There are the large areas of saline-alkali land around the world and this
problem is continuing to deteriorate. Salt stress is a limiting factor that
severely affects plant growth and development, the crop productivity is also
significantly inhibited (Munns and Tester 2008). Almost all plants or crops,
which are important to humans, are adversely affected by high concentration of
salt (Al-Maskri et al. 2010; Yang and
Guo 2018). Therefore, it is especially important for the research of salt-tolerant
plants.
Puccinellia tenuiflora is a monocotyledonous halophytic
species that extensively distributed in the saline land of the Songnen plain in
Northeastern China (Yu et al. 2011).
The leaves of P. tenuiflora were very
tender and more rich in nutrition, which is a good quality forage for livestock
(Wei 2016). Therefore, it is used as a typical material for the study of salt
tolerance mechanism, because it is the few species that can survive under
multiple salt stresses. The molecular mechanisms of P. tenuiflora which can adapt to
salt stress deserve further research.
The high affinity nitrate transporter
accessory protein (NAR2) plays a crucial role in nitrate absorption and
transport. Plant absorption of nitrogen can be classified as high-affinity
transport systems (HATS) and low-affinity transport systems (LATS) depending on
their absorptive capacity (Kotur and Glass 2015). NRT2 and NAR2 primarily regulate HATS and are responsible for the
transport of nitrates under lower nitrogen concentration in plant
growing media (Laugier et al. 2012). NRT2.1 is the major
contributor to total HATS activity in NRT2 family (Li et al. 2007). CrNRT2.1 did
not transport nitrate alone in Chlamydomonas
reinhartii, and required CrNAR2 to co-regulate in transport (Quesada et al. 1994; Zhou et al. 2000). A similar result was found in Arabidopsis
thaliana, which has NAR2-like genes named as AtNRT3.1. AtNRT3.1; abundantly expressed and proved to
be highly sensitive to nitrate induction (Okamoto et al. 2006; Orsel et al.
2006). In
Hordeum vulgare, three CrNAR2-like genes were cloned: HvNAR2.1, HvNAR2.2 and HvNAR2.3; while NO3- is
transported only if HvNRT2.1 and HvNAR2.3 are simultaneously present;
neither HvNRT2.1 nor HvNAR2.3 show transport activity when
present alone (Tong et al. 2005). These findings indicated that NAR2 is widely found in a variety of
higher plants and it will specifically
interact with NRT2 in the same
species to participate the HATS. Recent studies have shown that OsNAR2.1 overexpression manifested better drought tolerance in rice (Chen et al. 2019).
Salt stress can seriously
affect the plant uptake of nitrogen. Transcriptional analyses of P. tenuiflora treated with saline-alkali water showed
that PutNAR2 was strongly
up-regulated. This indicated the significance of PutNAR2 in P. tenuiflora; due to which the adaptation of this species in the saline-alkali lands
can never be ignored (Ye et al. 2019).
The expression of PutNAR2.1 may affect the salt tolerance
of P. tenuiflora. Through the
analysis of changes in salt tolerance of yeast, protein, and plants due to PutNAR2.1 overexpression, we have a
preliminary understanding that PutNAR2.1
improving the salt tolerance of organisms. This research gives further insight
into the study of salt-tolerant molecular mechanism P. tenuiflora.
Seeds of P. tenuiflora were obtained from saline-alkali land in Northeast China. The cDNA was obtained
from total RNA of P. tenuiflora using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and the reverse
transcription PCR kit (Takara, Tokyo, Japan). The specific primers (PutNAR2.1F:
5'-ATGGCTCGGCAAGGAATGGT-3' and PutNAR2.1R: 5'-TTAGTTGTTCTTCTTGCGCTTC-3')
were designed by analyzing the P. tenuiflora transcriptome sequence. The
PCR product was amplified with cDNA by connecting the template to plasmid
pMD18-T Vector (Takara, Tokyo, Japan), and then sequenced.
