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Article

Comparative Physiological and Gene Expression Analyses Provide Insights into Ion Transports and Osmotic Adjustment of Sweet Sorghum under Salt Stress

by
Jie Kang
†,
Xiao-Long Wang
†,
Shi-Jie Yan
†,
Huan Guo
* and
Yan-Nong Cui
*
College of Grassland Agriculture, Northwest A&F University, Yangling, Xianyang 712100, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(8), 1849; https://doi.org/10.3390/agronomy14081849 (registering DOI)
Submission received: 1 August 2024 / Revised: 18 August 2024 / Accepted: 19 August 2024 / Published: 21 August 2024
(This article belongs to the Special Issue Advances in Stress Biology of Forage and Turfgrass)
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Abstract

:
Sweet sorghum is an important sugar crop and forage with a strong tolerance to soil salinity. We have previously analyzed the ion accumulation traits and transcriptome of a sweet sorghum cultivar under NaCl treatments. However, the mechanisms underlying Na+, K+, Cl−, and NO3− transports and the osmotic adjustment of sweet sorghum under salt stresses need further investigations. In this study, the growth, photosynthesis, inorganic ion and organic solute contents, and leaf osmotic adjustment ability of the sweet sorghum cultivars “Lvjuren” and “Fengtian” under NaCl treatments were determined; meanwhile, the expressions of key genes associated with the Na+, K+, Cl−, and NO3− transport were analyzed using the qRT-PCR method. The results showed that NaCl treatments more severely inhibited the growth and photosynthesis of “Lvjuren” than those of “Fengtian”. After NaCl treatments, “Fengtian” could more efficiently restrict the overaccumulation of Na+ and Cl− in leaf blades than “Lvjuren” by withholding large amounts of Na+ in the roots or reserving high quantities of Cl− in the leaf sheaths, which could be attributed to the upregulated expressions of SbNHX2, SbHKT1;4, SbHKT1;5, SbCLCc, and SbCLCg or the downregulated expression of SbNPF6.4. “Fengtian” exhibited significantly lower leaf osmotic potential but higher leaf water potential and turgor pressure under NaCl treatments, suggesting that the former possessed a stronger osmotic ability than the latter. The contents of K+, NO3−, soluble sugar, and betaine in leaf blades, as well as the contributions of these osmolytes to the leaf osmotic potential, in “Fengtian” were significantly higher than those in “Lvjuren”. In addition, the upregulated expressions of SbAKT1, SbHAK5, SbSKOR, SbNPF3.1, SbNPF6.3, and SbNPF7.3 should be responsible for maintaining K+ and NO3− homeostasis under NaCl treatment. These results lay a foundation for uncovering the salt tolerance mechanisms of sweet sorghum and large-scale cultivation of this species in saline areas.

