Cotyledon removal decreases salt tolerance during seedling establishment of Ricinus communis, an oilseed energy crop species

Industrial Crops & Products 142 (2019) 111857

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Cotyledon removal decreases salt tolerance during seedling establishment of Ricinus communis, an oilseed energy crop species

T

Yingnan Wanga,b, Yao Xua, Xiaoyuan Penga, Junxin Yana, Xiufeng Yana, Zhiqiang Zhoub, Jixiang Lina,⁎ a

College of Landscape Architecture, Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education, Northeast Forestry University. Harbin 150040, China b Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, 150040 China

A R T I C LE I N FO

A B S T R A C T

Keywords: Ricinus communis Salt tolerance Cotyledon removal Photosynthesis Leaf ultrastructure

Early seedling growth is the most sensitive and critical periods for most plant establishment in saline environments. Castor bean (Ricinus communis) is an important oilseed crop species worldwide and has good salt tolerance. However, the specific functions of cotyledons during early seedling establishment of this species under salt stress are still not clearly understood. Here, the biomass, gas exchange, chlorophyll fluorescence, leaf ultrastructure, inorganic ion and organic solute contents of Ricinus communis seedlings whose cotyledons were partially or completely removed were measured in both salt and non-salt treatments. The results indicated that cotyledon removal decreased both the growth and photosynthesis of the seedlings under salt stress. The ultrastructure of the chloroplasts was greatly altered and distorted in the leaves of plants whose cotyledons were removed. With increasing salinity, cotyledon removal dramatically increased the Na+ content and simultaneously reduced the K+ content in the castor bean leaves. In addition, cotyledon removal also increased the contents of malondialdehyde (MDA), proline and soluble sugars in the leaves of plants under salt stress. These results suggest that cotyledons can alleviate the reduction in photosynthetic capability caused by stomatal closure in the leaves of castor bean plants and can maintain the structural integrity of chloroplasts under salt stress. Moreover, cotyledons can also counteract Na+ toxicity and alleviate osmotic stress caused by salt stress. Together, these findings provide an improved understanding of cotyledon function in young castor bean seedlings under salt stress.

1. Introduction Soil salinization is considered one of the most severe environmental hazards to agricultural and grassland systems and inhibits plant growth and productivity worldwide (Dorra et al., 2019; Wang et al., 2019a). It is estimated that saline soils cover almost 10% of the total global land area (approximately one billion hectares), and this area continues to expand (El-Ramady et al., 2018). Soil salinity causes low water potential and specific ion toxicity effects on plants, including Na+ and Cl− toxicity. These adverse factors can affect cellular structure, seedling growth, and photosynthetic capability, which then reduce plant yields. In addition, if the salinity in the soil is beyond tolerance limits, plants

will progressively die (Niu et al., 2016). Early seedling growth is the most sensitive stage and is highly susceptible to adverse environmental conditions such as salt-alkali stress, drought stress, flooding stress and extreme temperature stress for most species (Zhou et al., 2014). Cotyledons are the first photosynthetic organs of plants to develop and play an important role in seedling establishment (Marshall and Kozlowski, 2010). Previous studies have reported that cotyledons contain a considerable amount of storage substances, including proteins, lipids, and polysaccharides, in some species such as Glycine max (Golombek et al., 2001), Helianthus annuus (Yadav and Bhatla, 2015) and Phaseolus vulgaris (Tan-Wilson and Wilson, 2012). These types of cotyledons can provide starch (for

Abbreviations: NR, removal of no cotyledons; OR, removal of one cotyledon; TR, removal of both cotyledons; DW, dry weight; FW, fresh weight; WC, Water content; PN, net photosynthetic rate; Ci, intercellular CO2 concentration; gs, stomatal conductance; Tr, transpiration rate; Chl a, chlorophyll a; Chl b, chlorophyll b; ɸPSⅡ, actual quantum yield of photochemical energy conversion in PSⅡ; qp, photochemical quenching coefficient; Fv/Fm, maximal quantum yield of PSⅡ photochemistry; MDA, malondialdehyde; ROS, reactive oxygen species ⁎ Corresponding author: No. 26 Hexing Road, Xiangfang District, Harbin, Heilongjiang Province, China. E-mail address: [email protected] (J. Lin). https://doi.org/10.1016/j.indcrop.2019.111857 Received 21 June 2019; Received in revised form 3 September 2019; Accepted 8 October 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Model of the cotyledon removal experiment.

2. Materials and methods

respiration), fatty acids (for energy) and amino acids (for de novo protein synthesis) to plants via mobilization of stored substances (Wang et al., 2019b). In addition, the cotyledons of other plant species (e.g., Acer negundo, Cucumis sativus and Ricinus communis) contain inadequate amounts of storage substances and mainly function to photosynthesize and provide photoassimilates for subsequent seedling growth (Zhang et al., 2008; Ampofo et al., 2010). Castor bean (Ricinus communis L.) is one of the most important oilseed crop species and belongs to the Euphorbiaceae family. It widely planted in many areas, such as Brazil, India and China (Ribeiro et al., 2014). The species has tremendous commercial value because the oil content of its seeds is between 40% and 60%; this oil can be used for biorefinery and other industrial applications. The unique ricinoleic acid in the oil of this species is different from that of oil produced by other oil crop species (Lima et al., 2011). In addition, castor bean is a fastgrowing species and is highly tolerant to salt; thus, this species can be used in the restoration of salt-degraded soils in Northeast China (Zhang et al., 2018). Castor bean plants have a pair of large cotyledons that can stay in seedlings for more than a month. These cotyledons vastly differ from those of other dicotyledonous plant species such as pea (Pisum sativum; Hanley et al., 2010) and soybean (Glycine max; Sebastián et al., 2009). In our previous studies, the cotyledons and leaves of castor bean seedlings have different physiological responses under salt stress and further proteomic analysis reflected that castor bean cotyledons accumulated a large number of Na+ and provided more energy to help leaves cope with salt stress (Wang et al., 2019a,b). However, the functions of cotyledons in adaptation to salt stress in young castor bean seedlings are still not clearly understood. A cotyledon excision test can be carried out to determine the effect of cotyledons on the seedling growth and physiological responses to salt stress by comparison the difference between no cotyledons removed seedling and cotyledons removed seedling. Studies showed that cotyledon removal significantly decreased the biomass and delayed the flowering of six grass species (Trifolium pratense, Leontodon hispidus, Trifolium repens, Plantago lanceolata, Leontodon autumnalis and Plantago major) during the early seedling stage, and the dry weights (DWs) in the removal treatment decreased by 60–75% in these species (Hanley and Fegan, 2007). However, the contributions of cotyledons to seedling growth and physiological responses under salt stress have rarely been explored. In the present study, we hypothesized that cotyledons play an important role in the salt tolerance of castor bean seedlings and that the growth and physiological responses of the leaves are affected by the removal of cotyledons. To test the hypotheses, the effects of salt stress on castor bean seedlings was measured whose cotyledons were partially or completely removed in terms of seedling growth, photosynthesis, chlorophyll fluorescence, leaf ultrastructure, osmotic adjustment and ion changes.