Conserved domain of PutNAR2.1
in the NCBI database
The sequence of conserved domain of PutNAR2.1 of
cloned gene was analyzed by blast search in NCBI, and the amino acid sequence
of PutNAR2.1 protein was found highly
similar with other species. The homologous amino acid sequence of protein was
compared by DNAMAN software, and the phylogenetic tree was constructed by MEGA7 to observe the relationship between PutNAR2.1 and NAR2.1
of other species.
The expression pattern of PutNAR2.1 under salt stress in
P. tenuiflora was examined. Seeds were sown onto half-strength MS
medium. The seedlings of one-month age were subjected to various stress
treatments (300 mM NaCl, 100 mM Na2CO3 or
150 mM NaHCO3) for 0, 6, 12, 24 or 36 h. The PutNAR2.1 expression in P.
tenuiflora seedlings under different stress
treatments was detected by RT-qPCR analysis, total RNA was isolated from
P. tenuiflora and cDNA was synthesized.
Subsequently RT-qPCR analyses were carried out by SYBR green (Takara, Tokyo,
Japan) and IQ5 real-time PCR equipment (Bio-Rad, Hercules, CA, USA) with the
steps: 95℃ for 30 s, 30 cycles of 95℃
for 5 s, and 55℃ for 30 s. The next steps were
added to melt-curve analysis: 95℃ for 15 s, followed by
continuously increased from 60 to 95℃. The PutAct2 gene expression was used as control. The forward primer
sequence was ActinF: 5′-GGTAACATTGTGCTCAGTGGTGG-3′ and
reverse primer sequence was
ActinR: 5′-AACGACCTTAATCTTCATGCTGC
-3′). PutNAR2.1
RT-qPCR primers were designed by Quant Prime Tool.
To examine the
expression pattern of the PutNAR2.1 in P. tenuiflora among the different organs, total RNA was extracted from roots, shoots, leaves, flowers and seeds respectively and cDNA was synthesized. Subsequent RT-qPCR procedure was same as above.
The open reading frame (ORF) of the PutNAR2.1
gene was amplified from pMD18T-PutNAR2.1
plasmid DNA with BamH I forward
primer 5'-GGATCCATGGCTCGGCAAGGAA-3' (restriction site underlined
for all restriction enzymes) and XhoI
reverse primer 5-' CTCGAGTTAGTTGTTCTTCTTGCG-3'. The PCR amplified fragment
with BamH I and XhoI was recovered and then ligated to the corresponding
restriction enzyme site of the pYES2, pGEX-6p-3 and pBI121 vector,
respectively. It was finally verified by
double enzyme digestion and binary expression vector generating the plasmid
pYES2-PutNAR2.1, pGEX-PutNAR2.1 and pBI121-PutNAR2.1. The pGEX-PutNAR2.1 and pGEX-6p-3 plasmid
transformed E. Coli BL21 cells
were used for the expression of PutNAR2.1
fusion protein in E. coli BL21,
and plasmid pGEX-6p-3 was used as the control.
The plasmid DNAs of pYES2-PutNAR2.1 and pYES2 were transformed into the
yeast strain INVSC1 (Saccharomyces cerevisiae) using the electric
impulse method. The plasmid DNAs of pBI121-PutNAR2.1 was transformed into the Agrobacterium
tumefaciens strain EHA105 (Takara, Tokyo, Japan) by electro-transformation,
and the A. thaliana (ecotype Columbia) was infected with floral dip
method (Clough and Bent 1998).
Control strain pGEX-6p-3 vector and the transformant strain expressing PutNAR2.1 were grown in Luria-Bertani
(LB) liquid medium at 37℃ until the absorbance at 600 nm was 0.5 (OD600=0.5).
Expression of the PutNAR2.1 protein
was induced by 1 mM IPTG for 1 h, and separately added with 0.8 M NaCl,
0.1 M Na2CO3 or 0.2 M NaHCO3. The
concentrations were chosen based on the information available from a large
number of previous experiments. All the strains grow well in the selected
concentration). The cultures were grown with rotary shaking (160 rpm) at
37℃ for 1, 2, 3, 4 and 5 h. The growth rate of strains was monitored by
absorbance change at 600 nm using Spectrometer. Data are preliminary for three
replicate experiments.
