Cl − More Detrimental Than Na + in Rice Under Long-Term Saline Conditions

Rice is considered sensitive to salinity and hence, improving the salinity tolerance of rice is desired to increase productivity. Most research on salinity tolerance for the growth and grain yield of rice focuses on the toxicity of sodium (Na) and not chloride (Cl); the information on the negative effects of Cl on rice is still limited. To learn the difference in the long-term effects of Na and Cl on the whole plant and grain productivities, three rice varieties differing in salinity tolerance were grown in pots irrigated by water with NaCl and KCl in the same molar concentration. The whole plant dry weight and grain yield in all varieties decreased to the same extent after NaCl and KCl treatments compared to the control during the full heading and maturity stages. However, Na content in shoots of all varieties under NaCl treatment were higher at all stages than that under KCl treatment and the control. In the most cases, the Cl content in plants was similar in the NaCl and KCl treatment groups. There was a negative significant correlation between the relative dry weight and grain yield (treated/control) and the Cl content in the plants. From these results, it was suggested that plant dry weight and grain yield under long-term salinity conditions was reduced by Cl toxicity rather than Na toxicity.


Introduction
Soil salinity is one of the most severely limiting environmental factors for crop production.Globally, the total area of saline soils covers more than 800 million ha, and 45 million ha of irrigated land (19.5% of irrigated land) were salt-affected soils (FAO, 2018).Rice is a major staple food for hundreds of millions of people, but it is the most sensitive among cereal crops (Munns & Tester, 2008).As such, improving the salinity tolerance of rice is desired to increase productivity on salt-affected soil.
Plant growth responds to salinity in two phases: a rapid, osmotic phase that inhibits growth of young leaves, and a slower, ionic phase that accelerates senescence of mature leaves (Munns & Tester, 2008).The salinity tolerance of rice is thought to be closely related to Na + accumulation in shoots because there is overall a negative correlation between sodium content in plants and salinity tolerance in terms of survival (Flowers & Yeo, 1981;Yeo & Flowers, 1983), chlorophyll content (Yeo & Flowers, 1983), photosynthesis (Yeo, Caporn, & Flowers, 1985;Maegawa, Usii, Uchida, Yasud, & Yamaguchi, 1987), and plant growth (Akita & Cabuslay, 1990).There is a positive relationship between Na + and Cl − in roots and leaves (Maegawa et al., 1987;Ammar et al., 2009;Teakle & Tyerman, 2010), so Cl − content in plants also correlates to salinity tolerance (Flowers & Yeo, 1981).Several reports suggested that Na + toxicity is higher than Cl − toxicity by analyzing the contribution of Na + and Cl − (Kingsbury & Epstrin, 1986;Yamanouchi, 1989;Tsai, Hong, Liu, & Kao, 2004;Khare, Kumar, & Kishor, 2015).It is generally accepted that Na + appears to reach a toxic concentration before Cl − does in most species (Munns & Tester, 2008).Because of this, most research on salinity tolerance in cereals focuses on the toxicity of Na + and not Cl − (Teakle & Tyerman, 2010).As a result, the information on the effect of Cl − on rice is still limited.
The contribution of Na + and Cl − to plant growth was mainly investigated under short-term salinity stress (Lin & Kao, 2001;Tsai et al., 2004;Luo, Yu, & Liu, 2005).However, the ionic effect in plants under salinity stress appears when salt accumulates to toxic levels within the plant (Munns, Schachtman, & Condon, 1995).Therefore, the contribution of each ion to plant growth should be evaluated under long-term saline conditions.To separate the effects of Na + and Cl − on plant growth, equimolar concentrations of Na + and Cl − of various salts were made and increasing particular counter-anions or counter-cations were avoided (Kingsbury & Epstein, 1986;Luo et al., 2005;Tavakkoli, Fatehi, Coventry, Rengasamy, & McDonald, 2011;Khare et al., 2015).However, the ameliorative and/or deteriorative effects of counter-anions and counter-cations may not be completely excluded in such experiments.On the other hand, potassium (K + ) is an essential macronutrient which exists in relatively high concentrations in plants (Leigh & Jones, 1984), and its toxicity in plant is lower than that of Na + (Lynch, Cramer & Lauchli, 1987;Martin & Koebner, 1995).Therefore, the use of KCl equivalent to NaCl concentration is possible to detect the harmful effects of Cl − on the whole plant and grain productivities.We were to study the difference in the long-term effects of Na + and Cl − on the whole plant and grain productivities in three rice varieties and to clarify the toxicity of Cl − from its saline impact.

