsa from plants cultured at 1 mM NaCl (A) and
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500 mM NaCl (B), and embryos of brown seeds at 1 mM NaCl (C) and 500 mM NaCl (D); black
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seeds at 1 mM NaCl (E) and 500 mM NaCl (F), and embryos of black seeds at 1 mM NaCl (G)
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and 500 mM NaCl (H). Bar in each graph indicates 250 µm.
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Fig. 2. The brown seeds/black seeds ratio (A), seed weight (B) and seed protein content (C) of S.
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salsa cultured in 1 or 500 mM NaCl during plant growth. Values are means + SD, n = 3. For each
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pair of bars, means with different letters are significantly different at P ≤ 0.05.
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Fig. 3. Germination index (A), final germination (B) and total germination (C) of brown and black
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seeds from plants cultured in 1 or 500 mM NaCl at 0 or 600 mM NaCl during germination. Values
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are means + SD, n = 3. For each pair of bars, means with different letters are significantly different
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at P ≤ 0.05.
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Fig. 4. Concentration of IAA (A), ZR (B), GA (C), and ABA (D) in brown and black seeds from
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plants cultured in 1 or 500 mM NaCl. Values are means + SD, n = 3. For each pair of bars, means
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with different letters are significantly different at P ≤ 0.05.
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Fig. 2.
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2.00 1.00 0.00 1 mM 500 mM NaCl during plant growth
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0.5 0.4
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Seed weight (g 1000 seed -1)
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Germination index
A
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0 mM NaCl 600 mM NaCl
a
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100
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ABA concentration (ng g-1 FW)
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ACCEPTED MANUSCRIPT Highlights: High salt during plant growth can improve the brown seeds/black seeds ratio and the weight of brown and black seeds in Suaeda salsa plants. Salinity during plant growth increased salt tolerance of brown seeds, but decreased salt tolerance of black seeds in Suaeda salsa.
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Endogenous hormone accumulation in dimorphic seeds of Suaeda salsa during plant growth may be related to seed development and to salt tolerance of dimorphic seeds.
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The work presented here was carried out in collaboration with all authors. Dr. Jie Song designed the experiments and revised the paper. Ms. Fengxia Wang performed the experiments, analyzed the data, and wrote the paper. Ms. Yange Xu and Mr. Shuai Wang assisted in analyzing the brown seeds/black seeds ratio, seed weight and endogenous hormone production. Ms.Weiwei Shi helped in measuring endogenous hormone concentrations. Ms. Ranran Liu helped finishing the determination of germination of brown and black seeds. Prof. Gu Feng assisted in writing the paper.