The pYES2-PutNAR2.1 transgenic yeast and pYES2 were
cultivated in Yeast extract peptone dextrose medium (YPD) overnight at
30℃. When the bacterial culture concentration reached OD600 =
0.6, culture solutions with serial dilutions (10, 10-1, 10-2,
10-3 and 10-4) were dripped onto YPD agar plates with no
treatment (CK), 0.6 M NaCl, 0.8 M NaCl, 10 mM Na2CO3,
20 mM Na2CO3, 30 mM Na2CO3,
20 mM NaHCO3, 40 mM NaHCO3 or 60 mM
NaHCO3, respectively.
To observed the effects of
seeds germination of T3 generation homozygous transgenic
lines #1, #3 and #5 A. thaliana and
wild-type (Fig. 7),
disinfected seeds were directly placed on half-strength MS
agar medium supplemented with nothing else (CK), 100 mM NaCl, 125 mM
NaCl, 150 mM NaCl, 3 mM Na2CO3, 5 mM
Na2CO3, 7 mM Na2CO3, 3 mM
NaHCO3, 5 mM NaHCO3 or 7 mM NaHCO3.
The experiment was performed in triplicate independently and
photographed after 14 days.
Two-week-old seedlings with
similar size were arranged onto half-strength MS medium supplemented with
nothing and numerous stress (125 mM NaCl, 150 mM NaCl, 175 mM
NaCl, 3 mM Na2CO3, 5 mM Na2CO3,
7 mM Na2CO3, 3 mM NaHCO3,
5 mM NaHCO3 or 7 mM NaHCO3,). Each stress
treatment (included control) had three replications mentioned above. The Petri
plates were vertically positioned in order to visualize the root growth. Plants
were photographed after stress treatment for 7 days.
Statistical
analysis
All
treatments were performed in triplicates and data were treated for analysis of
variance using SPSS for Windows version 11.5.
The ORF of PutNAR2.1 was obtained from the cDNA in the P. tenuiflora.
The full-length sequence of PutNAR2.1
was 597 bp and encoded 199 amino acids; it contained the conserved domains of
the NAR2.1 gene family (Fig. 1). The
alignment of PutNAR2.1 amino acid sequence illustrated that it had the highest
similarity (88.94%) with
TaNAR2.1 protein from T. aestivum (Fig. 2). Phylogenetic tree
analysis was used to compare PutNAR2.1 protein with others known homologous
NAR2.1 protein from a variety of plants, which revealed that PutNAR2.1 was most
closely related to TaNAR2.1 from Triticum aestivum
(Fig. 3).
The
expression of PutNAR2.1 under different salt stresses was analyzed by
RT-qPCR. The results verified that the expression of PutNAR2.1 was
increased gradually and reached its the highest at 24 h under 300 mM NaCl, 100 mM
Na2CO3 or 150 mM NaHCO3, nearly 2.3 times higher than untreated (0 h) under 300
mM NaCl in (Fig. 4A), almost 2.7
times higher than untreated (0 h) under
100 mM Na2CO3 in (Fig. 4B), over 3.1 times higher than untreated (0 h) under 150 mM NaHCO3
in (Fig. 4C). PutNAR2.1 had the
highest expression in roots, followed by leaves and seeds, but was quite less
in shoots and flowers (Fig. 4D).
To investigate the salt response of PutNAR2.1
in E. coli, the E. coli growth
with only pGEX vector and transformants expressing pGEX-PutNAR2.1 were compared. PutNAR2.1 expressing strain and the
pGEX vector were inoculated into LB liquid medium, adding different salt
stresses respectively when both cell density was measured as OD600=0.5.
Fig. 1: Analyzing the conservative domain
of PutNAR2.1 in the NCBI database
Fig. 3: Phylogenetic tree of 10 selected plant NAR2.1 protein. The MEGA7 program
was used for the construction of phylogenetic trees. Bar represents 0.2 amino
acid substitutions per site
Fig. 4: Real-time Quantitative PCR
analysis for PutNAR2.1 expression in P. tenuiflora. (A) PutNAR2.1 expression at different times under 300 mM NaCl. (B) PutNAR2.1 expression at different times
under 100 mM Na2CO3. (C) PutNAR2.1 expression at different times under 150 mM NaHCO3.