Materials and Methods
Three rice varieties-NSICRc106 (salt tolerant), Nipponbare (salt sensitive), and IR28 (salt sensitive)-were used.The experiment was conducted in a greenhouse at Okayama University.Pre-germinated seeds were seeded on 19 May, 2013 in nursery boxes (Minoru pot 448, Minoru Industrial Co. Ltd., Japan) with 448 holes (16 diameter, 25 mm depth).One seed was seeded in each hole.All holes were filled with nursery soil for rice seedlings (Kumiai Ube Ryu-joh Baido, Ube Industries, Ltd., Japan).The seedlings were transplanted manually at the 6-leaf stage on June 13 into 5 L plastic buckets of 135 pots filled with grey lowland soil.Slow-release compound (NPK 14-14-14) fertilizer was mixed with the soil (5 g per pot).Different salts, such as NaCl and KCl, were mixed with soil as salt treatments; soil without salt was considered as the control.The equimolar concentration (60 mM) of NaCl and KCl was applied by adding 12.0 and 15.31 g of the salt per pot, respectively.Tap water (3.4 L) was supplied, and plans were irrigated every day to keep a stable water level.The additional 5g of compound fertilizer ) was supplied at 30 days after treatment (DAT) and at flowering stage.Pots were arranged randomly in the plastic covered house and the experimental design was completely random.
Five plants in each treatment group were harvested at three developmental stages: 30 DAT, the full heading stage (7 days after head emergence), and the maturity stage (45 days after head emergence).The dry weights of the leaf blades, stems (including the leaf sheath), and panicles during the three stages were measured after being dried in an oven at 80 °C for 72 hours.The grains were separated into ripened and immature grains by sinking them in a NaCl 1.06 specific gravity solution.Grain yield was divided into four components: panicle number, number of grains per panicle, percentage of ripened grain, and 1000 grain weight.The dried samples were extracted in a 1% HNO 3 solution at 80 °C for 24 h.The concentrations of Na + and K + in the samples were determined using a flame photometer (BWB XP, BWB technologies UK Ltd., UK).The concentration of Cl − was determined using an ion meter (D-53, Horiba, Japan).
The visual salt damage of a panicle was scored on a 0 to 4 scale.A score of 0 indicates that deformed spikelets are not observed in the panicle.A score of 1 indicates that the percentage of deformed spikelets of the total number is under 10%.A score of 2 indicates that the percentage of deformed spikelets of the total number ranges from 10 to 25%.A score of 3 indicates that the percentage of deformed spikelets of the total number ranges from 25 to 50%.A score of 4 indicates that the percentage of deformed spikelets of the total number is over 50%.
The results were analyzed using JMP software version 9.0.The difference among treatments were determined by a one-way analysis of variance (ANOVA) based on Tukey's multiple range test (p < 0.05).

Whole Plant Growth, Grain Production, and Yield Components
The heading dates in the control group were 4 August (52 DAT) in IR28, 8 August (56 DAT) in NSICRc106, and August 15 (63 DAT) in Nipponbare.Under NaCl and KCl treatments, the heading dates in IR28 and NSICRc106 were delayed by 3-5 days, but that in Nipponbare was almost same in control.
At 30 DAT, the whole plant dry weight of Nipponbare decreased by 44% in the NaCl treatment group and 24% in the KCl treatment group (Figure 1).At the full heading stage and maturity stage, the plant dry weight in the NaCl and KCl treatment groups decreased by around 30%; there was no difference between both treatments.The plant dry weight of IR28 and NSICRc106 in the NaCl and KCl treatment groups decreased by 21 to 43%, but there was no difference between both treatments.The reduction of grain weight in all varieties under NaCl and KCl treatments ranged from 58 to 87%, where there was no difference between NaCl and KCl treatments (Figure 2). Figure 2 shows that none of the varieties could be described as salinity-tolerant.Although IR28 had the highest relative grain yield (grain weight at salinity-stress/grain weight at control, as %) of 43%, NSICRc106 had maginally higher grain yield under NaCl (though not significant different), but a low relative grain yield of 30% (as it grew better than IR28 under good condition).The percentage of ripened grains was most affected by indicates that the percentage of deformed spikelets of the total number ranges from 10 to 25%.A score of 3 indicates that the percentage of deformed spikelets of the total number ranges from 25 to 50%.A score of 4 indicates that the percentage of deformed spikelets of the total number is over 50%.