2.1. Experimental design This study was conducted in a completely randomized design pot experiment in a greenhouse (Northeast Forestry University, Heilongjiang Province, China, in 2018) and included two factors: cotyledon removal (removal of no cotyledons, NR; removal of one cotyledon, OR; removal of both cotyledons, TR) and salt stress (0 mM NaCl, CK; 40 mM NaCl, S1; 80 mM NaCl, S2; 120 mM NaCl, S3). Eight seedlings constituted one replicate per pot, and four replicates were applied per set. Surface-sterilized Ricinus communis seeds (Fen Bi 10, growing period: 96 d; hundred-grain weight: 35 g; seed length: 9–14 mm) were sown in plastic pots (15 cm diameter) filled with 2 kg of soil. After seedling emergence, all the pots were irrigated with a Hoagland nutrient solution once a day. Hoagland nutrition solution used in this research contained 5.00 mM Ca2+, 2.00 mM Mg2+, 6.04 mM K+, 22.20 μM EDTA-Fe2+, 6.72 μM Mn2+, 3.16 μM Cu2+, 0.77 μM Zn2+, 2.10 mM SO42−, 1.00 mM H2PO4−, 46.3 μM H3BO3, 0.56 μM H2MoO4, and 15.04 mM NO3− (Lin et al., 2017). With respect to the cotyledon removal treatment, the cotyledons were removed from 42 d-old seedlings as described in the experimental design; the model of the cotyledon removal treatment is shown in Fig. 1. With respect to the salt stress treatments, seedlings whose cotyledons were removed were irrigated with 300 mL of the corresponding NaCl solution for 7 d, after which the seedlings were harvested, and the fresh sample was frozen in liquid nitrogen and stored at −80℃. 2.2. Growth measurements The leaves of castor bean plants were separated immediately and individually weighed to determine their fresh weight (FW) per pot. The leaves were dried for 15 min at 105℃ in an oven and then dried at 65℃ until a constant DW was obtained. The water content (WC) was calculated according to the method described by Lin et al. (2016). 2.3. Leaf gas exchange parameter and chlorophyll fluorescence measurements The gas exchange parameters were measured via an LI-6400XT portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA) between 09:00 a.m. and 11:00 a.m. The parameters were measured under constant photosynthetically active radiation (PAR; 1000 μmol m−2·s-1), and the air temperature and ambient CO2 concentration were maintained at 25℃ and 420 μmol mol-1, respectively. Fresh leaf tissues (100 mg) were extracted with 7 mL dimethyl sulfoxide (DMSO) and incubated at 4℃ for 72 h. After incubation, the extract was diluted to 10 mL with DMSO. The absorbance of the extraction solution was measured at 649 and 665 nm by a UV-vis spectrophotometer. (BioMate 3S UV–visible, Thermo Scientific, USA). 2

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leaves were postfixed in 1% osmium tetroxide (OsO4) for 4 h. The leaves were then dehydrated in a graded acetone series (50%, 70%, 90%, and 100% for 15 min each) and saturated in an acetone/Epon 812 mixture [1/1 (v/v) for 30 min and then 1:2 (v/v) for 1 h] and subsequently in pure Epon 812 for 2 h. Ultrathin sections (60 nm) were cut with an LKB-V ultramicrotome (Bromma, Sweden) and then poststained with uranyl acetate and lead citrate. The leaves were examined via transmission electron microscopy (TEM; H-7650, Hitachi, Japan) at 100 kV.

Table 1 Two-way analysis of variance (ANOVA) of the effects of salinity (S), cotyledon removal (CR) and their interactions on the growth, photosynthesis, photosynthetic pigments, chlorophyll fluorescence, stomatal characteristics, Na+ and K+ contents, lipid peroxidation and osmolyte accumulation of Ricinus communis seedlings. Source of variation

Variable

−1

Dry weight (g·plant ) Water content (%) PN (μmol CO2 m−2·s−1) Ci (μmol CO2 mol−1) Gs (μmol H2O m−2·s−1) Tr (mmol H2O m−2·s−1) Chl a (mg·g−1) Chl b (mg·g−1) Fv/Fm ɸPSⅡ qp NPQ Stomatal length (μm) Stomatal width (μm) Stomatal aperture (μm) Stomatal density (mm−2) Na+ (μmol·g−1) K+ (μmol·g−1) K+/Na+ ratio MDA (mmol·g−1) Proline (mmol·g−1) Soluble sugar (mmol·g−1)

S

CR

S × CR

27.83*** 33.97*** 199.97*** 141.45*** 91.64*** 192.37*** 33.80*** 0.83NS 8.57*** 40.37*** 62.88*** 106.65*** 60.19*** 162.73*** 136.41*** 54.00*** 513.27*** 1.04NS 3635.69*** 50.65*** 28.62*** 58.92***

4.72** 27.84*** 14.02*** 4.93** 7.26* 10.39** 11.34*** 19.27*** 13.71*** 16.50*** 2.08NS 2.03NS 5.32* 6.07* 5.22NS 16.67** 61.10*** 14.04*** 8.66** 35.65*** 48.52*** 19.20***

0.47NS 0.37NS 9.67*** 6.86** 3.58** 8.32** 3.84*** 0.31NS 0.86NS 14.33*** 3.84** 8.82*** 9.95** 5.85* 7.21* 10.67** 17.62*** 4.03** 3.85** 4.38** 0.57NS 2.20NS

2.6. Na+ and K+ content measurements Na+ and K+ were extracted from leaf samples (30 mg DW) in 10 mL of deionized water at 100℃ for 1 h. The extractions were measured by atomic absorption spectrometry (TAS-990, Purkinje General, Beijing, China) at 589 nm and 766.5 nm, respectively. 2.7. MDA, proline and total soluble sugar content measurements Malondialdehyde (MDA) was extracted in fresh leaf using 5% trichloroacetic acid, and the absorbance was measured with 0.6% thiobarbituric acid at 450, 532 and 600 nm (Meng et al., 2016). Proline was extracted with 3% sulfosalicylic acid for 1 h, and the absorbance was measured with ninhydrin at 520 nm (Bates et al., 1973). The total soluble sugar content was extracted with absolute ethanol and freshly prepared anthrone reagent for 15 min, after which the absorbance was measured at 620 nm (Maswada and Abd El-Kader, 2016). 2.8. Data analysis

The data represent F-values at the 0.05 level. *, **, *** and NS indicate significance at P < 0.05, P < 0.01, P < 0.001, and P > 0.05, respectively. PN, net photosynthetic rate; Ci, intercellular CO2 concentration; gs, stomatal conductance; Tr, transpiration rate; Chl a, chlorophyll a; Chl b, chlorophyll b; ɸPSⅡ, actual quantum yield of photochemical energy conversion in PSⅡ; qp, photochemical quenching coefficient; Fv/Fm, maximal quantum yield of PSⅡ photochemistry; MDA, malondialdehyde;

The data were analyzed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Two-way analysis of variance (ANOVA) was used to test the effects of cotyledon removal, salinity and their interactions on physiological changes in the leaves of the castor bean plants. Tukey’s test was applied to determine significant differences comparison of means (P < 0.05).