(D) PutNAR2.1 expression in different organs of P. tenuiflora
Fig.
2:
Alignment of PutNAR2.1 deduced amino acid
sequence with other plant species NAR2.1 protein. The amino acid sequence of this transcript had the similarity with that of
the TaNAR2.1 protein (GenBank:
AAV35210.1, 88.94%) from Triticum aestivum, BdNAR2.1 protein (XP_003575282.1, 84.08%)
from Brachypodium distachyon, AtaNAR2.2 protein (XP_020163322.1, 85.43%) from Aegilops
tauschii subsp. Tauschii, HvNAR2.1 protein (AAP31850.1, 86.43%) from Hordeum vulgare subsp. Vulgare, DoNAR2.1 protein (OEL38054.1, 71.51%) from Dichanthelium
oligosanthes, PmNAR2.1 (GenBank: RLM78325.1, 70.39%) protein from Panicum miliaceum, SiNAR2.1 protein (XP_004952978.1,
65.84%) from Setaria italica, OsNAR2.1 protein
(XP_015623791.1, 65.12%) from Oryza sativa Japonica Group, ZmNAR2.1 protein (GenBank: AAY40796.1, 67.05%) from Zea mays, SbNAR2.1 protein (XP_002454118.1 , 68.00%)
Fig. 5: The bacterial concentration
of pGEX and pGEX-PutNAR2.1 at
different times in OD 600 nm under salt stresses. CK: no salt treatment
Fig. 6: Growth of PutNAR2.1
transgenic yeast cells under salt stress. Ten-fold dilutions of yeast cells
containing pYES2 (upper line) and pYES2-PutNAR2.1
vector (lower line) were spotted onto solid YPG media supplemented with the
indicated stresses. No treatment is a control (CK)
Sorghum bicolor
When both of the stains
inoculated in LB liquid medium (CK), the OD600
values of the control strain and the transgenic strain after culture for 1 h
were 0.68 and 0.67, respectively, both of them had the maximum OD600
of 2.0 after 5 h of incubation (Fig. 5A). Under 0.8 M NaCl treatment, the OD600
values of the control strain was decreased to 0.57. However, the transgenic
strain did not decrease after 1 h incubation, while the OD600 values
of the control strain and the transgenic strain after 5 h of culture were noted
as 1.38 and 1.62, respectively (Fig. 5B). Under the treatment of 0.1 M Na2CO3,
the OD600 values of the control strain and the transgenic strain
were 0.29 and 0.48 after 1 h, the OD600 values were 0.69 and 1.45
after 5 h, respectively (Fig. 5C). Under 0.2 M NaHCO3 treatment,
control and transgenic strain were cultured, with OD600 values of
0.41 and 0.65 after 1 h, these values were 0.98 and 1.67 after 5 h,
respectively (Fig. 5D).
Salt stress types induced expression of PutNAR2.1 in transgenic yeast was
investigated. The growth of PutNAR2.1 transgenic yeast cell and pYES2 was compared at five
serial dilutions for different salt treatments (corresponding to five columns
in each panel in Fig. 6). The control was no treatment (CK). The growth of both
pYES2 (upper line) and pYES2-PutNAR2.1
vector (lower line) transgenic yeasts showed no significant difference.
However, the growth of yeasts had the most drastic change when salt treatment
was applied. The transgenic yeasts grew significantly better than control in
0.8 M NaCl, 20 mM Na2CO3 and 40 mM NaHCO3
treatments. When the concentration was increased to 30 mM
Na2CO3 or 60 mM NaHCO3 the transgenic
yeasts grew as before, but non-transgenic yeasts could hardly grow.
