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Adaptation of euhalophyte Suaeda salsa to nitrogen starvation under salinity
Journal Pre-proof Adaptation of euhalophyte Suaeda salsa to nitrogen starvation under salinity Yanchun Ma, Yang Yang, Ranran Liu, Qiang Li, Jie Song PII:
S0981-9428(19)30490-5
DOI:
https://doi.org/10.1016/j.plaphy.2019.11.025
Reference:
PLAPHY 5940
To appear in:
Plant Physiology and Biochemistry
Received Date: 6 October 2019 Revised Date:
13 November 2019
Accepted Date: 14 November 2019
Please cite this article as: Y. Ma, Y. Yang, R. Liu, Q. Li, J. Song, Adaptation of euhalophyte Suaeda salsa to nitrogen starvation under salinity, Plant Physiology et Biochemistry (2019), doi: https:// doi.org/10.1016/j.plaphy.2019.11.025. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Masson SAS.
Title page
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Title：Adaptation of euhalophyte Suaeda salsa to nitrogen starvation under salinity
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Author names and affiliations：
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Yanchun Ma, Yang Yang, Ranran Liu, Qiang Li, Jie Song*
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Yanchun Ma and Yang Yang contributed equally to this work
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Shandong Provincial Key Laboratory of Plant Stress, College of Life Science, Shandong Normal
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University, Jinan 250014, P.R. China
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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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Nitrogen utilization efficiency, NUE; Nitrate transporter, NRT; Chloride channel, CLC; Net
19
photosynthetic rate, Pn; Quantitative real-time PCR, qRT-PCR.
20 21 22
1
1
ABSTRACT: Suaeda salsa L. (S. salsa) is an annual euhalophyte with high salt tolerance. The
2
NO3- content in soils where S. salsa populations occur are very low, especially in intertidal habitat.
3
However, it remains unclear how S. salsa populations adapt to low nitrogen environments. Plants
4
of two S. salsa populations were pre-cultured with nitrate nitrogen (1 mM of NO3--N) for 30 days.
5
Then, the seedlings were cultured with 1 mM of NO3--N and N-free solution (N starvation) at 200
6
mM of NaCl for an additional 14 days. The expression of two genes in S. salsa, nitrate transporter
7
1.7 (SsNRT1.7) and nitrate transporter 2.5 (SsNRT2.5) in old and mature leaves, was markedly
8
upregulated during N starvation in the intertidal population, when compared to the inland
9
population, but this was not the case in young leaves. After N starvation, the decrease in NO3- and
10
chlorophyll content, net photosynthetic rate in young leaves, and shoot dry weight in the intertidal
11
population were lower than those in the inland population. In conclusion, SsNRT1.7 and
12
SsNRT2.5 may play a role in NO3- remobilization, especially in the intertidal population, during N
13
starvation. This trait may benefit the intertidal population for adapting to low nitrogen
14
environments.
15
Keywords: Adaptation; Halophyte; Nitrogen remobilization; Nitrogen starvation; Suaeda salsa
16 17
1. Introduction
18
It is estimated that the global annual loss in agricultural production from salt-affected land are
19
in excess of 12 billion US$ and rising (Shabala, 2013), while N fertilization to provide the
20
promotive level of N might help to correct the nutritional imbalance in plants exposed to salinity
21
(Gómez et al., 1996). It has been regarded that nitrogen-availability is a major limiting factor that
22
restricts plant growth in saline soils (Liu et al., 2005; Song et al., 2009a; Li et al., 2019). For
2
1
higher plants, NO3- and NH4+ are the major N sources, and these are also important signal
2
molecules that affect plant growth and metabolism (Maninoia et al., 1981; Krouk et al., 2010; Xu
3
et al., 2016; Ashraf et al., 2018). Nitrogen utilization efficiency (NUE) includes the absorption,
4
assimilation and reuse of nitrogen (Kant et al., 2010), while salinity can inhibit nitrogen
5
utilization efficiency. For example, salinity can limit the acquisition of NO3- through the roots. It
6
also restricts the ability to reduce and assimilate N by inhibiting the synthesis and activities of
7
certain N assimilation enzymes, including nitrate reductase (NR), nitrite reductase (NiR),
8
glutamine synthetase (GS), and glutamate synthase (GOGAT) (Ashraf et al., 2018).
9
Three types of nitrate transporters, namely, nitrate transporter 1 (NRT1), nitrate transporter 2
10
(NRT2) and the chloride channel (CLC) family, are mainly involved in NO3- uptake, storage and
11
allocation (Tasy et al., 2007). Recently, it has been reported that NPF (NRT1/PTR FAMILY)
12
members display sequence and structural homologies with peptide transporter (PTR) proteins
13
involved in the uptake of di- and tri-peptides in most other organisms, and these are initially
14
characterized as nitrate or peptide transporters in plants (Corratgé-Faillie and Lacombe, 2017).
15
NO3- uptake is regulated by NRT1 and NRT2 transporters in root cells via a low-affinity transport
16
system (LATS) and a high-affinity transport system (HATS), and these activities depend on
17
external NO3- concentrations (Wang et al., 2014). The expression of AtNRT2.1 and AtNRT2.2
18
genes, which belong to the inducible system (iHATS), is strongly induced by NO3- (Filleur et al.,
19
2001), especially for AtNRT2.1 (Li et al., 2007). In Arabidopsis, AtNRT1.1, AtNRT1.2 and
20
AtNRT2.1 are responsible for the uptake of NO3- from the soil into the roots (Cerezo et al., 2001;
21
Filleur et al., 2001; Li et al., 2007; Little et al., 2005; Orsel et al., 2002). AtNRT1.7 is involved in
22
the phloem loading of NO3- in the source leaf to allow NO3- transport out of older leaves and into
3
1
younger leaves (Fan et al., 2009). It has been reported that the nitrogen limitation adaptation (nla)
2
mutant is hypersensitive to N limitation. In the nla mutant,
3
preferentially accumulated in the youngest leaves. The results of the bimolecular fluorescence
4
complementation,
5
demonstrated that NLA interacts with NRT1.7 in the plasma membrane in Arabidopsis (Liu et al.,
6
2017). Recently, it was found that AtNRT2.5 also plays an essential role in NO3- remobilization
7
during long-term N starvation (Lezhneva et al., 2014). When the absorption of NO3- exceeds the
8
metabolic requirements, the excess NO3- would be stored in the vacuole of leaves, and the stored
9
NO3- in the vacuole would be released for plant growth when the NO3- around the roots is
10
depleted (Blom-Zandstra and Lampe, 1983). Therefore, the reuse of NO3- is very important for
11
plants in natural habitats that are low in nitrogen.
split-ubiquitin
yeast
two-hybrid
and
15
N-nitrate spotted on old leaves
co-immunoprecipitation
assays
12
Suaeda salsa L. (S. salsa) is an annual herbaceous halophyte with high salt tolerance (Chen et
13
al., 2016; Song et al., 2011; Sui et al., 2017; Wang et al., 2015). This species occurs in both
14
intertidal zones and inland saline sites in China (Liu et al., 2018; Song et al., 2009b; Wang et al.,
15
2018). S. salsa is economically important, because its fresh branches have high value as a
16
vegetable, and it can be used as traditional Chinese medicine (Song and Wang, 2015). Recently, it
17
was reported that salinity increased the content of certain metabolites, such as organic acids, the
18
content of amino acids, lipids such as α-linolenic acid, and certain antioxidants such as quercetin.
19
The investigators suggest that these substances may be correlated to osmotic tolerance, increased
20
antioxidant activity, and medical and nutritional value in this species (Li and Song, 2019).