Chlorophyll (Chl) a and b were calculated according to the described by Alan (1994). The chlorophyll fluorescence of detached whole leaves was imaged with a FluorCam FC 800-O (Photon System Instruments, Czech Republic) in each treatment. The FluorCam program was subsequently used to analyze the fluorescence images to determine the fluorescence parameters.

3. Results 3.1. Growth Two-way ANOVA tests indicated that the DW and WC of castor bean seedlings were affected by salt concentration and cotyledon removal but were not affected by the interaction of these two factors (Table 1). The DW of the NR, OR and TR seedlings significantly decreased with increasing salinity (P < 0.05). For example, compared with the control seedlings treatment, the DW of the NR, OR and TR seedlings decreased by 23.39%, 24.87% and 34.08% at the highest salinity (120 mM), respectively (Fig. 2A). A similar trend was also found for the WC under salt stress (Fig. 2B). Moreover, the WC and DW of the TR seedlings were much lower than those of the NR seedlings at each salt concentration (40, 80 and 120 mM). In details, the water content of the TR seedlings were decreased 1.71%, 2.54% and 1.85% compared with the NR seedlings, the dry weight of the TR seedlings were decreased 11.97%, 14.00% and 14.88% compared with the NR seedlings under 40, 80 and 120 mM NaCl treatment, respectively.

2.4. Leaf ultrastructural observations The stomatal structure of the leaves in the control and 120 mM NaCl treatments were observed with scanning electron microscopy (SEM; Hitachi S-3400 N, Japan). Fresh samples were chopped into pieces (5 mm × 3 mm) in fixative solution (2.5% glutaric dialdehyde, pH 6.8) at 4℃ for 4 h under darkness and then washed twice for 10 min with phosphate buffer (pH 6.8) to remove any excess fixative solution. After the samples were washed, an ethanol gradient was applied to dehydrate the samples in a step-by-step process for 15 min each time [50% →70% →90% →100% (twice)], after which the samples were immersed in a 100% ethanol/tertiary butyl alcohol (1/1) solution for 15 min, followed immersion in tertiary butyl alcohol for 15 min. The samples were then freeze-dried (Hitachi WS-2030, Japan) for 4 h. After gold sputtering was applied to the samples, the samples were imaged by SEM.

3.2. Photosynthesis and photosynthetic pigments Two-way ANOVA tests indicated that the net photosynthetic rate (PN), intercellular CO2 concentration (Ci), stomatal conductance (gs) and transpiration rate (Tr) of castor bean seedlings were affected by salinity, cotyledon removal and their interactions (Table 1). Cotyledon removal increased the PN, Ci, gs and Tr of castor bean seedlings under the control conditions, and the increases were much greater in the TR seedlings than in the other seedlings (Fig. 3 A–D). For example,

2.5. Chloroplast ultrastructural observations The control and 120 mM NaCl treatments fresh leaf samples (3 mm × 1 mm) without veins were collected randomly and fixed in 2.5% glutaraldehyde (v/v) for 2 h at 4℃. After the samples were rinsed with 0.1 M phosphate buffer (pH 6.8, 3 times for 15 min each), the 3

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Fig. 2. Dry weight (DW) (A) and water content (WC) (B) of Ricinus communis whose cotyledons were not removed (NR), seedlings from which one cotyledon was removed (OR) and seedlings from which both cotyledons were removed (TR) under salt stress. The letters denote significant differences among the different degrees of cotyledon removal from seedlings under the same salinity. The differences in each parameter were tested by one-way analysis of variance (ANOVA) at the P < 0.05 level. The bars represent the means ± SEs (n = 4).

seedlings compared with the NR seedlings under salt stress. For example, the ɸPSⅡ values were 0.57 and 0.52 for the NR and TR seedlings, respectively, at 120 mM salinity (Fig. 4 B). However, cotyledon removal significantly increased the ɸPSⅡ in the control group. Compared with that of the NR seedlings, the ɸPSⅡ of OR and TR seedlings increased by 3.5% and 6.4%, respectively. The qp in the NR, OR and TR seedlings significantly decreased with increasing salinity (P < 0.05, Fig. 4 C). Cotyledon removal significantly decreased the qp in the TR seedlings compared with the NR seedlings under salt stress (40 and 80 mM NaCl). In addition, cotyledon removal significantly increased the qp in the control group. increased by 8.45%. The NPQ values of the NR, OR and TR seedlings significantly increased with increasing For instance, compared with that of the NR seedlings, the qp of the TR seedlings salinity (P < 0.05, Fig. 4 D). Cotyledon removal significantly increased the NPQ under salt stress (40 and 80 mM NaCl) and significantly decreased the NPQ in the control group. For instance, compared with that of the NR seedlings at the highest salinity (120 mM), the NPQ of the TR seedlings increased by 16.58%.

compared with that of the NR seedlings under the control conditions, the PN of the OR and TR seedlings increased by 6.83% and 13.87%. In addition, the PN, Ci, gs and Tr of the NR, OR and TR seedlings significantly decreased with increasing salinity (P < 0.05), and the PN, Ci, gs and Tr of the TR seedlings were much lower than those of the NR seedlings under salt stress (40, 80 and 120 mM NaCl). The PN value of the NR seedlings was 10.13 μmol CO2 m−2·s−1, but the PN value was only 4.42 μmol CO2 m−2·s−1 for the TR seedlings at the highest salinity (120 mM). Two-way ANOVA tests indicated that the Chl a content in castor bean seedlings was affected by salinity, cotyledon removal and their interactions and that the Chl b content was affected by salinity and cotyledon removal (Table 1). The Chl a content in the NR, OR and TR seedlings decreased with increasing salinity (P < 0.05, Fig. 3 E). The Chl a contents in the NR, OR and TR seedlings were much greater under salt stress than under the control conditions. In addition, the Chl a content in the TR seedlings was much lower than that in the NR seedlings under salt stress (P < 0.05). For example, the Chl a content in the NR seedlings was 4.35 mg g−1 but was only 3.84 mg g−1 in the TR seedlings at the highest salinity (120 mM, Fig. 3E). Unlike the Chl a content, the Chl b content in all the seedlings remained unchanged as the salinity increased (Fig. 3F). Moreover, cotyledon removal decreased the Chl b content, which was much lower in the TR seedlings than in the other seedlings under salt stress. For example, compared with that in the NR seedlings at 120 mM salinity, the Chl b content in TR seedlings decreased by 31.68% (Fig. 2 F).