1. Introduction

Soil salinity has been a major threat to agricultural productivity worldwide [1,2]. Most varieties of crops are not capable of achieving adequate productions when grown in saline soils [3]. Differently, plant species that are utilized for other aspects such as bioenergy and feeding can generally acclimatize well to harsh habitats [3,4,5]. Therefore, understanding how these plants adapt to salt stress is a high priority for food security and ecological restoration.
Sweet sorghum [Sorghum bicolor (L.) Moench] is a C4 plant within the Poaceae family. This species shows many advantages in agronomic traits: for example, it contains high fermentable sugars in stalks and leaves, which makes it a desirable sugar crop [6,7]; due to the fast growth rate, large biomass, and abundant dietary nutrient contents, sweet sorghum also serves as a valuable forage [8,9]. In addition, unlike most traditional cereals, sweet sorghum can tolerate various abiotic stresses well, and its cultivation requires relatively low fertilizer and irrigation inputs [10,11]. Therefore, this species is regarded as a source for understanding stress resistance mechanisms and discovering genetic resources to improve crop adaptabilities to adverse environments [12,13].
Salinity primarily exerts osmotic stress on plants, which inhibits water and nutrient acquisitions in roots [14,15]. It has been demonstrated that osmotic adjustment (OA) is the most effective strategy of plants to cope with osmotic stress, and it is achieved by accumulating large amounts of soluble osmolytes, including inorganic ions (e.g., K+ and NO3−) and organic metabolites (e.g., free proline, soluble sugars, and betaine) in cells to enhance water influx and maintain tissue hydration status [16]. With the continuous uptake of salt ions (e.g., Na+ and Cl−) by roots, these ions in plant tissues unavoidably accumulate to toxic levels that deteriorate the ultrastructure of cells and hamper photosynthesis, nutrient balance, and cellular metabolism [1,17]. Osmotic stress and ion toxicity eventually lead to the overproduction of reactive oxygen species (ROS) that cause damages to cell membranes, proteins, and lipids, leading to an increased content of malondialdehyde (MDA) in plant tissues [18]. It has been discovered that restricting Na+ transport into shoots or leaves is a key strategy of Poaceae plants to avoid Na+ toxicity [19,20]. However, the strategies of these species to deal with Cl− toxicity have not been well documented. We have preliminarily investigated the ion accumulation traits of sweet sorghum and found that the Na+ content in its shoots is substantially lower than in the roots, while the shoot Cl− content is much higher than the root Cl− content [13]. Thus, the adaptative strategies of sweet sorghum to Na+ and Cl− toxicity are probably different. Nevertheless, the mechanisms involved in OA and alleviating ion toxicity of sweet sorghum still need further investigations.
The ion transporters/channels play important roles in plants to alleviate Na+ and Cl− toxicities. For example, the Na+/H+ antiporter NHX1 can transport excessive Na+ into vacuoles to relieve the effects of Na+ on the metabolic processes in cells [21]; the high-affinity potassium transporter HKT1;5 mediates the root xylem Na+ unloading to restrict Na+ transport into shoots [22]. The chloride channel (CLC) and nitrate transporter 1/peptide transporter (NPF) are involved in Cl− or NO3− transport [23,24,25]. Particularly, CLCc and CLCg mediate the vacuolar sequestration of Cl−; NPF6.4 mediates Cl− uptake into cells. The maintenance of high K+ and NO3− levels in tissues is essential for plants’ adaptation to salinity [15]. The potassium transporter HAK5 and shaker-type potassium channel AKT1 constitute important pathways for K+ absorption; the potassium outward rectifier SKOR governs the translocation of K+ into shoots [26,27]; and the NPF6.3 and NPF7.3 mediate root NO3− uptake and reallocation of NO3−, respectively [28,29]. Although the transcription factor WRKY50 and Casparian strip membrane domain protein CASP4 have been found to play important roles in Na+ exclusion in sweet sorghum [30,31], the functions of ion transporters and channels in the salt adaptation of sweet sorghum are still unclear. Our previous transcriptome study has preliminarily identified the transcripts possibly associated with ion transports in sweet sorghum [13]; these genes might play essential roles in sweet sorghum to adapt to saline environments.
In this study, we first compared the growth between sweet sorghum cultivars “Lvjuren” and “Fengtian” under NaCl treatments, then analyzed ion accumulation traits and organic osmolytes (free proline, soluble sugar, and betaine) contents in the tissues of both cultivars. We also compared their OA abilities under salt stress by determining leaf osmotic potential, water potential, and turgor pressure. To preliminarily uncover the molecular basis underlying ion transports in sweet sorghum, we finally analyzed the expressions of key genes related to Na+, K+, Cl−, and NO3− transports in “Lvjuren” and “Fengtian” under salt treatment using the qRT-PCR method.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