21
Therefore, this species has high value in understanding salt tolerance and developing saline
22
agriculture (Song and Wang, 2015). S. salsa from both the intertidal zone and saline inland
4
1
produces dimorphic seeds (Guo et al., 2015; Xu et al., 2016; Guo et al., 2018). The seed weight of
2
brown and black seeds for S. salsa in the intertidal zone was 4.2 and 5.5 times greater,
3
respectively, than that of S. salsa from inland saline soils (Song et al., 2008). The stems and
4
leaves are rich in nutrients, and the seeds contain high-quality edible oil (Song et al., 2016; Zhao
5
et al., 2018a, b, 2019). The content of NO3- is lower in soils in the intertidal zone (1.3 g kg-1 dry
6
soil) than that at the inland site (3.3 g kg-1 dry soil). However, NO3- content is higher in the leaves
7
of plants from the intertidal zone than that at the inland saline site (Song et al., 2009a). The
8
investigators suggest that S. salsa from the intertidal zone may be better at accumulating NO3-
9
even with low soil NO3-, ensuring plant growth when the NO3- around the roots is depleted (Song
10
et al., 2009a). However, the mechanism involved in this process remains unknown. Therefore, the
11
physiological and molecular mechanisms for NO3- reuse in two S. salsa populations were
12
investigated in the present experiment. It was found that SsNRT1.7 and SsNRT2.5 may play a role
13
in NO3- remobilization, especially in the intertidal population, during N starvation in the present
14
study. This trait may benefit the intertidal population for adapting to changeable low nitrogen
15
environments under salinity.
16 17
2. Materials and Methods
18
2.1. Plant culture and experimental design
19
Mature seeds of S. salsa were randomly collected from inland and intertidal habitats along
20
the Yellow River Delta in Shandong province of China in November 2017. Soil water content (%),
21
total soluble salt content, and the content of Na+ and Cl- (g kg-1 dry soil) for the intertidal
22
population was 22.4, 11.8, 3.9 and 6.1, respectively, while for the inland population, the value was
5
1
18.5, 5.4, 1.6 and 2.1, respectively (Song et al., 2009a). The dry seeds were stored in a refrigerator
2
at <4°C for three months before use.
3
In late March 2018, brown seeds of two S. salsa populations were sown in 2-L plastic pots
4
with drainage holes. Each pot was filled with 2.0 kg of washed river sand and 30 seedlings.
5
Seedlings were cultured in a glasshouse in Shandong Normal University under natural light. The
6
temperature was 23 ± 5°C during the day and 19 ± 5°C at night in the glasshouse. The seedlings
7
were watered every day with 1 mM of NO3--N nutrient solution. The nutrient solution contained
8
0.5 mM of Ca(NO3)2, 1 mM of MgSO4, 1 mM of KH2PO4, 2 mM of K2SO4, 2 mM of CaCl2, 45
9
μM of Fe-EDTA, 23 μM of H3BO3, 4.55 μM of MnCl2, 0.16 μM of CuSO4, 0.38 μM of ZnSO4
10
and 0.28 μM of Na2MoO4, which was modified according to the study conducted by Liu et al.
11
(2018). The pH was regulated to 6.2 ± 0.1 with KOH or H2SO4. The seedlings were watered daily
12
with the nutrient solution. After the seedlings were pre-cultured for 30 days, 20 seedlings in each
13
pot were left. After that, seedlings were treated with 200 mM of NaCl, which was prepared with
14
the nutrient solution described above. In a previous study, it was found that the optimal NaCl
15
concentration for S. salsa growth is approximately 200 mM of NaCl (Song et al., 2009b).
16
Therefore, 200 mM of NaCl was used to meet the demand of salinity for the plants in the present
17
study. In order to avoid osmotic shock, NaCl was provided in increments of 50 mM per day. Four
18
days later, these seedlings were irrigated either with 1 mM of NO3--N (four pots for each
19
population as control, that is, the N normal treatment), or N-free nutrient solution (four pots for
20
each population as the N starvation treatment). The N-free nutrient solution contained 1 mM of
21
MgSO4, 1 mM of KH2PO4, 2 mM of K2SO4, 2.5 mM of CaCl2, 45 μM of Fe-EDTA, 23 μM of
22
H3BO3, 4.55 μM of MnCl2, 0.16 μM of CuSO4, 0.38 μM of ZnSO4 and 0.28 μM of Na2MoO4.
6
1
The nutrient solution of both the N normal treatment and N starvation treatment contained 200
2
mM of NaCl. The pH was regulated to 6.2 ± 0.1 with KOH or H2SO4. After these seedlings were
3
cultured for an additional 14 days when the old leaves began to become yellow, the net
4
photosynthetic rate, chlorophyll and nitrate content, shoot dry weight, and expression of
5
SsNRT1.7 and SsNRT2.5 were measured from the old, mature and young leaves. Four replicates
6
were set for each treatment.
7 8
2.2. Total RNA extraction and cDNA synthesis
9
Total RNA was extracted from the leaves of two S. salsa populations (0.2 g per sample),
10
using a Quick RNA Isolation Kit (Waryong, Beijing, China), according to manufacturer’s
11
instructions.
12
The cDNA was synthesized using a PrimeScript™RT reagent Kit with gDNA Eraser (TaKaLa,
13
China), according to manufacturer’s instructions, and 1 μg total RNA was used as a template for
14
the real-time quantitative polymerase chain reaction (RT-qPCR) analysis.
15 16
2.3. Bioinformatic analysis of SsNRT1.7 and SsNRT2.5
17
The gene of S. salsa NRT1.7 and NRT2.5 was cloned based on the previous transcriptome
18
data in the leaves of the species. BLASTp online and software such as DNAman were used to
19
translate the nucleic acid sequences into protein sequences, as well as for homologous sequence
20
alignment and homology analysis. The phylogenetic tree was constructed by MEGA5 using
21
Neighbor-Joining (NJ). Bootstrap analysis, in which 1,000 replicates was performed to assess the
22
statistical reliability of the tree topology. The MEGA5 software service was used for the analysis
7
1
of the phylogenetic relationships of the amino acid residues of SsNRT1.7 and SsNRT2.5 between
2
different plants.
3 4
2.4. Real-time quantitative polymerase chain reaction analysis
5
The gene of S. salsa NRT1.7 was cloned based on the previous transcriptome data in the
6
leaves of the species. The similarity of the CDS sequence (coding sequence) of the gene was
7
55.8% in a comparative analysis with AtNRT1.7 in Arabidopsis thaliana (A. thaliana), and the
8
protein sequence was 98% similar to the NRT1 family of Beta vulgaris subsp. Vulgaris (B.
9
vulgaris). The gene of S. salsa NRT2.5 was also cloned based on the same transcriptome data in
10
the leaves of the species. The similarity of the CDS sequence of the gene was 60.4% in a
11
comparative analysis with AtNRT2.5 in A. thaliana, and the protein sequence was 74.3% similar
12
to NRT2.5 in B. vulgaris. These two genes were temporarily named as SsNRT1.7 and SsNRT2.5 in
13
the present study. The primers were designed using the Beacon Designer software (version 7.9,
14
Table 1). The ACTIN (GenBank ID: EU429457) was used as an internal standard (Ma et al.,
15
2009). For the qRT-PCR, 20 μl of the system contained 1 μl of cDNA, 0.5 μl of primer pairs
16
(ACTIN and target gene), 10 μl of SYBR Premix Ex Taq and 8 μl of sterile water. The qRT-PCR
17
was performed at an initial denaturation step at 95°C for 300 seconds, followed by 40 cycles at
18
95°C for 15 seconds, 54°C for 20 seconds, and 72°C for 20 seconds. This program was performed
19
in an RT-qPCR instrument (LightCycler® 96, Roche). The results were calculated using the 2−ΔΔCT
20
method (Liu et al., 2018).
21 22
2.5. Determination of NO3- content
8
1
The leaf samples (0.3 g for each replicate) were boiled in distilled water for three hours. Then,
2
the solution was filtered, and distilled water was added to obtain a final volume of 25 ml.
3
Afterwards, the content of NO3- was determined with the Dionex ICS-1100 Ion Chromatography
4
System (Dionex Corp., Sunnyvale, CA, USA) (Song et al., 2017).
5 6
2.6. Determination of chlorophyll
7
After washing the leaves, the water on the samples was absorbed by tissue paper. Then, the
8
leaves (0.3 g for each replicate) were grounded in 80% acetone, and the chlorophyll content was
9
determined spectrophotometrically (UV-120-02 Spectrophotometer, Shimadzu, Kyoto, Japan)
10
(Duan et al., 2018; Li et al., 2012).
11 12 13 14
2.7. Determination of photosynthetic rate The net photosynthetic rate (Pn) was measured using the Li-6400 photosynthesis measurement system (LI 6400; LI-COR Inc., Lincoln, NE, USA) (Duan et al., 2018).