3.4. Stomatal characteristics and chloroplast ultrastructural analysis Two-way ANOVA tests indicated that, with the exception of the stomatal aperture in the cotyledon removal treatment, the stomatal length, width, aperture and density in castor bean seedlings were affected by salinity, cotyledon removal and their interactions (Table 1). On the basis of the analyses of photosynthesis and chlorophyll fluorescence, the differences between control and 120 mM NaCl treatment were very obvious in NR and TR seedlings. Hence, the stomata of the NR and TR seedlings were observed by SEM in the highest salinity (120 mM) and control groups. The results showed that the stomatal length, width, aperture and density did not differ between the TR and NR seedlings in the control group (Table 2, Fig. 5). However, compared with those of the NR seedlings, the stomatal length, width and aperture of the TR seedlings decreased significantly at 120 mM salinity. In addition, the stomatal density of the TR seedlings increased at 120 mM salinity. The chloroplast ultrastructure showed no obvious differences between the NR and TR seedlings in the control group. The chloroplast envelope was well defined, and the shape was the original shuttle-type shape in the NR and TR seedlings. In addition, the chloroplasts had well-developed grana, stroma thylakoids and a few small plastoglobules

3.3. Chlorophyll fluorescence Two-way ANOVA tests indicated that the actual quantum yield of photochemical energy conversion in PSⅡ (ɸPSⅡ), the photochemical quenching coefficient (qp) and the nonphotochemical quenching coefficient (NPQ) of the NR, OR and TR seedlings of castor bean were affected by the interactions of salinity and cotyledon removal (Table 1). The maximal quantum yield of PSⅡ photochemistry (Fv/Fm) of all seedlings significantly decreased with increasing salinity (P < 0.05, Fig. 4 A). Cotyledon removal significantly decreased the Fv/Fm of the TR seedlings compared with the NR seedlings under salt stress (40, 80 and 120 mM NaCl). The ɸPSⅡ of both the OR and TR seedlings decreased significantly with increasing salinity, but the ɸPSⅡ of the NR seedlings increased first but then decreased. The highest value of ɸPSⅡ (0.61) of the NR seedlings occurred at 40 mM salinity (Fig. 4 B). In addition, cotyledon removal significantly decreased the ɸPSⅡ in the TR 4

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Fig. 3. Net photosynthetic rate (PN; A), intercellular CO2 concentration (Ci; B), stomatal conductance (gs; C), transpiration rate (Tr; D), chlorophyll a (Chl a) content (E) and chlorophyll b (Chl b) content (E) of Ricinus communis seedlings whose cotyledons were not removed (NR), seedlings from which one cotyledon was removed (OR) and seedlings from which both cotyledons were removed (TR) under salt stress. The letters denote significant differences among the different degrees of cotyledon removal from seedlings under the same salinity. The differences in each parameter were tested by one-way analysis of variance (ANOVA) at the P < 0.05 level. The bars represent the means ± SEs (n = 4).

in the castor bean seedlings. In addition, the K+ content was not affected by cotyledon removal (Table 1). The Na+ contents in the NR, OR and TR seedlings significantly increased with increasing salinity (P < 0.05, Fig. 7 A). Cotyledon removal significantly increased the Na+ content in the OR and TR seedlings compared with the NR seedlings at each salt concentration. For instance, the Na+ content in the OR and TR seedlings was 62.99 μmol g−1 and 85.85 μmol g−1, respectively, but only 46.98 μmol g−1 in the NR seedlings at 120 mM salinity. The K+ content in the NR and OR seedlings remained unchanged with increasing salinity (Fig. 7 B). However, the K+ content in the TR seedlings decreased significantly (P < 0.05). Cotyledon removal significantly decreased the K+ content in the TR seedlings compared with the NR seedlings under salt stress.

(Fig. 6 A and B). However, observable alterations were visible in the chloroplasts of the NR and TR seedlings under 120 mM salinity. The chloroplasts were expanded, spindle-shaped or oblong round instead of the original shuttle-type shape and presented increased numbers of plastoglobules in the NR seedlings at 120 mM salinity. Additionally, chloroplasts accumulated starch grains and were almost completely full (Fig. 6 C). The chloroplast envelopes were relatively more swelled with a destroyed thylakoid system in the TR seedlings compared with the other seedlings under 120 mM salinity (Fig. 6 D).

3.5. Na+ and K+ contents Two-way ANOVA tests indicated that the Na+ content and K+/ Na+ ratio were affected by salinity, cotyledon removal and their interactions 5

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Fig. 4. Maximal quantum yield of PSⅡ photochemistry (Fv/Fm; A), actual quantum yield of photochemical energy conversion in PSⅡ (ɸPSⅡ; B), photochemical quenching coefficient (qp; C) and nonphotochemical quenching coefficient (NPQ; D) of Ricinus communis whose cotyledons were not removed (NR), seedlings from which one cotyledon was removed (OR) and seedlings from which both cotyledons were removed (TR) under salt stress. The letters denote significant differences among the different degrees of cotyledon removal from seedlings under the same salinity. The differences in each parameter were tested by one-way analysis of variance (ANOVA) at the P < 0.05 level. The bars represent the means ± SEs (n = 4).

The K+/Na+ ratio in the NR, OR and TR seedlings significantly decreased with increasing salinity (P < 0.05, Fig. 7 C). Cotyledon removal significantly increased the K+/Na+ ratio in the TR seedlings compared with the NR seedlings under salt stress. For example, compared with that in the NR seedlings at the highest salinity (120 mM), the K+/Na+ ratio in the TR seedlings decreased by 58.62%.

peroxidation levels in the NR, OR and TR seedlings under salt stress. The MDA contents in the NR, OR and TR seedlings significantly increased with increasing salinity (P < 0.05, Fig. 8 A). The MDA contents in the TR seedlings were much greater in the NR seedlings than in the other seedlings under salt stress (40, 80 and 120 mM NaCl). For example, the MDA content in the TR seedlings was 36.19 mmol·g−1, but in the NR seedlings, it was 27.29 mmol·g−1 at the highest salinity (120 mM). The soluble sugar and proline contents in the NR, OR and TR seedlings significantly increased with increasing salinity (P < 0.05, Fig. 8 B and C). Cotyledon removal significantly increased the total soluble sugar and proline contents in the TR seedlings compared with the NR seedlings under salt stress. In addition, compared with no cotyledon removal, cotyledon removal significantly increased the proline

3.6. Lipid peroxidation and osmolyte accumulation Two-way ANOVA tests indicated that MDA, soluble sugars and proline were affected by salinity concentration and cotyledon removal in the castor bean seedlings. Moreover, the interactions of these two factors were significant only for MDA (Table 1). Malondialdehyde contents were measured to evaluate lipid