Ion Content in Plants
The Na + content in shoots of all varieties in the NaCl treatment group at all stages was higher than that in the KCl treatment and control groups (Figures 5a, 5d, and 5g).The K + contents in shoots of all varieties at all stages in the NaCl treatment group was lower than that in the KCl treatment and control groups (Figures 5b, 5e, and 5h).
There was no difference in the K + content in shoots of all varieties between the KCl treatment and control groups at 30 DAT (Figure 5b); after that, the K + content in shoots in the KCl treatment group was higher or the same as the control (Figures 5e and 5h).The Cl − content in shoots of all varieties in the NaCl and KCl treatment groups at all stages was higher than that in the control group (Figures 5c, 5f, and 5i).The Cl − content in shoots of IR28 in the KCl treatment group at 30 DAT and at the full heading stage was higher than that in the NaCl treatment group (Figures 5c and 5f).The Cl − content in shoots of NSICRc106 in the KCl treatment group at 30 DAT was also higher than that in the NaCl treatment group (Figure 5c).There was no difference in the Cl − content in shoots of Nipponbare between the NaCl and KCl treatment groups (Figures 5c, 5f, and 5i).1a).Th Figure 2).Thes reasons that the uss whether K + ng KCl treatmen ng KCl treatm ment does not r addition, the K 5).Therefore, reported that K us, it is conclud of IR28 and N milar in both io ive value of dr y weight and gr Ion toxicity in rice, barley, and wheat has been thought to be due to Na + rather than Cl − .However, in recent years, excess Cl − was implicated in the reduction of chlorophyll content (Tavakkoli et al., 2011).Cl − toxicity cannot be ignored in these crops (Tavakkoli et al., 2011;Khare et al., 2015;Kumar & Khare, 2015).Furthermore, Na + and Cl − have an additive effect to suppress the yield of rice (Kumar & Khare, 2016).However, in our study, the degree of suppression of the grain yield and dry matter in KCl and NaCl treatment were similar, thus, the additive effect of Na + and Cl − was not observed.One possible reason for this discrepancy is the existence of counter ions.It is impossible to make ions other than Na + and Cl − equal.Several studies were done using Na + dominant and Cl − dominant treatments, which are designed to give equimolar concentrations of Na + and Cl − (generated from various salts of Na + and Cl − ) to avoid increasing particular counter-anions or counter-cations (Kingsbury & Epstein, 1986;Luo et al., 2005;Tavakkoli et al., 2011;Khare et al., 2015;Kumar & Khare, 2016).However, the ameliorative and/or deteriorative effects of counter-anions or counter-cations may not be completely removed in such experiments.For example, Ca 2+ availability can change the salinity tolerance in plants (Muhammed, Akbar, & Neue, 1987;Anil et al., 2005).However, K + is the only counter ion in this study and, as mentioned above, it is unlikely that K + has the toxicity in this study.The other possible reason is the length of the salt treatment.The results in the short-term salt treatment at the early growth stage indicate that Na + toxicity is the main factor (Lin & Kao, 2001;Tsai et al., 2004;Luo et al., 2005).On the other hand, the additive effect of Na + and Cl − was observed in the salt treatments from early-reproductive to maturity stages (Kumar & Khare, 2016).This indicates the possibility that Cl − toxicity appears more clearly under long-term salt treatment.The reason why the additive effect of Na + and Cl − was not observed in this study is that long-term salt treatments from the seedling stage to the maturity stage are carried out, Cl − toxicity was more severe and Na + toxicity was not detected.
In this experiment, three varieties differing in salinity tolerance at the seedling stage were used, and it did not coincide with the salinity tolerance at 30 DAT, the full heading stage, and the maturity stage.The relative whole plant dry weight at 30 DAT under NaCl decreased by 44% for Nipponbare, 36% for IR28, and 27% for NSICRc106 (Figure 1a).However, there was no difference among the varieties relating to the reduction of whole plant dry weight under NaCl treatment, with the range from 25 to 27% and 24 to 30% at the full heading stage and the maturity stage, respectively (Figures 1b and 1c).The salinity tolerance in rice is known to differ during the growing stage (Heenan, Lewin, & Maccaffery, 1988;Makihara, Tsuda, Morita, Hirai, & Kuroda, 1999), and their tolerance at different stages seems to be controlled by independent genes (Singh & Flowers, 2010).The different mechanism of salinity tolerance at different growth stages may relate to the result that the Cl − toxicity is critical more than Na + toxicity under long-term salinity conditions.Individual research on Cl − toxicity at different growth stages would help in understanding the mechanism of salinity tolerance in rice.
Salinity tolerance can be classified into three main mechanisms: ion exclusion, osmotic tolerance, and tissue tolerance (Munns & Tester, 2008), and our results suggest the importance of Cl − exclusion in salinity-stressed rice.
Recently, it was shown that osmotic tolerance and tissue tolerance to Na + and/or Cl − should be the main foci for further improvement of salinity tolerance in barley, wheat and durum wheat (Genc et al., 2016).However, in rice, it is not clear which mechanism is more critical in salinity tolerance.Further study on this is necessary to understand salinity tolerance in rice.

Conclusion
The results of long-term NaCl and KCl treatments using three rice varieties with different salinity tolerances suggested that plant growth and grain productivity of rice under long-term salinity conditions is reduced by Cl − toxicity rather than Na + toxicity.The understanding of the mechanism to reduce Cl − accumulation in plants is necessary to improve salinity tolerance in rice.