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Research Article

Low-Affinity Cation Transporter 1 Improves Salt Stress Tolerance in Japonica Rice

Marjohn C. Niño1, Franz Marielle Nogoy1, Kwon-Kyoo Kang2, Yong-Gu Cho1,*
Plant Breeding and Biotechnology 2018;6(1):82-93.
Published online: March 1, 2018

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*Corresponding author Yong-Gu Cho, ygcho@cbnu.ac.kr, Tel: +82-43-261-2514, Fax: +82-43-273-1598
• Received: February 20, 2018   • Revised: February 21, 2018   • Accepted: February 21, 2018

Copyright © 2018 The Korean Society of Breeding Science

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Low-Affinity Cation Transporter 1 Improves Salt Stress Tolerance in Japonica Rice
Plant Breed. Biotech.. 2018;6(1):82-93.   Published online March 1, 2018
DOI: https://doi.org/10.9787/PBB.2018.6.1.82
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Low-Affinity Cation Transporter 1 Improves Salt Stress Tolerance in Japonica Rice
Plant Breed. Biotech.. 2018;6(1):82-93.   Published online March 1, 2018
DOI: https://doi.org/10.9787/PBB.2018.6.1.82
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Low-Affinity Cation Transporter 1 Improves Salt Stress Tolerance in Japonica Rice
Image Image Image Image Image Image Image Image Image
Fig. 1 Schematic diagram of pCAMbia1300 containing the full-length cDNA of OsLCT1 from japonica rice ‘Jinbaek’. The ORF was ligated into the SacI and XbaI enzyme sites of the vector.
Fig. 2 Secondary structure of protein OsLCT1. The protein contains 10 transmembrane helices.
Fig. 3 Evolutionary relationship of 13 representative members of monocot plants based on the amino acid sequence of OsLCT1. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 3.44 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling 1965) and are in the units of the number of amino acid substitutions per site. The analysis involved 13 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 659 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016).
Fig. 4 Genomic PCR amplification of the OsLCT1 gene. Bands of approximately 1.6 kb indicate OsLCT1 amplicon. The first lane was wild-type, the second lane (pDNA) was plasmid DNA as a positive control containing the gene, and 3rd to 26th lanes were regenerated T0 plants.
Fig. 5 The calculated copy number (CN) of T0 OsLCT1-overexpression rice plants assayed using TaqMan RT-qPCR. Data presented were means ± SD from three replications. Plants with asterisk (*) were selected for further experiments. NC = negative control, PC = double copy positive control.
Fig. 6 Expression of OsLCT1 in different tissues and growth stages of the rice plant.
Fig. 7 The phenotype of wild-type ‘Dongjin’ and representative of three overexpression plants in T1 generation during the mature stage in the field.
Fig. 8 Phenotypic responses of OsLCT1–overexpression rice evaluated against salinity stress (200 mM NaCl). Hwayeong = tolerant (T) control; Dongjin = susceptible (S), wild-type (WT).
Fig. 9 Induction of transcripts level of OsLCT1 after treatment with 100 mM salicylic acid and 100 mM jasmonic acid. Data presented were relative expression means ± SD from three replicates after normalization by Ubiquitin (Ub) expression. Mock = water, SA = salicylic acid, JA = jasmonic acid.

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Fig. 8

Fig. 9

Low-Affinity Cation Transporter 1 Improves Salt Stress Tolerance in Japonica Rice

Estimates of evolutionary divergence between OsLCT1 amino acid sequences of representative members of grass family. The number of amino acid substitutions per site from between sequences are shown. Standard error estimate(s) are shown above the diagonal. Analyses were conducted using the Poisson correction model (Zuckerkandl and Pauling 1965). The analysis involved 13 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 659 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016).

T. aestivum H. vulgare S. officinarum O. nivara O. punctata O. meridionalis O. barthii O. glaberrima S. italica S. bicolor Nipponbare Kasalath Jinbaek
T. aestivum 0.071 0.060 0.069 0.046 0.066 0.065 0.065 0.069 0.126 0.065 0.064 0.065
H. vulgare 1.180 0.103 0.073 0.074 0.042 0.066 0.066 0.072 0.090 0.066 0.068 0.066
S. officinarum 0.588 1.174 0.108 0.067 0.098 0.097 0.097 0.095 0.130 0.098 0.102 0.098
O. nivara 1.127 1.125 1.1204 0.065 0.071 0.015 0.015 0.053 0.134 0.014 0.011 0.014
O. punctata 0.717 1.242 0.661 1.069 0.074 0.059 0.060 0.065 0.123 0.060 0.060 0.060
O. meridionalis 1.153 0.573 1.109 1.071 1.269 0.068 0.068 0.073 0.078 0.069 0.069 0.069
O. barthii 1.168 1.144 1.184 0.081 1.135 1.124 0.003 0.049 0.109 0.005 0.011 0.006
O. glaberrima 1.175 1.144 1.184 0.081 1.142 1.124 0.004 0.049 0.109 0.005 0.011 0.006
S. italica 1.053 1.129 0.999 0.730 1.032 1.099 0.680 0.680 0.135 0.050 0.049 0.050
S. bicolor 1.305 0.845 1.116 1.070 1.262 0.707 1.142 1.142 1.079 0.111 0.113 0.111
Nipponbare 1.161 1.137 1.199 0.075 1.129 1.117 0.014 0.014 0.685 1.180 0.01 0.002
Kasalath 1.153 1.169 1.210 0.045 1.124 1.126 0.063 0.061 0.677 1.200 0.057 0.011
Jinbaek 1.161 1.137 1.199 0.078 1.129 1.117 0.016 0.016 0.690 1.180 0.002 0.059