Table 2 Stomatal length, width, aperture and density of Ricinus communis seedlings whose cotyledons were not removed (NR), seedlings from which one cotyledon was removed (OR) and seedlings from which both cotyledons were removed (TR) under salt stress. The data are presented as means ± SEs. 0 mM NaCl

Stomatal Stomatal Stomatal Stomatal

length (μm) width (μm) aperture(μm) density (mm−2)

120 mM NaCl

Removal of no cotyledons

Removal of both cotyledons

Removal of no cotyledons

Removal of both cotyledons

19.36 ± 0.81 a 9.41 ± 0.04 a 3.88 ± 0.18 a 176.72 ± 7.68 a

19.78 ± 0.43 a 9.39 ± 0.67 a 3.95 ± 0.23 a 184.40 ± 13.31 a

17.08 ± 0.38 a 5.59 ± 0.07 a 2.23 ± 0.21 a 215.13 ± 10.16 a

14.37 ± 0.03 b 3.79 ± 0.31 b 1.32 ± 0.07 b 284.28 ± 3.84 b

One-way analysis of variance (ANOVA) was used to test the differences between NR seedlings and TR seedlings in the control and highest salinity treatments. The differences were considered significant when P < 0.05, as indicated by the different letters. 6

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Fig. 5. The stomata of adaxial leaf surfaces obtained using scanning electron microscopy (SEM) of Ricinus communis seedlings whose cotyledons were not removed (A), seedlings from which both cotyledons were removed (B), seedlings whose cotyledons were not removed at 120 mM NaCl (C), and seedlings from which both cotyledons were removed at 120 mM NaCl (D).

However, this compensation cannot completely fill the vacancy of that produced by the cotyledons. This notion can explain why the DW of the TR seedlings was lower than that of the NR seedlings. Moreover, photosynthesis is an extremely sensitive biological process influenced by salinity, and a reduction in photosynthesis is always a response to the osmotic shock of salt stress because of stomatal closure (Ashraf and Harris, 2013). Jiang et al. (2017) reported that reductions in the PN, gs and Tr were accompanied by a decrease in Ci under salt stress, indicating the effects of stomatal limitation on the photosynthesis of tomato (Solanum lycopersicum) seedlings. In the present study, the PN, Ci, gs and Tr values of the NR, OR and TR seedlings decreased significantly with increasing salinity (Fig. 3). Therefore, the PN reduction in castor bean seedlings was largely due to stomatal factors. In addition, the PN, Ci, gs and Tr of the NR seedlings were greater than those of the TR seedlings under salt stress, indicating that cotyledons could alleviate the reduction in the photosynthetic capability of castor bean leaves caused by stomatal closure under salt stress. The results of the analysis of stomatal characteristics in this study also confirm this conclusion. Compared with those of the TR seedlings, the leaf stomata of the NR seedlings were longer, wider and had a greater aperture under salt stress (Table 2, Fig. 5 C and D). Chlorophyll a and chlorophyll b are essential components of the plant photosynthetic apparatus (Stefanov et al., 2018). The roles in light harvesting, stabilization of thylakoid membranes and energy transduction have been well established (Syed et al., 2017). In this study, cotyledon removal increased the Chl a content in castor bean seedlings under non-saline conditions, indicating that cotyledon removal improved light harvesting and energy transduction in the photosynthetic apparatus of the leaves, which may explain how castor bean leaves improve the photosynthetic capability to compensate for the

content in the control group. 4. Discussion Early seedling growth is the most sensitive period and is highly susceptible to salt stress for most plant species (Zhou et al., 2014). Our previous works have revealed different response mechanisms between cotyledons and leaves of castor bean plants under salt stress (Wang et al., 2019a,b). However, the specific functions of the cotyledons of this species during seedling establishment under salt stress are still not clearly understood. Thus, in the present study, a cotyledon excision test was carried out, and different responses of NR, OR and TR seedlings to salt stress were explored. The results clearly showed that, compared with that of the NR seedlings, the DW of the TR seedlings significantly decreased in response to cotyledon removal under salt stress at each concentration, indicating that cotyledons play a positive role in castor bean seedlings under salt stress (Fig. 2A). In addition, the WC of the TR seedlings was significantly lower than that of the NR seedlings under salt stress (Fig. 2B). In general, water loss is a typical response of plants to osmotic stress. The relatively low WC in the TR seedlings indicated that the effects of osmotic stress caused by salinity were much stronger on the TR seedlings than on the other seedlings, suggesting that cotyledons can alleviate stress effects in castor bean seedlings under salt stress. As photosynthetic organs, cotyledons can provide energy and assimilates for castor bean seedling growth during the seedling establishment stage. In this study, cotyledon removal increased the PN, Ci, gs and Tr of seedlings under non-stress conditions (Fig. 3), indicating that castor bean leaves could improve the photosynthetic capability to compensate for the photoassimilate loss caused by cotyledon removal. 7

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Fig. 6. Micrographs of the ultrastructure of leaf mesophyll cells obtained via transmission electron microscopy (TEM) of Ricinus communis seedlings whose cotyledons were not removed (A), seedlings from which both cotyledons were removed (B), seedlings whose cotyledons were not removed at 120 mM NaCl (C), and seedlings from which both cotyledons were removed at 120 mM NaCl (D). CW: cell wall, Chl: chloroplast, Mit: mitochondrion, PG: plastoglobule, SG: starch grain, SL: stroma lamella.

results showed that the photochemical activities of castor bean seedlings were significantly limited under salt stress and that the cotyledons could increase the activities of the castor bean leaves under salt treatment to some extent (Fig. 4). Song et al. (2011) reported that the reduction in Fv/Fm always suggested damage to PSⅡ in plants. The reductions in Fv/Fm combined with ΦPSⅡ and the PN in the leaves of castor bean under salt stress showed that the photosynthetic apparatus was damaged and that the photon use decreased. Similar results were also found in Populus euphratica by Li et al. (2013). The relatively high Fv/Fm and ΦPSⅡ values of the NR seedlings implied that the cotyledons could alleviate the damaging effects of the photosynthetic apparatus caused by salinity. The results of the chloroplast ultrastructural analysis also supported this viewpoint. From Fig. 6 C and D, the ultrastructure of the chloroplasts was greatly altered and distorted in the TR seedlings under salt stress. The accumulation of starch, an osmoticum, plays an important role in maintaining the structural integrity of chloroplasts (Goussi et al., 2018). In the NR seedlings, the chloroplasts were expanded, spindle-shaped or oblong round instead of the original shuttletype shape, but the NR seedlings also accumulated starch grains in chloroplasts to cope with salt stress. In addition, swelled and destroyed thylakoid systems in chloroplasts were found in the TR seedlings, indicating that cotyledons can maintain the structural integrity of chloroplasts under salt stress.