15 16 17 18
2.8. Determination of plant growth At the end of the experiment, the shoots were sampled, and the shoot dry weight was measured after drying at 80°C for three days (Duan et al., 2018).
19 20
2.9. Statistical analysis
21
The data of Pn, chlorophyll and NO3- content in the leaves of two populations were subjected
22
to two-way ANOVA, and the shoot dry weight in the inland or intertidal population were analyzed
9
1
by one-way ANOVA using the SAS™ software (SAS Institute Inc., 1989).
2 3
3. Results
4
3.1. Shoot dry weight
5
N starvation decreased shoot dry weight in both populations. The decline in the intertidal
6
population was significantly lower than that in the inland population. That is, the value for N
7
starvation plants was 74.47% and 91.65% of the control for the inland and intertidal population,
8
respectively (Fig. 1).
9 10
3.2. Bioinformatic analysis of SsNRT1.7 and SsNRT2.5
11
The similarity of the CDS sequence (coding sequence) of SsNRT1.7 was 55.8% in a
12
comparative analysis with AtNRT1.7 in A. thaliana, and the protein sequence was 72.78% similar
13
to the NRT1 family of Chenopodium quinoa (C. quinoa) and 71.57% similar to B. vulgaris. The
14
similarity of the CDS sequence of SsNRT2.5 was 60.4% in the comparative analysis with
15
AtNRT2.5 in A. thaliana, and the protein sequence was 71.04 % similar to NRT2.5 in C. quinoa
16
and 71.81 % in B. vulgaris.
17
In order to investigate the evolutionary relationship among NRTs in plants, a phylogenetic
18
tree of the full-length amino acid sequences was constructed using the Neighbor–Joining method.
19
It was found that SsNRT1.7 had the highest identities with NRTs from C. quinoa, followed
20
through B. vulgaris and Spinacia oleracea. Similarly, SsNRT2.5 had the highest identities with
21
NRTs from C. quinoa (Fig. 2).
22
10
1
3.3. Relative expression of SsNRT1.7 and SsNRT2.5 in the leaves
2
It was reported that AtNRT1.7 and AtNRT2.5 play an essential role in NO3- remobilization in
3
the leaves during N starvation (Fan et al., 2009; Lezhneva et al., 2014). Therefore, SsNRT1.7 and
4
SsNRT2.5 were selected in the present study to primarily evaluate their possible role in NO3-
5
remobilization in the leaves of S. salsa during N starvation. N starvation upregulated the
6
expression of SsNRT1.7, when compared to that in the control (plants watered with 1 mM of NO3-
7
during the whole time course of the experiment), in old, mature and young leaves, especially in
8
old leaves in the intertidal population (Fig. 3A). Furthermore, N starvation upregulated the
9
expression of SsNRT1.7, when compared to that in the control, for old and young leaves, but this
10
had no effect on mature leaves in the inland population (Fig. 3A). It appears that N starvation
11
more effectively upregulated the expression of SsNRT1.7 in old leaves in the intertidal population,
12
when compared to the inland population. That is, the expression of SsNRT1.7 in the old leaves of
13
N starvation plants was 2.88 and 1.48 times of that of the control, for the intertidal and inland
14
population, respectively (Fig. 3A).
15
Except for young leaves of the intertidal population, the expression of SsNRT2.5 was also
16
upregulated, when compared to that in the control in both populations, especially in mature leaves
17
in the intertidal population during N starvation (Fig. 3B).
18 19
3.4. NO3- content in the leaves
20
N starvation has no significant effect on the content of NO3- in the leaves, when compared to
21
that in the control, regardless of the leaf position in the intertidal population. Furthermore, N
22
starvation has no significant effect on the content of NO3- in old and mature leaves, when
11
1
compared to that in the control, but this significantly decreased the NO3- content in young leaves
2
in the inland population (Fig. 4, Table 2).
3 4
3.5. Content of chlorophyll in the leaves
5
N starvation significantly decreased the content of chlorophyll (a+b), when compared to that
6
in the control, regardless of the leaf position in both populations (Fig. 5A, Table 2). Furthermore,
7
N starvation more severely decreased the content of chlorophyll (a+b) in young leaves in the
8
inland population than in the intertidal population. That is, the value in young leaves of N
9
starvation plants was 46.63% and 73.89% of the control for the inland and intertidal population,
10
respectively (Fig. 5A, Table 2).
11 12
3.6. Pn in the leaves
13
N starvation inhibited the net photosynthetic rate in both populations. However, the decline in
14
intertidal population was significantly lower, when compared to that in the inland population,
15
especially in mature and young leaves. That is, the value in mature and young leaves of N
16
starvation plants was 58.25% and 62.43 % of the control for the inland population, respectively,
17
and this was 91.95% and 96.29 % of the control for the intertidal population, respectively (Fig.
18
5B, Table 2).
19 20
4. Discussion
21
Nitrogen is an essential nutrient for plants, and is closely correlated to the salt tolerance of
22
certain halophytes (Liu et al., 2005; Song et al., 2017). Based on the requirements of ecological
12
1
environment protection and economic benefits, the study of crop nitrogen uptake and utilization,
2
and the selection of nitrogen efficient varieties have become closely watched by agronomists
3
(Zhang et al., 2013). It has been considered that when the absorption of NO3- exceeds its
4
metabolism, the excess NO3- would be stored in the vacuole of the leaf (Blom-Zandstra and
5
Lampe, 1983), while the stored NO3- would be reused during the N starvation of plants (Fan et al.,
6
2009).
7
In Arabidopsis, AtNRT1.7 is involved in nitrate remobilization, and the properties of
8
AtNRT1.7 indicate that NO3- remobilization from source to sink tissues is mediated by phloem
9
transport (Fan et al., 2009). In the present study, the expression of SsNRT1.7 was significantly
10
upregulated by N starvation in old leaves, particularly in the intertidal population (Fig. 3A). This
11
indicates that SsNRT1.7 may be involved in the loading of nitrate in the phloem of old leaves.
12
Meanwhile, the result indicates that S. salsa in the intertidal population can better remobilize
13
stored NO3- in the old leaves, when compared with the inland population. In Arabidopsis,
14
AtNRT2.5 is expressed in the minor veins of mature leaves, and is together involved in delivering
15
NO3- into the phloem for remobilization during long-term N starvation (Lezhneva et al., 2014).
16
That is, AtNRT2.5 plays an essential role in plants under severe N starvation by taking part in
17
NO3- loading into the phloem during NO3- remobilization (Lezhneva et al., 2014). In the present
18
study, the expression of SsNRT2.5 was more effectively upregulated by N starvation in mature
19
leaves in the intertidal population than in the inland population (Fig. 3B). Meanwhile, the
20
expression in young leaves was very low in the intertidal population, but this was not the case in
21
the inland population (Fig. 3B). It appears that SsNRT2.5 is involved in NO3- loading into the
22
phloem, which assists SsNRT1.7 in the remobilization of NO3- from source to sink tissues during
13
1
N starvation, especially in the intertidal population of S. salsa. Nitrate transporter 2.4 (NRT2.4) is
2
one of seven NRT2 family genes in A. thaliana, and NRT2.4 expression is induced under N
3
starvation. It has been reported that NRT2.4 is a plasma membrane transporter expressed in the
4
epidermis of lateral roots, and that it is in or close to the shoot phloem (Kiba et al., 2012). In
5
addition, the spatiotemporal expression pattern of NRT2.4 in the roots is complementary with
6
NTR2.1 (Kiba et al., 2012). In the absence of NRT2.1 and NRT2.2, the loss of function of
7
NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. This
8
indicates that NRT2.4 is a nitrate transporter that has a role in both the roots and shoots under N
9
starvation in Arabidopsis (Kiba et al., 2012). In further studies, the role of NRT2.1, NRT2.2 and
10
NRT2.4 in the roots, as well as NRT2.4 in the shoots, under N starvation of S. salsa should be
11
evaluated.
12
Salinity can decrease NO3- accumulation in both nonhalophyte such as Ricinus communis L.
13
(Peuke et al., 1996) and halophytes such as Plantago maritima L. (Rubinigg et al., 2003).