Fig. 7: Identification
of transgenic A. thaliana lines by
contrasting the expression of PutNAR2.1 in the wild-type and transgenic lines using RT-qPCR. WT:
wild-type. #1–#7: PutNAR2.1 transgenic A. thaliana lines
Fig. 8: Seed germination in A. thaliana wild-type, PutNAR2.1 transgenic plants under
different stresses. Seed germination on medium supplemented with 0 mM (CK), 100
mM NaCl, 125 mM NaCl, 150 mM NaCl, 3 mM NaHCO3, 5 mM NaHCO3
and 7 mM NaHCO3, 3 mM Na2CO3, 5 mM Na2CO3
and 7 mM Na2CO3.WT: Wild-type A. thaliana. #1, #3 and #5: PutNAR2.1
transgenic lines
The expression of PutNAR2.1
transgenic A. thaliana plant was identified via RT-qPCR
analysis (Fig. 7). The expression level of PutNAR2.1 in seven randomly selected T3 transgenic A.
thaliana was higher than its wild-type counterparts. PutNAR2.1 expression in the transgenic lines #1- #7 had 31,
29, 42, 25, 30, 12 and 13 times higher than that in wild-type plants,
respectively. Among transgenic lines, #1, #3 and #5 indicated higher expression
level of PutNAR2.1,
and were selected for further research.
The T3 PutNAR2.1 transgenic A. thaliana
which exhibited higher levels of PutNAR2.1 (#1, #3, #5) and wild-type seeds were placed on half-strength MS supplemented
with no stress (CK) and salt stresses (Fig. 8).
Seeds of transgenic and wild-type
plants exhibited no difference when they were germinated on half-strength MS
directly (CK). With 100 mM NaCl, 3 mM NaHCO3 or 5 mM
NaHCO3 treatment, seeds of transgenic plants had bigger leaves than
the wild-type,
but germination of both was similar. Transgenic
plant seeds were germinated 1–2 days earlier than wild-type on the medium
containing 125 mM NaCl, 3 mM Na2CO3 5 mM
Na2CO3 or 7 mM NaHCO3, and the
transgenic A. thaliana growth was
obviously better. The germination of the wild-type plants was suppressed
significantly under 150 mM NaCl and 7 mM Na2CO3; a few
seeds did not germinate and the leaves of wild-type seedlings
were severely curled with light color.
However, all the seeds of the transgenic A. thaliana
lines germinated and remained green. These results demonstrated
that transgenic lines had significantly higher salt tolerance compared to
wild-type plants.
Fig. 9: Seedlings growth between A. thaliana wild-type and PutNAR2.1 transgenic plants under
different stresses. Seedlings growth on half-strength MS medium supplemented with
0 mM (CK), 125 mM NaCl, 150 mM NaCl, 175 mM NaCl, 3 mM NaHCO3, 5 mM
NaHCO3 and 7 mM NaHCO3, 3 mM Na2CO3,
5 mM Na2CO3 and 7 mM Na2CO3. WT:
Wild-type A. thaliana. #1, #3 and #5:
PutNAR2.1 transgenic lines
Furthermore, PutNAR2.1
transgenic line and wild-type grown on half-strength MS
medium with no treatment (CK) or salt stresses for 2 weeks were tested at the
seedling stage (Fig. 9). Under normal growth condition, the PutNAR2.1
transgenic lines and wild-type seedlings showed no significant morphological or
developmental abnormalities. Under increased NaCl treatment, in the wide-type
plants, the leaf margins turned brown and the color became darker. When
seedlings were grown on half-strength MS
medium containing Na2CO3 stress, the cotyledons of
wild-type seedlings were smaller compared with PutNAR2.1 transgenic
lines and most wild-type leaves turned white. Data further showed that PutNAR2.1
was induced by salt stress and exhibited a positive response to salt stress.
Therefore, the PutNAR2.1 gene is involved in the response to salt
stress, expression of PutNAR2.1 gene can increase plant tolerance to
salt stress.
Discussion
Reportedly P. tenuiflora is one
of the few plants that can survive on saline-alkali land (Zhang et al. 2013). The gene related to salt
and alkali stress was cloned from the P.
tenuiflora and the study of its gene function is helpful to explore the
molecular mechanism of salt and alkali resistance of the species (Ye et al. 2019).