photoassimilate loss caused by cotyledon removal. In addition, the chlorophyll fluorescence results could also clarify why the photosynthetic capability improved in the NR seedlings under non-saline conditions. The ΦPSⅡ value reflects the utilization of photons absorbed by PSⅡ antennae, and qp reflects the share of light energy absorbed by PSⅡ antennae used for photochemical electron transfer (Ding et al., 2016; Zai et al., 2012). In the present study, cotyledon removal significantly increased the ΦPSⅡ and qp in the TR seedlings compared with the NR seedlings in the absence of salt stress, indicating that leaves of castor bean could improve photochemical electron transfer activity and the use of photons in PSⅡ to compensate for the photoassimilate loss caused by cotyledon removal. Moreover, salt stress can increase the activity of the chlorophyll-degrading enzyme chlorophyllase, resulting in a reduction in the amount of photosynthetic pigments (Liu and Shi, 2010). The amount of chlorophyll was greater in the NR seedlings than in the TR seedlings under salt stress, indicating that the cotyledons could inhibit the activity of chlorophyllase in the leaves of castor bean. This result was consistent with the conclusion that cotyledons play a positive role in the response to salt stress during the early seedling stage. It is well known that nonstomatal factors also affect photosynthesis under salt stress, resulting from impairment of photochemical activities (Hanachi et al., 2014; Souza et al., 2004). The chlorophyll fluorescence 8

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Fig. 7. Na+ content (A), K+ content (B) and the K+/Na+ ratio (C) in Ricinus communis seedlings whose cotyledons were not removed (NR), seedlings from which one cotyledon was removed (OR) and seedlings from which both cotyledons were removed (TR) under salt stress. The letters denote significant differences among the different degrees of cotyledon removal from seedlings under the same salinity. The differences in each parameter were tested by oneway analysis of variance (ANOVA) at the P < 0.05 level. The bars represent the means ± SEs (n = 4).

Fig. 8. Malondialdehyde (MDA) content (A), proline content (B) and soluble sugar content (C) in Ricinus communis seedlings whose cotyledons were not removed (NR), seedlings from which one cotyledon was removed (OR) and seedlings from which both cotyledons were removed (TR) under salt stress. The letters denote significant differences among the different degrees of cotyledon removal from seedlings under the same salinity. The differences in each parameter were tested by one-way analysis of variance (ANOVA) at the P < 0.05 level. The bars represent the means ± SEs (n = 4).

The use of light energy in the leaves of castor bean decreased, and a portion of the light energy was dissipated in the form of heat, which was absorbed by PSⅡ antenna pigments and thus could not be used for photosynthetic electron transport. This phenomenon can be reflected by the decreased qp and increased NPQ values in this research. Previous studies have reported that the NPQ pathway cannot completely dissipate excess energy under salt stress and that the PSⅠ reaction center was completely reduced to produce triplet P700, which can easily react with O2 to generate reactive oxygen species (ROS; Qin et al., 2011; Wang et al., 2015a,b). In general, the accumulation of lipid peroxidation is indicative of increased production of ROS, which can be estimated by the production of MDA (Soares et al., 2016). The

improvement of lipid peroxidation (increased MDA content) in the leaves of castor bean under salt stress may be related to the accumulation of ROS induced by severe photoinhibition of PSⅡ. In addition, the relatively low NPQ value and MDA content combined with a relatively high qp value for the NR seedlings indicated that cotyledons could improve the use of light energy and ultimately reduce the generation of ROS in the leaves of castor bean under salt stress. Most plants accumulate abundant amounts of Na+ and inhibit K+ absorption in saline environments, which induces physiological disorders such as leaf cell dehydration (Flowers et al., 1991), stomatal closure (Niu et al., 2018) and photosynthesis inhibition (Li et al., 2010; Afaq et al., 2011). Here, salinity induced the accumulation of Na+ and 9

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Fig. 9. Diagram of the central roles of cotyledons in the salt tolerance of Ricinus communis seedlings. The polar plots show the effects of cotyledon removal on the parameters of the controls and under different salinity concentrations (40, 80 and 120 mM NaCl). All the data were normalized to those of the reference treatment (removal of no cotyledons), and each reference variable was standardized by receiving a numerical value of 1.

the reduction in K+ in the leaves of castor bean together with growth inhibition, stomatal closure, chloroplast damage and reduced photosynthesis. In our previous study, the accumulation of Na+ in castor bean leaves was lower than that in the cotyledons, and we speculated that cotyledons had the ability to compartmentalize Na+ and reduce Na+ levels in the leaves of plants under salt stress. Similarly, in the present study, compared with the OR and TR seedlings, the NR seedlings had the lowest Na+ content and the highest K+/Na+ ratio and K+ content, further indicating that cotyledons can counteract toxic Na+ and protect leaves under stress conditions. A previous study reported that plants could accumulate proline and soluble sugars as osmotic regulators in the protoplasm under salt stress to maintain osmotic balance (Lin et al., 2014). In the present study, the proline and soluble sugar contents in the NR, OR and TR seedlings increased significantly with increasing salinity (Fig. 8 B, C). Kianipouya et al. (2017) reported that the synthesis of osmotic regulators for resisting stress might consume carbohydrates such as glucose and fructose-6-phosphate, both of which are also needed for seedling growth. Compared with the other seedlings, the NR seedlings had lower proline and soluble sugar contents under salt stress, suggesting that cotyledons could alleviate osmotic stress caused by salinity and conserve carbon structures for leaf growth. This phenomenon is another reason why cotyledon removal significantly decreased the DW of the TR seedlings

compared with the NR seedlings in the present study. 5. Conclusion This paper first reported the effects of cotyledon removal on the salt tolerance of Ricinus communis during seedling establishment and revealed the specific functions of cotyledons during this specific period. Cotyledons alleviated the reduction in the photosynthetic capability of castor bean leaves caused by stomatal closure and maintained the structural integrity of chloroplasts under salt stress. In addition, the cotyledons could also counteract toxic Na+, alleviate osmotic stress caused by salinity and increase leaf growth (Fig. 9). These findings provide an improved understanding of cotyledon function in young Ricinus communis seedlings under salt stress. Authors’ contributions Yingnan Wang wrote the manuscript and performed the experiments. Yao Xu and Xiaoyuan Peng performed part of the experiments. Junxin Yan analyzed the data. Xiufeng Yan revised the grammar. Zhiqiang Zhou gave some good suggestions to revise the manuscript. Jixiang Lin conceived and directed the study. All authors contributed to manuscript revision and gave final approval for publication. 10