14
Therefore, maintaining an appropriate nitrogen supply is essential for plant growth in halophytes.
15
After N starvation, the change in NO3- content in the leaves of the two populations differed in the
16
present experiment (Fig. 4). That is, the decline in NO3- content in young leaves was lower in the
17
intertidal population than in the inland population (Fig. 4). In a previous glasshouse experiment,
18
mature leaves of the intertidal S. salsa population accumulated more NO3-, even with low soil
19
NO3- concentration (0.1 mM of NO3--N), when compared to the inland population. The present
20
result confirms the hypothesis that this trait may ensure plant growth when the NO3- around the
21
roots is depleted for the intertidal population (Song et al., 2009a). Therefore, SsNRT1.7 and
22
SsNRT2.5 may play a more important role in NO3- loading into the phloem of old leaves or
14
1
mature leaves in the intertidal population, when compare to the inland population, during
2
long-term N starvation.
3
Salinity can inhibit plant growth, which is associated with a decrease in photosynthetic
4
capacity (Lu et al., 2002; Sui et al., 2010). Salinity often decreases the chlorophyll content in
5
plants, such as tomato (Solanum lycopersicum L.) (Li et al., 2015). Nitrogen levels significantly
6
affect the content of chlorophyll, which in turn affects photosynthesis capacity (Wang et al., 2014).
7
For example, the actual PSII efficiency (ΦPSII) increased with the increase in NO3--N supply in
8
the leaves of S. salsa (Song et al., 2009a). In the present study, the decrease in NO3- and
9
chlorophyll content, and the net photosynthetic rate in leaves was lower in the intertidal
10
population, when compared to the inland population, during N starvation (Figs. 4 and 5). This
11
trait may be due to the higher expression of SsNRT1.7 and SsNRT2.5 in old and mature leaves in
12
the intertidal population, which contribute to the higher of NO3- in young leaves by loading NO3-
13
into the phloem and transferring these to young leaves for NO3- reuse, when compared with the
14
inland population. In addition to N uptake and remobilization, N assimilation is another important
15
process that influences N use efficiency in plants. N assimilated in the form of NO3- has to be
16
reduced into NH4+ before being incorporated into an organic compound (Ashraf et al., 2018;
17
Ranjan and Yadav, 2019). Therefore, the activity of NR, NiR, GS and GOGAT needs to be
18
measured under N starvation in the leaves of S. salsa.
19
In conclusion, SsNRT1.7 and SsNRT2.5 may play more important role in NO3- remobilization
20
in the intertidal population, when compared to the inland population, during N starvation.
21
Therefore, more NO3- in young leaves allocated from old or mature leaves can better maintain the
22
chlorophyll content and other physiological traits in the intertidal population, when compared to
15
1
the inland population, in fluctuating low nitrogen environments. This trait may benefit the
2
intertidal population for adapting to changeable low nitrogen environments.
3 4
Author contributions
5
Yanchun Ma and Yang Yang performed the experiments, and analyzed the data. Yanchun Ma
6
wrote the paper. Qiang Li and Ranran Liu assisted in analyzing the data. Jie Song designed the
7
experiments and revised the paper.
8 9 10 11
Acknowledgments The work is supported from National Natural Science Research Foundation of China (31570392, U1803233).
12 13
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Arabidopsis. Front. Plant Sci. 8, 1337-1350.
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Wang, F.X., Xu, Y.G., Wang, S., Shi, W.W., Liu, R.R., Feng, G., Song. J., 2015. Salinity affects
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endogenous hormones and seed coat phenolics during seed storage of two Suaeda salsa
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21
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22
511-516.
22
1 2
Table 1
3
Primer pairs for relative expression of SsNRT1.7, SsNRT2.5 and actin by qRT-PCR. Genes
Sense primer(5’-3’)
Antisense primer(3’-5’)
SsNRT1.7
GAACCTCATCAACATCTAC
CACAGAACCAATAGCAATA
SsNRT2.5
CTTCTAAGGTGTTATATCA
TAAGTAGTAAGCAATGAC
Actin
GCTCTACCCCATGCAATCCT
TGCTCTTGGCAGTCTCTGATT
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
23
1 2
Table 2
3
Results of a two-way ANOVA describing how NO3-, chl(a+b) content and net photosynthetic rate
4
(Pn) in leaves of Suaeda salsa were affected by (A) leaf position (old, mature or young), (B)
5
nitrogen treatments (N normal or N starvation) and their interactions. After the seedlings were
6
pre-cultured with 1 mM NO3--N for 30 days, all seedlings were treated with 200 mM NaCl which
7
was prepared with 1 mM NO3--N or N-free nutrient solution for additional 14 days. Populations
Parameters
A
B
A*B
Inland
NO3- content
7.36*
5.23*
2.49NS
Chl (a+b) content
271.42***
560.44***
39.35***
Pn
106.51***
44.75***
3NS
NO3- content
58.12***
1.46NS
1.22NS
Chl (a+b) content
827.41***
473.33***
18.54***
Pn
243.13***
3.83NS
0.41NS
Intertidal
8
Significant differences at *P ≤ 0.05 and ***P ≤ 0.001 are indicated;
9
difference. Data represent F values.
NS
indicates no significant
10 11 12 13 14 15 16
24
1
Figure legends
2
Fig. 1 The effect of N starvation on the shoot dry weight of two S. salsa populations. After the
3
seedlings were pre-cultured with 1 mM of NO3--N for 30 days, these were treated with 200 mM
4
of NaCl, which was prepared with 1 mM of NO3--N or N-free nutrient solution for an additional
5
14 days. For each group of two bars, the means with different letters are significantly different at
6
P<0.05. The vertical bars indicate standard errors of the means, n=4.
7
Fig. 2 The phylogenetic tree of NRT1.7 (A) and NRT2.5 (B) proteins from different plant species.
8
Fig. 3 The effect of N starvation on the relative expression of SsNRT1.7 (A), SsNRT2.5 (B) in the
9
leaves of S. salsa from inland saline soil and the intertidal zone. After the seedlings were
10
pre-cultured with 1 mM of NO3--N for 30 days, these were treated with 200 mM of NaCl, which
11
was prepared with 1 mM of NO3--N or N-free nutrient solution for an additional 14 days. The
12
vertical bars indicate the standard errors of the means, n=3.
13
Fig. 4 Effect of N starvation on the NO3- content in the old, mature and young leaves of two S.
14
salsa populations. After the seedlings were pre-cultured with 1 mM of NO3--N for 30 days, these
15
were treated with 200 mM of NaCl, which was prepared with 1 mM of NO3--N or N-free nutrient
16
solution for an additional of 14 days. For each group of two bars, the means with different letters
17
were significantly different at P<0.05. The vertical bars indicate standard errors of the means,
18
n=4.
19
Fig. 5 Effect of N starvation on chlorophyll (a+b) (A) content and net photosynthetic rate (B) in
20
the leaves of two S. salsa populations. After the seedlings were pre-cultured with 1 mM of
21
NO3--N for 30 days, these were treated with 200 mM of NaCl, which was prepared with 1 mM of
22
NO3--N or N-free nutrient solution for an additional of 14 days. For each group of two bars, the
25
1
means with different letters are significantly different at P<0.05. The vertical bars indicate the
2
standard errors of the means, n=4.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
26
1 2
Fig. 1.
3 4 5 6 7 8 9 10 11 12 13 14
27
1 2
Fig. 2.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
28
1 2
Fig. 3.
3 4 5 6 7 8 9 10
29
1 2
Fig. 4.
3 4 5 6 7 8 9 10 11 12 13 14
30
1 2
Fig. 5.
3
4 5 6 7 8 9 10 11
31
Yanchun Ma and Yang Yang performed the experiments, and analyzed the data. Yanchun Ma wrote the paper. Qiang Li and Ranran Liu assisted in analyzing the data. Jie Song designed the experiments and revised the paper.
Highlights: The expression of SsNRT1.7 and SsNRT2.5 in the old and mature leaves was upregulated markedly during N starvation in the intertidal population compared to the inland population in the euhalophyte Suaeda salsa. After N starvation, the decrease of NO3- and chlorophyll content in the young leaves in the intertidal population was lower than those in the inland population. SsNRT1.7 and SsNRT2.5 may play a role in NO3- remobilization, especially in the intertidal population, during N starvation.