Evaluation of morpho-agronomic characteristics of OsLCT1-overexpression and wild-type plants under field condition.

Plants Height (cm) Culm length (cm)  Panicle length (cm)  Tiller  Grain length (mm)  Grain width (mm)
Dongjin 1,098.17 ± 43.76 841 ± 45.18 199.33 ± 3.88 11.5 ± 0.00 7.35 ± 0.14 3.09 ± 0.06
OsLCT1-7 1,053.67 ± 38.26 750.33 ± 55.5* 188.17 ± 12.98 11.17 ± 1.26 7.45 ± 0.11 3.15 ± 0.09
OsLCT1-18   1,169.83 ± 21.51*  876.67 ± 19.05 200 ± 3.28 8.83 ± 1.26 7.65 ± 0.05* 3.22 ± 0.11
OsLCT1-19 1,212.33 ± 42.03* 877.5 ± 43.37 201 ± 31.75 12 ± 2.78 7.79 ± 0.15* 3.16 ± 0.04
CV (%) 3.30 5.13 8.79 15.19 1.55 2.54

Dunnett’s multiple comparison was used to determine the significance (0.05 probability level) of OsLCT1-OX plants with the wild-type ‘Dongjin’. Asterisk (*) indicates significant difference from the wild-type at the 0.05 probability level. CV = coefficient of variation.

Response of two-week-old seedlings of OsLCT1-overexpression and wild-type plants seven days after treatments with 200 mM NaCl stress.

Plants  Shoot Biomass (g)   % Moisture Content   EC (μS/cm)  Salinity Score
7 DAT 7 DAT 7 DAT 7DAT  Reaction 
Dongjin 1.84 ± 0.44c 61.20 ± 4.03c 89.89 ± 0.23a 8.27 ± 0.21a S
Hwayeong 5.82 ± 0.94a 75.27 ± 0.26a 33.16 ± 0.82d 1.77 ± 0.25c HT
OsLCT1-7 3.11 ± 0.10b 69.12 ± 1.27b 52.91 ± 0.39c 4.63 ± 0.25b MT
OsLCT1-18 3.08 ± 0.19b 70.97 ± 1.71b 53.74 ± 0.26bc 4.27 ± 0.32b MT
OsLCT1-19  2.98 ± 0.34b 71.48 ± 1.70b 54.22 ± 0.38b 4.43 ± 0.35b MT
CV (%) 9.94 3.13 0.82 6.83

Means followed by a common letter in a column are not significantly different at the 0.05 probability level using Duncan’s multiple range test (DMRT); S = Susceptible; HT = High tolerant; MT = Moderately tolerant (Based on standard evaluation system for rice, IRRI 2007).

Table 1 Estimates of evolutionary divergence between OsLCT1 amino acid sequences of representative members of grass family. The number of amino acid substitutions per site from between sequences are shown. Standard error estimate(s) are shown above the diagonal. Analyses were conducted using the Poisson correction model (Zuckerkandl and Pauling 1965). The analysis involved 13 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 659 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016).
Table 2 Evaluation of morpho-agronomic characteristics of OsLCT1-overexpression and wild-type plants under field condition.

Dunnett’s multiple comparison was used to determine the significance (0.05 probability level) of OsLCT1-OX plants with the wild-type ‘Dongjin’. Asterisk (*) indicates significant difference from the wild-type at the 0.05 probability level. CV = coefficient of variation.

Table 3 Response of two-week-old seedlings of OsLCT1-overexpression and wild-type plants seven days after treatments with 200 mM NaCl stress.

Means followed by a common letter in a column are not significantly different at the 0.05 probability level using Duncan’s multiple range test (DMRT); S = Susceptible; HT = High tolerant; MT = Moderately tolerant (Based on standard evaluation system for rice, IRRI 2007).

Table 1

Table 2

Table 3

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