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Declaration of interest

stress. Photosynthetica. 48 (1), 127–134. https://doi.org/10.1007/s11099-0100017-4. Marshall, P.E., Kozlowski, T.T., 2010. Importance of photosynthetic cotyledons for early growth of woody angiosperms. Physiol. Plant. 37 (4), 336–340. https://doi.org/10. 1111/j.1399-3054.1976.tb03981.x. Maswada, H.F., Abd El-Kader, N.I.K., 2016. Redox halopriming: a promising strategy for inducing salt tolerance in bread wheat. J. Agron. Crop Sci. 202 (1), 37–50. https:// doi.org/10.1111/jac.12123. Meng, X.J., Zhao, Q., Jin, Y.D., Yu, J.J., Yin, Z.P., Chen, S.X., Dai, S.J., 2016. Chillingresponsive mechanisms in halophyte Puccinellia tenuiflora seedlings revealed from proteomics analysis. J. Proteomics 143, 365–381. https://doi.org/10.1016/j.jprot. 2016.04.038. Niu, M.L., Huang, Y., Sun, S.T., Sun, J.Y., Cao, H.S., Shabala, S., Bie, Z.L., 2018. Root respiratory burst oxidase homologue-dependent H2O2 production confers salt tolerance on a grafted cucumber by controlling Na+ exclusion and stomatal closure. J. Exp. Bot. 69 (14), 3465–3476. https://doi.org/10.1093/jxb/erx386. Niu, X.P., Xu, J.T., Chen, T., Tao, A.F., Qi, J.M., 2016. Proteomic changes in kenaf (Hibiscus cannabinus L.) leaves under salt stress. Ind. Crops Prod. 9, 255–263. https:// doi.org/10.1016/j.indcrop.2016.07.034. Qin, L.Q., Li, L., Bi, C., Zhang, Y.L., Wan, S.B., Meng, J.J., 2011. Damaging mechanisms of chilling and salt stress Toarachis hypogaeal leaves. Photosynthetica. 49 (1), 37–42. https://doi.org/10.1007/s11099-011-0005-3. Ribeiro, P.R., Fernandez, L.G., Castro, R.D., Ligterink, W., Hilhorst, H.W., 2014. Physiological and biochemical responses of Ricinus communis seedlings to different temperatures: a metabolomics approach. BMC Plant Biol. 14 (1), 223–237. https:// doi.org/10.1186/s12870-014-0223-5. Sebastián, J., Galatro, A., Villordo, J.J., Puntarulo, S., Simontacchi, M., 2009. Role of nitric oxide in soybean cotyledon senescence. Plant Sci. 176 (5), 662–668. https:// doi.org/10.1016/j.plantsci.2009.02.007. Soares, C., Sousa, A.D., Pinto, A., Azenha, M., Teixeira, J., Azevedo, R.A., 2016. Effect of 2,4-epibrassinolide on ROS content, antioxidant system, lipid peroxidation and Ni uptake in Solanum nigrum L. Under Ni stress. Environ. Exp. Bot. 122 (5), 115–125. https://doi.org/10.1016/j.envexpbot.2015.09.010. Song, X.S., Shang, Z.W., Yin, Z.P., Ren, J., Sun, M.C., Ma, X.L., 2011. Mechanism of xanthophyll-cycle-mediated photoprotection in Cerasus humilis seedlings under water stress and subsequent recovery. Photosynthetica. 49 (4), 523–530. https://doi.org/ 10.1007/s11099-011-0065-4. Souza, R.P., Machado, E.C., Silva, J.A.B., Lagôa, A.M.M.A., Silveira, J.A.G., 2004. Photosynthetic gas exchange, chlorophyll fluorescence and some associated metabolic changes in cowpea (Vigna unguiculata) during water stress and recovery. Environ. Exp. Bot. 51 (1), 45–56. https://doi.org/10.1016/s0098-8472(03)00059-5. Stefanov, M., Yotsova, E., Markovska, Y., Apostolova, E.L., 2018. Effect of high light intensity on the photosynthetic apparatus of two hybrid lines of Paulownia grown on soils with different salinity. Photosynthetica. 56 (3), 832–840. https://doi.org/10. 1007/s11099-017-0735-y. Syed, H.S., Rasmus, H., Matthew, F.M., 2017. Response of chlorophyll, carotenoid and SPAD-502 measurement to salinity and nutrient stress in wheat (Triticum aestivum L.). Agron. J. 7 (3), 61–82. https://doi.org/10.3390/agronomy7030061. Tan-Wilson, A.L., Wilson, K.A., 2012. Mobilization of seed protein reserves. Physiol. Plant. 145, 140–153. https://doi.org/10.1111/j.1399-3054.2011.01535.x. Wang, L., Pan, D., Li, J., Tan, F., Hoffmann-Benning, S., Liang, W., 2015a. Proteomic analysis of changes in the Kandelia Candel chloroplast proteins reveals pathways associated with salt tolerance. Plant Sci. 231, 159–172. https://doi.org/10.1016/j. plantsci.2014.11.013. Wang, X.P., Jiang, P., Ma, Y., Geng, S.J., Wang, S.C., Shi, D.C., 2015b. Physiological strategies of sunflower exposed to salt or alkali stresses: restriction of ion transport in the cotyledon node zone and solute accumulation. Agron. J. 107, 2181–2192. https://doi.org/10.2134/agronj15.0012. Wang, Y.N., Jie, W.G., Peng, X.Y., Hua, X.Y., Yan, X.F., Zhou, Z.Q., Lin, J.X., 2019b. Physiological adaptive strategies of oil seed crop Ricinus communis early seedlings (cotyledon vs. True leaf) under salt and alkali stresses: from the growth, photosynthesis and chlorophyll fluorescence. Front. Plant Sci. 9, 1–15. https://doi.org/10. 3389/fpls.2018.01939. Wang, Y.N., Peng, X.Y., Fernanda, S., Wang, Y.C., Yan, X.F., Zhou, Z.C., Lin, J.X., 2019a. Salt-adaptive strategies in oil seed crop Ricinus communis early seedlings (cotyledon vs. True leaf) revealed from proteomics analysis. Ecotoxicol. Environ. Saf. 171 (15), 12–25. https://doi.org/10.1016/j.ecoenv.2018.12.046. Yadav, M., Bhatla, S.C., 2015. Photomodulation of fatty acid composition and lipoxygenase activity in sunflower seedling cotyledons. Acta Physiol. Plant. 37, 1–13. https://doi.org/10.1007/s11738-015-1891-y. Zai, X.M., Zhu, S.N., Qin, P., Wang, X.Y., Che, L., Luo, F.X., 2012. Effect of Glomus mosseae on chlorophyll content, chlorophyll fluorescence parameters, and chloroplast ultrastructure of beach plum (Prunus maritima) under NaCl stress. Photosynthetica. 50 (3), 323–328. https://doi.org/10.1007/s11099-012-0035-5. Zhang, H.Y., Wu, Y., Matthew, C., Zhou, D.Y., Wang, P., 2008. Contribution of cotyledons to seedling dry weight and development in Medicago dalcata L. N. Z. J. Agric. Res. 51 (2), 107–114. https://doi.org/10.1080/00288230809510440. Zhang, T., Hu, Y.J., Zhang, K., Tian, C.Y., Guo, J.X., 2018. Arbuscular mycorrhizal fungi improve plant growth of Ricinus communis by altering photosynthetic properties and increasing pigments under drought and salt stress. Ind. Crops Prod. 117, 13–19. https://doi.org/10.1016/j.indcrop.2018.02.087. Zhou, J.C., Zhao, W.W., Yin, C.H., Song, J., Wang, B.S., Fan, J.L., 2014. The role of cotyledons in the establishment of Suaeda physophora seedlings. Plant Biosyst. 148 (4), 584–590. https://doi.org/10.1080/11263504.2013.788574.