Declaration of Interest Statement: The authors declare that they have no competing interests.
S0981-9428(19)30490-5
DOI:
https://doi.org/10.1016/j.plaphy.2019.11.025
Reference:
PLAPHY 5940
To appear in:
Plant Physiology and Biochemistry
Received Date: 6 October 2019 Revised Date:
13 November 2019
Accepted Date: 14 November 2019
Please cite this article as: Y. Ma, Y. Yang, R. Liu, Q. Li, J. Song, Adaptation of euhalophyte Suaeda salsa to nitrogen starvation under salinity, Plant Physiology et Biochemistry (2019), doi: https:// doi.org/10.1016/j.plaphy.2019.11.025. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Masson SAS.
Title page
1 2
Title：Adaptation of euhalophyte Suaeda salsa to nitrogen starvation under salinity
3 4
Author names and affiliations：
5
Yanchun Ma, Yang Yang, Ranran Liu, Qiang Li, Jie Song*
6
Yanchun Ma and Yang Yang contributed equally to this work
7 8
Shandong Provincial Key Laboratory of Plant Stress, College of Life Science, Shandong Normal
9
University, Jinan 250014, P.R. China
10 11
Corresponding author：
12
Jie Song
13
Tel.: +86 531 86182568; Fax: +86 531 86180107.
14
E-mail address: [email protected]
15
Postal address: Wenhua East Road 88, Jinan China, 250014
16 17
Abbreviations
18
Nitrogen utilization efficiency, NUE; Nitrate transporter, NRT; Chloride channel, CLC; Net
19
photosynthetic rate, Pn; Quantitative real-time PCR, qRT-PCR.
20 21 22
1
1
ABSTRACT: Suaeda salsa L. (S. salsa) is an annual euhalophyte with high salt tolerance. The
2
NO3- content in soils where S. salsa populations occur are very low, especially in intertidal habitat.
3
However, it remains unclear how S. salsa populations adapt to low nitrogen environments. Plants
4
of two S. salsa populations were pre-cultured with nitrate nitrogen (1 mM of NO3--N) for 30 days.
5
Then, the seedlings were cultured with 1 mM of NO3--N and N-free solution (N starvation) at 200
6
mM of NaCl for an additional 14 days. The expression of two genes in S. salsa, nitrate transporter
7
1.7 (SsNRT1.7) and nitrate transporter 2.5 (SsNRT2.5) in old and mature leaves, was markedly
8
upregulated during N starvation in the intertidal population, when compared to the inland
9
population, but this was not the case in young leaves. After N starvation, the decrease in NO3- and
10
chlorophyll content, net photosynthetic rate in young leaves, and shoot dry weight in the intertidal
11
population were lower than those in the inland population. In conclusion, SsNRT1.7 and
12
SsNRT2.5 may play a role in NO3- remobilization, especially in the intertidal population, during N
13
starvation. This trait may benefit the intertidal population for adapting to low nitrogen
14
environments.
15
Keywords: Adaptation; Halophyte; Nitrogen remobilization; Nitrogen starvation; Suaeda salsa
16 17
1. Introduction
18
It is estimated that the global annual loss in agricultural production from salt-affected land are
19
in excess of 12 billion US$ and rising (Shabala, 2013), while N fertilization to provide the
20
promotive level of N might help to correct the nutritional imbalance in plants exposed to salinity
21
(Gómez et al., 1996). It has been regarded that nitrogen-availability is a major limiting factor that
22
restricts plant growth in saline soils (Liu et al., 2005; Song et al., 2009a; Li et al., 2019). For
2
1
higher plants, NO3- and NH4+ are the major N sources, and these are also important signal
2
molecules that affect plant growth and metabolism (Maninoia et al., 1981; Krouk et al., 2010; Xu
3
et al., 2016; Ashraf et al., 2018). Nitrogen utilization efficiency (NUE) includes the absorption,
4
assimilation and reuse of nitrogen (Kant et al., 2010), while salinity can inhibit nitrogen
5
utilization efficiency. For example, salinity can limit the acquisition of NO3- through the roots. It
6
also restricts the ability to reduce and assimilate N by inhibiting the synthesis and activities of
7
certain N assimilation enzymes, including nitrate reductase (NR), nitrite reductase (NiR),
8
glutamine synthetase (GS), and glutamate synthase (GOGAT) (Ashraf et al., 2018).
9
Three types of nitrate transporters, namely, nitrate transporter 1 (NRT1), nitrate transporter 2
10
(NRT2) and the chloride channel (CLC) family, are mainly involved in NO3- uptake, storage and
11
allocation (Tasy et al., 2007). Recently, it has been reported that NPF (NRT1/PTR FAMILY)
12
members display sequence and structural homologies with peptide transporter (PTR) proteins
13
involved in the uptake of di- and tri-peptides in most other organisms, and these are initially
14
characterized as nitrate or peptide transporters in plants (Corratgé-Faillie and Lacombe, 2017).
15
NO3- uptake is regulated by NRT1 and NRT2 transporters in root cells via a low-affinity transport
16
system (LATS) and a high-affinity transport system (HATS), and these activities depend on
17
external NO3- concentrations (Wang et al., 2014). The expression of AtNRT2.1 and AtNRT2.2
18
genes, which belong to the inducible system (iHATS), is strongly induced by NO3- (Filleur et al.,
19
2001), especially for AtNRT2.1 (Li et al., 2007). In Arabidopsis, AtNRT1.1, AtNRT1.2 and
20
AtNRT2.1 are responsible for the uptake of NO3- from the soil into the roots (Cerezo et al., 2001;
21
Filleur et al., 2001; Li et al., 2007; Little et al., 2005; Orsel et al., 2002). AtNRT1.7 is involved in
22
the phloem loading of NO3- in the source leaf to allow NO3- transport out of older leaves and into
3
1
younger leaves (Fan et al., 2009). It has been reported that the nitrogen limitation adaptation (nla)
2
mutant is hypersensitive to N limitation. In the nla mutant,
3
preferentially accumulated in the youngest leaves. The results of the bimolecular fluorescence
4
complementation,
5
demonstrated that NLA interacts with NRT1.7 in the plasma membrane in Arabidopsis (Liu et al.,
6
2017). Recently, it was found that AtNRT2.5 also plays an essential role in NO3- remobilization
7
during long-term N starvation (Lezhneva et al., 2014). When the absorption of NO3- exceeds the
8
metabolic requirements, the excess NO3- would be stored in the vacuole of leaves, and the stored
9
NO3- in the vacuole would be released for plant growth when the NO3- around the roots is
10
depleted (Blom-Zandstra and Lampe, 1983). Therefore, the reuse of NO3- is very important for
11
plants in natural habitats that are low in nitrogen.
split-ubiquitin
yeast
two-hybrid
and
15
N-nitrate spotted on old leaves
co-immunoprecipitation
assays
12
Suaeda salsa L. (S. salsa) is an annual herbaceous halophyte with high salt tolerance (Chen et
13
al., 2016; Song et al., 2011; Sui et al., 2017; Wang et al., 2015). This species occurs in both
14
intertidal zones and inland saline sites in China (Liu et al., 2018; Song et al., 2009b; Wang et al.,
15
2018). S. salsa is economically important, because its fresh branches have high value as a
16
vegetable, and it can be used as traditional Chinese medicine (Song and Wang, 2015). Recently, it
17
was reported that salinity increased the content of certain metabolites, such as organic acids, the
18
content of amino acids, lipids such as α-linolenic acid, and certain antioxidants such as quercetin.
19
The investigators suggest that these substances may be correlated to osmotic tolerance, increased
20
antioxidant activity, and medical and nutritional value in this species (Li and Song, 2019).