The NAR2 protein is
mostly studied as nitrogen transport (Yan et
al. 2011; Chen et al. 2019). In
recent years, NAR2.1 gene has been
found to be related to stress tolerance and NAR2.1
was described as novel potato drought-responsive genes (Pieczynski et al. 2018). NaCl salinity reduces the absorption of nitrogen by
roots (Rubinigg et al. 2003; Yousif et al. 2010). PutNAR2.1 transcript
level was upregulated significantly in transcriptome analyses of P. tenuiflora treated with water
extracts from the saline-alkali soils. In this study, the possibility
of PutNAR2.1 participating in
regulation of the response to salt stress was examined by using PutNAR2.1 E. coli transformants, PutNAR2.1 transgenic yeasts and PutNAR2.1
transgenic A. thaliana lines.
The PutNAR2.1 has been cloned from P. tenuiflora. The amino acid sequence of PutNAR2.1 had the
similarity with other plant species. Drought stress can induce OsNAR2.1 high expression (Chen et al. 2019). In this research, the
expression of PutNAR2.1 started to
increase gradually at 6 h and reached the highest value at 24 h in P. tenuiflora under 300
mM NaCl, 100 mM
Na2CO3 or 150 mM NaHCO3 stress, indicated its gradual
induction by salt stress. NAR2 was mainly expressed in root (Orsel et al.
2002; Lupini et al. 2016; Luo et al.
2018), while the highest expression of the PutNAR2.1 was also found in roots of P. tenuiflora, under salt stresses. Thus PutNAR2.1 may play a defense role when roots are exposed to salt
stress.
It takes a long time to identify
the related functions after the gene is transferred into the plant. The effect
of salt stress on the growth of control strain and PutNAR2.1 expressing
strain was examined in LB medium. The OD600 value of the PutNAR2.1
expressing strain after 5 h culture was higher than that of the control
strain. It is indicated that PutNAR2.1 can protect E. coli to
against the salt stress from the environment. However, the prokaryotes may not have the function of protein
completely consistent with that in eukaryotes. The growth of control yeasts was
inhibited by NaCl, Na2CO3 or NaHCO3 stress,
while PutNAR2.1 transgenic yeast grew
well. These results revealed that the function of
PutNAR2.1 in eukaryotes
was similar to that in the prokaryotes.
Chen et al.
(2019) reported that OsNAR2.1 overexpressing
plant line increased the grain yield by about 26.6% compared to wild-type in limited irrigation conditions.
To observe PutNAR2.1 response to salt
stress, comparison was made for seeds germination and seedlings growth between transgenic lines and wild-type counterparts. The
germination and seedlings growth of wild-type were quite more reduced than that
of transgenic lines under salt stress. PutNAR2.1
transgenic line showed better growth compared with wild-type under stress of various
salts at different plant development stages. The data proved that expression of PutNAR2.1
helped the plants to resist the salt stress better.
Nitrate uptake was closely
related to plant growth and development, and the ability of nitrate uptake by
roots was decreased under salt stress. PutNAR2.1 is the key gene for
nitrate uptake and transport in P. tenuiflora. Under salt stress, P. tenuiflora can regulate the expression of NAR
to improve the ability of nitrate uptake and utilization, thus enhanced the
resistance to salt stress.
Conclusion
Tolerance of E. coli, yeast and plant to salt stresses could be greatly enhanced
with the high expression of PutNAR2.1. Based on the previous research progress of NAR2.1 gene and the results of this experiment, it is conjectured
that PutNAR2.1 may be
used as an auxiliary protein to participate in the high-affinity nitrogen
absorption system of plants under different salinity treatment. Consequently, PutNAR2.1
overexpression can help plants to
resist abiotic stress in the environment, but determination of specific working
mechanism needs further studies.
Acknowledgments
This work was supported by the Heilongjiang Province Nature Science
Foundation (LH2019C011), Key Laboratory Open Fund of Saline-alkali Vegetation Ecology
Restoration (SAVER1701) and the National Natural Science Foundation of China
(No. 31070616, No. 31500317).
Author Contributions
JSM planned the experiments and contributed
reagents/materials/analysis tools; ZGQ, CSY, XY and HH performed the experiments;
ZGQ statistically analyzed the data and made illustrations; JSM and ZGQ wrote the manuscript.
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