The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities (2572019AA16 and 2572018BS02) and the National Natural Science Foundation of China (31800546). References Afaq, M., Ronald, O., Isayenkov, S., Sentenac, H., Maathuis, F.J.M., 2011. Over-expression of an Na+- and K+-permeable HKT transporter in barley improves salt tolerance. Plant J. 68 (3), 468–479. https://doi.org/10.1111/j.1365-313X.2011.04701.x. Alan, R.W., 1994. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144 (3), 307–313. https://doi.org/10.1016/S0176-1617(11) 81192-2. Ampofo, S.T., Moore, K.G., Lovell, P.H., 2010. Cotyledon photosynthesis during seedling development in Acer. New Phytol. 76 (1), 41–52. https://doi.org/10.1111/j.14698137.1976.tb01436.x. Ashraf, M., Harris, P., 2013. Photosynthesis under stressful environments: an overview. Photosynthetica. 51, 163–190. https://doi.org/10.1007/s11099-013-0021-6. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. https://doi.org/10.1007/BF00018060. Ding, X.T., Jiang, Y.P., Hao, T., Jin, H.J., Zhang, H.M., He, L.Z., Zhou, Q., Huang, D.F., Hui, D.F., Yu, J.Z., 2016. Effects of heat shock on photosynthetic properties, antioxidant enzyme activity, and Downy mildew of cucumber (Cucumis sativus L.). PLoS One 11 (4), 1–15. https://doi.org/10.1371/journal.pone.0152429. Dorra, S., Fatma, B.A., Sami, G., Souhir, K., Mohamed, T., Ferdinando, B., Neila, T., Sonia, M., 2019. An insight from tolerance to salinity stress in halophyte Portulaca oleracea L.: physio-morphological, biochemical and molecular responses. Ecotoxicol. Environ. Saf. 172, 45–52. https://doi.org/10.1016/j.ecoenv.2018.12.082. El-Ramady, H., et al., 2018. Plant nutrients and their roles under saline soil conditions. In: Hasanuzzaman, M., Fujita, M., Oku, H., Nahar, K., Hawrylak-Nowak, B. (Eds.), Plant Nutrients and Abiotic Stress Tolerance. Springer, Singapore. https://doi.org/10. 1007/978-981-10-9044-8_13. Flowers, T.J., Hajibagherp, M.A., Yeo, A.R., 1991. Ion accumulation in the cell walls of rice plants growing under saline conditions: evidence for the Oertli hypothesis. Plant Cell Environ. 14 (3), 319–325. https://doi.org/10.1111/j.1365-3040.1991. tb01507.x. Golombek, S., Rolletschek, H.U., Weber, H., 2001. Control of storage protein accumulation during legume seed development. J. Plant Physiol. 158, 457–464. https://doi. org/10.1078/0176-1617-00357. Goussi, R., Manaa, A., Derbali, W., Cantamessa, S., Abdelly, C., Barbato, R., 2018. Comparative analysis of salt stress, duration and intensity, on the chloroplast ultrastructure and photosynthetic apparatus in Thellungiella salsuginea. J. Photochem. Photobiol. B 183, 275–287. https://doi.org/10.1016/j.jphotobiol.2018.04.047. Hanachi, S., Labeke, M.C.V., Mehouachi, T., 2014. Application of chlorophyll fluorescence to screen eggplant (Solanum melongena L.) cultivars for salt tolerance. Photosynthetica. 52 (1), 57–62. https://doi.org/10.1007/s11099-014-0007-z. Hanley, M.E., Fegan, E.L., 2007. Timing of cotyledon damage affects growth and flowering in mature plants. Plant Cell Environ. 30 (7), 812–819. https://doi.org/10.1111/ j.1365-3040.2007.01671.x. Jiang, Y., Ding, X., Dong, Z., Qi, D., Yu, C.L., Zhou, S., 2017. Soil salinity increases the tolerance of excessive sulfur fumigation stress in tomato plants. Environ. Exp. Bot. 133, 70–77. https://doi.org/10.1016/j.envexpbot.2016.10.002. Kianipouya, A., Roessner, U., Jayasinghe, N.S., Lutz, A., Rupasinghe, T., Bazihizina, N., 2017. Epidermal bladder cells confer salinity stress tolerance in the halophyte quinoa and Atriplex species. Plant Cell Environ. 40 (9), 1900–1915. https://doi.org/10. 1111/pce.12995. Li, J.Y., Zhao, C.Y., Li, J., Yan, Y.Y., Yu, B., Han, M., 2013. Growth and leaf gas exchange in Populus euphratica across soil water and salinity gradients. Photosynthetica. 51 (3), 321–329. https://doi.org/10.1007/s11099-013-0028-z. Li, R., Shi, F., Fukuda, K., Yang, Y., 2010. Effects of salt and alkali stresses on germination, growth, photosynthesis and ion accumulation in alfalfa (Medicago sativa L.). J. Plant Nutr. Soil Sci. (1999) 56, 725–733. https://doi.org/10.1111/j.1747-0765.2010. 00506.x. Lima, R.L.S., Severino, L.S., Sampaio, L.R., Sofiatti, V., Jucélia, A.G., Beltrão, N.E.M., 2011. Blends of castor meal and castor husks for optimized use as organic fertilizer. Ind. Crops Prod. 33 (2), 364–368. https://doi.org/10.1016/j.indcrop.2010.11.008. Lin, J.X., Li, Z.L., Shao, S., Wang, Y.N., Mu, C.C., 2014. Effects of various mixed saltalkaline stress conditions on seed germination and early seedling growth of Leymus chinensis from Songnen Grassland of China. Not. Bot. Horti Agrobot. Cluj. 42 (1), 154–159. https://doi.org/10.15835/nbha4219388. Lin, J.X., Wang, Y.N., Sun, S.N., Mu, C.S., Yan, X.F., 2016. Effects of arbuscular mycorrhizal fungi on the growth, photosynthesis and photosynthetic pigments of Leymus chinensis seedlings under salt-alkali stress and nitrogen deposition. Sci. Total Environ. 576, 234–241. https://doi.org/10.1016/j.scitotenv.2016.10.091. Liu, J., Shi, D.C., 2010. Photosynthesis, chlorophyll fluorescence, inorganic ion and organic acid accumulations of sunflower in responses to salt and salt-alkaline mixed

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