21
Therefore, this species has high value in understanding salt tolerance and developing saline
22
agriculture (Song and Wang, 2015). S. salsa from both the intertidal zone and saline inland
4
1
produces dimorphic seeds (Guo et al., 2015; Xu et al., 2016; Guo et al., 2018). The seed weight of
2
brown and black seeds for S. salsa in the intertidal zone was 4.2 and 5.5 times greater,
3
respectively, than that of S. salsa from inland saline soils (Song et al., 2008). The stems and
4
leaves are rich in nutrients, and the seeds contain high-quality edible oil (Song et al., 2016; Zhao
5
et al., 2018a, b, 2019). The content of NO3- is lower in soils in the intertidal zone (1.3 g kg-1 dry
6
soil) than that at the inland site (3.3 g kg-1 dry soil). However, NO3- content is higher in the leaves
7
of plants from the intertidal zone than that at the inland saline site (Song et al., 2009a). The
8
investigators suggest that S. salsa from the intertidal zone may be better at accumulating NO3-
9
even with low soil NO3-, ensuring plant growth when the NO3- around the roots is depleted (Song
10
et al., 2009a). However, the mechanism involved in this process remains unknown. Therefore, the
11
physiological and molecular mechanisms for NO3- reuse in two S. salsa populations were
12
investigated in the present experiment. It was found that SsNRT1.7 and SsNRT2.5 may play a role
13
in NO3- remobilization, especially in the intertidal population, during N starvation in the present
14
study. This trait may benefit the intertidal population for adapting to changeable low nitrogen
15
environments under salinity.
16 17
2. Materials and Methods
18
2.1. Plant culture and experimental design
19
Mature seeds of S. salsa were randomly collected from inland and intertidal habitats along
20
the Yellow River Delta in Shandong province of China in November 2017. Soil water content (%),
21
total soluble salt content, and the content of Na+ and Cl- (g kg-1 dry soil) for the intertidal
22
population was 22.4, 11.8, 3.9 and 6.1, respectively, while for the inland population, the value was
5
1
18.5, 5.4, 1.6 and 2.1, respectively (Song et al., 2009a). The dry seeds were stored in a refrigerator
2
at <4°C for three months before use.
3
In late March 2018, brown seeds of two S. salsa populations were sown in 2-L plastic pots
4
with drainage holes. Each pot was filled with 2.0 kg of washed river sand and 30 seedlings.
5
Seedlings were cultured in a glasshouse in Shandong Normal University under natural light. The
6
temperature was 23 ± 5°C during the day and 19 ± 5°C at night in the glasshouse. The seedlings
7
were watered every day with 1 mM of NO3--N nutrient solution. The nutrient solution contained
8
0.5 mM of Ca(NO3)2, 1 mM of MgSO4, 1 mM of KH2PO4, 2 mM of K2SO4, 2 mM of CaCl2, 45
9
μM of Fe-EDTA, 23 μM of H3BO3, 4.55 μM of MnCl2, 0.16 μM of CuSO4, 0.38 μM of ZnSO4
10
and 0.28 μM of Na2MoO4, which was modified according to the study conducted by Liu et al.
11
(2018). The pH was regulated to 6.2 ± 0.1 with KOH or H2SO4. The seedlings were watered daily
12
with the nutrient solution. After the seedlings were pre-cultured for 30 days, 20 seedlings in each
13
pot were left. After that, seedlings were treated with 200 mM of NaCl, which was prepared with
14
the nutrient solution described above. In a previous study, it was found that the optimal NaCl
15
concentration for S. salsa growth is approximately 200 mM of NaCl (Song et al., 2009b).
16
Therefore, 200 mM of NaCl was used to meet the demand of salinity for the plants in the present
17
study. In order to avoid osmotic shock, NaCl was provided in increments of 50 mM per day. Four
18
days later, these seedlings were irrigated either with 1 mM of NO3--N (four pots for each
19
population as control, that is, the N normal treatment), or N-free nutrient solution (four pots for
20
each population as the N starvation treatment). The N-free nutrient solution contained 1 mM of
21
MgSO4, 1 mM of KH2PO4, 2 mM of K2SO4, 2.5 mM of CaCl2, 45 μM of Fe-EDTA, 23 μM of
22
H3BO3, 4.55 μM of MnCl2, 0.16 μM of CuSO4, 0.38 μM of ZnSO4 and 0.28 μM of Na2MoO4.
6
1
The nutrient solution of both the N normal treatment and N starvation treatment contained 200
2
mM of NaCl. The pH was regulated to 6.2 ± 0.1 with KOH or H2SO4. After these seedlings were
3
cultured for an additional 14 days when the old leaves began to become yellow, the net
4
photosynthetic rate, chlorophyll and nitrate content, shoot dry weight, and expression of
5
SsNRT1.7 and SsNRT2.5 were measured from the old, mature and young leaves. Four replicates
6
were set for each treatment.
7 8
2.2. Total RNA extraction and cDNA synthesis
9
Total RNA was extracted from the leaves of two S. salsa populations (0.2 g per sample),
10
using a Quick RNA Isolation Kit (Waryong, Beijing, China), according to manufacturer’s
11
instructions.
12
The cDNA was synthesized using a PrimeScript™RT reagent Kit with gDNA Eraser (TaKaLa,
13
China), according to manufacturer’s instructions, and 1 μg total RNA was used as a template for
14
the real-time quantitative polymerase chain reaction (RT-qPCR) analysis.
15 16
2.3. Bioinformatic analysis of SsNRT1.7 and SsNRT2.5
17
The gene of S. salsa NRT1.7 and NRT2.5 was cloned based on the previous transcriptome
18
data in the leaves of the species. BLASTp online and software such as DNAman were used to
19
translate the nucleic acid sequences into protein sequences, as well as for homologous sequence
20
alignment and homology analysis. The phylogenetic tree was constructed by MEGA5 using
21
Neighbor-Joining (NJ). Bootstrap analysis, in which 1,000 replicates was performed to assess the
22
statistical reliability of the tree topology. The MEGA5 software service was used for the analysis
7
1
of the phylogenetic relationships of the amino acid residues of SsNRT1.7 and SsNRT2.5 between
2
different plants.
3 4
2.4. Real-time quantitative polymerase chain reaction analysis
5
The gene of S. salsa NRT1.7 was cloned based on the previous transcriptome data in the
6
leaves of the species. The similarity of the CDS sequence (coding sequence) of the gene was
7
55.8% in a comparative analysis with AtNRT1.7 in Arabidopsis thaliana (A. thaliana), and the
8
protein sequence was 98% similar to the NRT1 family of Beta vulgaris subsp. Vulgaris (B.
9
vulgaris). The gene of S. salsa NRT2.5 was also cloned based on the same transcriptome data in
10
the leaves of the species. The similarity of the CDS sequence of the gene was 60.4% in a
11
comparative analysis with AtNRT2.5 in A. thaliana, and the protein sequence was 74.3% similar
12
to NRT2.5 in B. vulgaris. These two genes were temporarily named as SsNRT1.7 and SsNRT2.5 in
13
the present study. The primers were designed using the Beacon Designer software (version 7.9,
14
Table 1). The ACTIN (GenBank ID: EU429457) was used as an internal standard (Ma et al.,
15
2009). For the qRT-PCR, 20 μl of the system contained 1 μl of cDNA, 0.5 μl of primer pairs
16
(ACTIN and target gene), 10 μl of SYBR Premix Ex Taq and 8 μl of sterile water. The qRT-PCR
17
was performed at an initial denaturation step at 95°C for 300 seconds, followed by 40 cycles at
18
95°C for 15 seconds, 54°C for 20 seconds, and 72°C for 20 seconds. This program was performed
19
in an RT-qPCR instrument (LightCycler® 96, Roche). The results were calculated using the 2−ΔΔCT
20
method (Liu et al., 2018).
21 22
2.5. Determination of NO3- content
8
1
The leaf samples (0.3 g for each replicate) were boiled in distilled water for three hours. Then,
2
the solution was filtered, and distilled water was added to obtain a final volume of 25 ml.
3
Afterwards, the content of NO3- was determined with the Dionex ICS-1100 Ion Chromatography
4
System (Dionex Corp., Sunnyvale, CA, USA) (Song et al., 2017).
5 6
2.6. Determination of chlorophyll
7
After washing the leaves, the water on the samples was absorbed by tissue paper. Then, the
8
leaves (0.3 g for each replicate) were grounded in 80% acetone, and the chlorophyll content was
9
determined spectrophotometrically (UV-120-02 Spectrophotometer, Shimadzu, Kyoto, Japan)
10
(Duan et al., 2018; Li et al., 2012).
11 12 13 14
2.7. Determination of photosynthetic rate The net photosynthetic rate (Pn) was measured using the Li-6400 photosynthesis measurement system (LI 6400; LI-COR Inc., Lincoln, NE, USA) (Duan et al., 2018).
15 16 17 18
2.8. Determination of plant growth At the end of the experiment, the shoots were sampled, and the shoot dry weight was measured after drying at 80°C for three days (Duan et al., 2018).
19 20
2.9. Statistical analysis
21
The data of Pn, chlorophyll and NO3- content in the leaves of two populations were subjected
22
to two-way ANOVA, and the shoot dry weight in the inland or intertidal population were analyzed
9
1
by one-way ANOVA using the SAS™ software (SAS Institute Inc., 1989).
2 3
3. Results
4
3.1. Shoot dry weight
5
N starvation decreased shoot dry weight in both populations. The decline in the intertidal
6
population was significantly lower than that in the inland population. That is, the value for N
7
starvation plants was 74.47% and 91.65% of the control for the inland and intertidal population,
8
respectively (Fig. 1).
9 10
3.2. Bioinformatic analysis of SsNRT1.7 and SsNRT2.5
11
The similarity of the CDS sequence (coding sequence) of SsNRT1.7 was 55.8% in a
12
comparative analysis with AtNRT1.7 in A. thaliana, and the protein sequence was 72.78% similar
13
to the NRT1 family of Chenopodium quinoa (C. quinoa) and 71.57% similar to B. vulgaris. The
14
similarity of the CDS sequence of SsNRT2.5 was 60.4% in the comparative analysis with
15
AtNRT2.5 in A. thaliana, and the protein sequence was 71.04 % similar to NRT2.5 in C. quinoa
16
and 71.81 % in B. vulgaris.
17
In order to investigate the evolutionary relationship among NRTs in plants, a phylogenetic
18
tree of the full-length amino acid sequences was constructed using the Neighbor–Joining method.
19
It was found that SsNRT1.7 had the highest identities with NRTs from C. quinoa, followed
20
through B. vulgaris and Spinacia oleracea. Similarly, SsNRT2.5 had the highest identities with
21
NRTs from C. quinoa (Fig. 2).
22
10
1
3.3. Relative expression of SsNRT1.7 and SsNRT2.5 in the leaves
2
It was reported that AtNRT1.7 and AtNRT2.5 play an essential role in NO3- remobilization in
3
the leaves during N starvation (Fan et al., 2009; Lezhneva et al., 2014). Therefore, SsNRT1.7 and
4
SsNRT2.5 were selected in the present study to primarily evaluate their possible role in NO3-
5
remobilization in the leaves of S. salsa during N starvation. N starvation upregulated the
6
expression of SsNRT1.7, when compared to that in the control (plants watered with 1 mM of NO3-
7
during the whole time course of the experiment), in old, mature and young leaves, especially in
8
old leaves in the intertidal population (Fig. 3A). Furthermore, N starvation upregulated the
9
expression of SsNRT1.7, when compared to that in the control, for old and young leaves, but this
10
had no effect on mature leaves in the inland population (Fig. 3A). It appears that N starvation
11
more effectively upregulated the expression of SsNRT1.7 in old leaves in the intertidal population,
12
when compared to the inland population. That is, the expression of SsNRT1.7 in the old leaves of
13
N starvation plants was 2.88 and 1.48 times of that of the control, for the intertidal and inland
14
population, respectively (Fig. 3A).
15
Except for young leaves of the intertidal population, the expression of SsNRT2.5 was also
16
upregulated, when compared to that in the control in both populations, especially in mature leaves
17
in the intertidal population during N starvation (Fig. 3B).
18 19
3.4. NO3- content in the leaves
20
N starvation has no significant effect on the content of NO3- in the leaves, when compared to
21
that in the control, regardless of the leaf position in the intertidal population. Furthermore, N
22
starvation has no significant effect on the content of NO3- in old and mature leaves, when
11
1
compared to that in the control, but this significantly decreased the NO3- content in young leaves
2
in the inland population (Fig. 4, Table 2).
3 4
3.5. Content of chlorophyll in the leaves
5
N starvation significantly decreased the content of chlorophyll (a+b), when compared to that
6
in the control, regardless of the leaf position in both populations (Fig. 5A, Table 2). Furthermore,
7
N starvation more severely decreased the content of chlorophyll (a+b) in young leaves in the
8
inland population than in the intertidal population. That is, the value in young leaves of N
9
starvation plants was 46.63% and 73.89% of the control for the inland and intertidal population,
10
respectively (Fig. 5A, Table 2).
11 12
3.6. Pn in the leaves
13
N starvation inhibited the net photosynthetic rate in both populations. However, the decline in
14
intertidal population was significantly lower, when compared to that in the inland population,
15
especially in mature and young leaves. That is, the value in mature and young leaves of N
16
starvation plants was 58.25% and 62.43 % of the control for the inland population, respectively,
17
and this was 91.95% and 96.29 % of the control for the intertidal population, respectively (Fig.
18
5B, Table 2).
19 20
4. Discussion
21
Nitrogen is an essential nutrient for plants, and is closely correlated to the salt tolerance of
22
certain halophytes (Liu et al., 2005; Song et al., 2017). Based on the requirements of ecological
12
1
environment protection and economic benefits, the study of crop nitrogen uptake and utilization,
2
and the selection of nitrogen efficient varieties have become closely watched by agronomists
3
(Zhang et al., 2013). It has been considered that when the absorption of NO3- exceeds its
4
metabolism, the excess NO3- would be stored in the vacuole of the leaf (Blom-Zandstra and
5
Lampe, 1983), while the stored NO3- would be reused during the N starvation of plants (Fan et al.,
6
2009).
7
In Arabidopsis, AtNRT1.7 is involved in nitrate remobilization, and the properties of
8
AtNRT1.7 indicate that NO3- remobilization from source to sink tissues is mediated by phloem
9
transport (Fan et al., 2009). In the present study, the expression of SsNRT1.7 was significantly
10
upregulated by N starvation in old leaves, particularly in the intertidal population (Fig. 3A). This
11
indicates that SsNRT1.7 may be involved in the loading of nitrate in the phloem of old leaves.
12
Meanwhile, the result indicates that S. salsa in the intertidal population can better remobilize
13
stored NO3- in the old leaves, when compared with the inland population. In Arabidopsis,
14
AtNRT2.5 is expressed in the minor veins of mature leaves, and is together involved in delivering
15
NO3- into the phloem for remobilization during long-term N starvation (Lezhneva et al., 2014).
16
That is, AtNRT2.5 plays an essential role in plants under severe N starvation by taking part in
17
NO3- loading into the phloem during NO3- remobilization (Lezhneva et al., 2014). In the present
18
study, the expression of SsNRT2.5 was more effectively upregulated by N starvation in mature
19
leaves in the intertidal population than in the inland population (Fig. 3B). Meanwhile, the
20
expression in young leaves was very low in the intertidal population, but this was not the case in
21
the inland population (Fig. 3B). It appears that SsNRT2.5 is involved in NO3- loading into the
22
phloem, which assists SsNRT1.7 in the remobilization of NO3- from source to sink tissues during
13
1
N starvation, especially in the intertidal population of S. salsa. Nitrate transporter 2.4 (NRT2.4) is
2
one of seven NRT2 family genes in A. thaliana, and NRT2.4 expression is induced under N
3
starvation. It has been reported that NRT2.4 is a plasma membrane transporter expressed in the
4
epidermis of lateral roots, and that it is in or close to the shoot phloem (Kiba et al., 2012). In
5
addition, the spatiotemporal expression pattern of NRT2.4 in the roots is complementary with
6
NTR2.1 (Kiba et al., 2012). In the absence of NRT2.1 and NRT2.2, the loss of function of
7
NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. This
8
indicates that NRT2.4 is a nitrate transporter that has a role in both the roots and shoots under N
9
starvation in Arabidopsis (Kiba et al., 2012). In further studies, the role of NRT2.1, NRT2.2 and
10
NRT2.4 in the roots, as well as NRT2.4 in the shoots, under N starvation of S. salsa should be
11
evaluated.
12
Salinity can decrease NO3- accumulation in both nonhalophyte such as Ricinus communis L.
13
(Peuke et al., 1996) and halophytes such as Plantago maritima L. (Rubinigg et al., 2003).
14
Therefore, maintaining an appropriate nitrogen supply is essential for plant growth in halophytes.
15
After N starvation, the change in NO3- content in the leaves of the two populations differed in the
16
present experiment (Fig. 4). That is, the decline in NO3- content in young leaves was lower in the
17
intertidal population than in the inland population (Fig. 4). In a previous glasshouse experiment,
18
mature leaves of the intertidal S. salsa population accumulated more NO3-, even with low soil
19
NO3- concentration (0.1 mM of NO3--N), when compared to the inland population. The present
20
result confirms the hypothesis that this trait may ensure plant growth when the NO3- around the
21
roots is depleted for the intertidal population (Song et al., 2009a). Therefore, SsNRT1.7 and
22
SsNRT2.5 may play a more important role in NO3- loading into the phloem of old leaves or
14
1
mature leaves in the intertidal population, when compare to the inland population, during
2
long-term N starvation.
3
Salinity can inhibit plant growth, which is associated with a decrease in photosynthetic
4
capacity (Lu et al., 2002; Sui et al., 2010). Salinity often decreases the chlorophyll content in
5
plants, such as tomato (Solanum lycopersicum L.) (Li et al., 2015). Nitrogen levels significantly
6
affect the content of chlorophyll, which in turn affects photosynthesis capacity (Wang et al., 2014).
7
For example, the actual PSII efficiency (ΦPSII) increased with the increase in NO3--N supply in
8
the leaves of S. salsa (Song et al., 2009a). In the present study, the decrease in NO3- and
9
chlorophyll content, and the net photosynthetic rate in leaves was lower in the intertidal
10
population, when compared to the inland population, during N starvation (Figs. 4 and 5). This
11
trait may be due to the higher expression of SsNRT1.7 and SsNRT2.5 in old and mature leaves in
12
the intertidal population, which contribute to the higher of NO3- in young leaves by loading NO3-
13
into the phloem and transferring these to young leaves for NO3- reuse, when compared with the
14
inland population. In addition to N uptake and remobilization, N assimilation is another important
15
process that influences N use efficiency in plants. N assimilated in the form of NO3- has to be
16
reduced into NH4+ before being incorporated into an organic compound (Ashraf et al., 2018;
17
Ranjan and Yadav, 2019). Therefore, the activity of NR, NiR, GS and GOGAT needs to be
18
measured under N starvation in the leaves of S. salsa.
19
In conclusion, SsNRT1.7 and SsNRT2.5 may play more important role in NO3- remobilization
20
in the intertidal population, when compared to the inland population, during N starvation.
21
Therefore, more NO3- in young leaves allocated from old or mature leaves can better maintain the
22
chlorophyll content and other physiological traits in the intertidal population, when compared to
15
1
the inland population, in fluctuating low nitrogen environments. This trait may benefit the
2
intertidal population for adapting to changeable low nitrogen environments.
3 4
Author contributions
5
Yanchun Ma and Yang Yang performed the experiments, and analyzed the data. Yanchun Ma
6
wrote the paper. Qiang Li and Ranran Liu assisted in analyzing the data. Jie Song designed the
7
experiments and revised the paper.
8 9 10 11
Acknowledgments The work is supported from National Natural Science Research Foundation of China (31570392, U1803233).
12 13
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1 2
Table 1
3
Primer pairs for relative expression of SsNRT1.7, SsNRT2.5 and actin by qRT-PCR. Genes
Sense primer(5’-3’)
Antisense primer(3’-5’)
SsNRT1.7
GAACCTCATCAACATCTAC
CACAGAACCAATAGCAATA
SsNRT2.5
CTTCTAAGGTGTTATATCA
TAAGTAGTAAGCAATGAC
Actin
GCTCTACCCCATGCAATCCT
TGCTCTTGGCAGTCTCTGATT
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
23
1 2
Table 2
3
Results of a two-way ANOVA describing how NO3-, chl(a+b) content and net photosynthetic rate
4
(Pn) in leaves of Suaeda salsa were affected by (A) leaf position (old, mature or young), (B)
5
nitrogen treatments (N normal or N starvation) and their interactions. After the seedlings were
6
pre-cultured with 1 mM NO3--N for 30 days, all seedlings were treated with 200 mM NaCl which
7
was prepared with 1 mM NO3--N or N-free nutrient solution for additional 14 days. Populations
Parameters
A
B
A*B
Inland
NO3- content
7.36*
5.23*
2.49NS
Chl (a+b) content
271.42***
560.44***
39.35***
Pn
106.51***
44.75***
3NS
NO3- content
58.12***
1.46NS
1.22NS
Chl (a+b) content
827.41***
473.33***
18.54***
Pn
243.13***
3.83NS
0.41NS
Intertidal
8
Significant differences at *P ≤ 0.05 and ***P ≤ 0.001 are indicated;
9
difference. Data represent F values.
NS
indicates no significant
10 11 12 13 14 15 16
24
1
Figure legends
2
Fig. 1 The effect of N starvation on the shoot dry weight of two S. salsa populations. After the
3
seedlings were pre-cultured with 1 mM of NO3--N for 30 days, these were treated with 200 mM
4
of NaCl, which was prepared with 1 mM of NO3--N or N-free nutrient solution for an additional
5
14 days. For each group of two bars, the means with different letters are significantly different at
6
P<0.05. The vertical bars indicate standard errors of the means, n=4.
7
Fig. 2 The phylogenetic tree of NRT1.7 (A) and NRT2.5 (B) proteins from different plant species.
8
Fig. 3 The effect of N starvation on the relative expression of SsNRT1.7 (A), SsNRT2.5 (B) in the
9
leaves of S. salsa from inland saline soil and the intertidal zone. After the seedlings were
10
pre-cultured with 1 mM of NO3--N for 30 days, these were treated with 200 mM of NaCl, which
11
was prepared with 1 mM of NO3--N or N-free nutrient solution for an additional 14 days. The
12
vertical bars indicate the standard errors of the means, n=3.
13
Fig. 4 Effect of N starvation on the NO3- content in the old, mature and young leaves of two S.
14
salsa populations. After the seedlings were pre-cultured with 1 mM of NO3--N for 30 days, these
15
were treated with 200 mM of NaCl, which was prepared with 1 mM of NO3--N or N-free nutrient
16
solution for an additional of 14 days. For each group of two bars, the means with different letters
17
were significantly different at P<0.05. The vertical bars indicate standard errors of the means,
18
n=4.
19
Fig. 5 Effect of N starvation on chlorophyll (a+b) (A) content and net photosynthetic rate (B) in
20
the leaves of two S. salsa populations. After the seedlings were pre-cultured with 1 mM of
21
NO3--N for 30 days, these were treated with 200 mM of NaCl, which was prepared with 1 mM of
22
NO3--N or N-free nutrient solution for an additional of 14 days. For each group of two bars, the
25
1
means with different letters are significantly different at P<0.05. The vertical bars indicate the
2
standard errors of the means, n=4.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
26
1 2
Fig. 1.
3 4 5 6 7 8 9 10 11 12 13 14
27
1 2
Fig. 2.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
28
1 2
Fig. 3.
3 4 5 6 7 8 9 10
29
1 2
Fig. 4.
3 4 5 6 7 8 9 10 11 12 13 14
30
1 2
Fig. 5.
3
4 5 6 7 8 9 10 11
31
Yanchun Ma and Yang Yang performed the experiments, and analyzed the data. Yanchun Ma wrote the paper. Qiang Li and Ranran Liu assisted in analyzing the data. Jie Song designed the experiments and revised the paper.
Highlights: The expression of SsNRT1.7 and SsNRT2.5 in the old and mature leaves was upregulated markedly during N starvation in the intertidal population compared to the inland population in the euhalophyte Suaeda salsa. After N starvation, the decrease of NO3- and chlorophyll content in the young leaves in the intertidal population was lower than those in the inland population. SsNRT1.7 and SsNRT2.5 may play a role in NO3- remobilization, especially in the intertidal population, during N starvation.
Declaration of Interest Statement: The authors declare that they have no competing interests.