We have preliminarily analyzed the growth of 12 sweet sorghum cultivars under 100 mM NaCl treatment for 10 d. As shown in Figure S2, among these cultivars, the shoot growth of “Fengtian” was less affected under salt treatment, while the leaves of “Paka”, “Hainiu”, “Lvjuren”, “Keller”, and “Dale” were severely impaired by salt treatment. Moreover, the decrease in the shoot fresh weight in “Fengtian” was much lower than that in other cultivars under NaCl treatment (Figure S3), suggesting that “Fengtian” possessed a prominent salt tolerance. Therefore, in this study, we selected “Fengtian” and “Lvjuren” to compare their responses to NaCl treatments. The seeds of both cultivars were surface-sterilizing in 75% ethanol and sown in culture pots filled with coarse silica sands. After germination, seedlings were cultured with Hoagland solution in a growth chamber as described by Guo et al. [13].
After one week, the seedlings were thinned out (one seedling/pot). When seedlings of “Lvjuren” and “Fengtian” grew to three weeks old, uniform seedlings were selected and transferred to Hoagland solution containing 0 (control), 50, 100, and 200 mM NaCl for 15 d, and then tissue samples were harvested. Six replicates were used for each sampling (n = 6)
For the gene expression experiment, 3-week-old seedlings of “Lvjuren” and “Fengtian” were cultured as in the above-mentioned methods. Then, the seedlings were treated with Hoagland solution containing 200 mM NaCl for 0 (control), 6, and 48 h, and then the root, leaf sheath, and leaf blade samples were collected, respectively. Three replicates were used for each sampling (n = 3).

2.2. Determination of Growth-Related Parameters

The plant height (PH) and tissue fresh weight (FW) were measured first. Then, all samples were thoroughly dried at 80 °C to measure the dry weight (DW), and the shoot relative water content (RWC) was calculated as (FW − DW)/DW [15].

2.3. Measurements of Photosynthesis-Related Parameters

The LI-6800 Portable Photosynthesis System was used to measure the net photosynthesis rate (Pn) and stomatal conductance (Gs). The fully expanded leaf blades were settled in the leaf chamber, and during the measurement, in the leaf chamber, the temperature was set at 25 °C, relative humidity at 60%, photosynthetic photon flux density at 800 μmol m−2 s−1, and CO2 concentration at 600 μmol mol−1 [13].
Fresh leaf blade samples were crushed with 10 mL of 80% acetone and 95% ethanol (1:1, v/v). After centrifuging, the absorbances at 645 and 663 nm in the supernatant were determined to calculate the chlorophyll contents [32].

2.4. Determination of Malondialdehyde (MDA) Content in Leaf Blades

As MDA is a production of lipid peroxidation that can reflect the damages of cell membranes [18], we determined the MDA content in the leaf blades after salt treatments. Briefly, fresh leaf blade samples were homogenized with 10% trichloroacetic acid, and then the MDA in leaf blades was extracted with 0.6% thiobarbituric acid. Finally, the absorbances at 532 and 600 nm in the supernatant were determined to calculated the MDA content [33].

2.5. Measurement of Tissue Ion Contents

The oven-dried root, leaf sheath, and leaf blade samples were immersed in 100 mM glacial acetic acid and incubated at 100 °C for 2 h, and then the tissue Na+ and K+ contents were determined using a flame spectrophotometer (Model 410, Sherwood Scientific, Ltd., Cambridge, UK) [34]. Tissues samples were immersed in deionized water at 100 °C for 2 h, and then the tissue Cl− content was determined using a chloride analyzer (Model 926, Sherwood Scientific Ltd., Cambridge, UK) [35], and the tissue NO3− content was detected using the colorimetric methods with salicylic acid [27].

2.6. Determination of Free Proline, Soluble Sugar, and Betaine Contents

Fresh leaf blades were crushed with 3% sulfosalicylic acid and treated with 2.5% acid-ninhydrin and glacial acetic acid at 100 °C. After leaching with toluene, the absorbance at 520 nm was measured to calculate the free proline content [36].
Fresh leaf blades were immersed in 80% ethanol at 100 °C. Then, the soluble sugar content was determined by the classic anthrone colorimetric method [37].
The betaine content in leaf blades was determined with a Reinecke Salt Kit (Comin Biotechnology, Co., Ltd., Suzhou, China) [38].

2.7. Determination of Leaf Water Potential, Osmotic Potential, and Turgor Pressure

The leaf blade was placed in a pressure chamber (C-52, Wescor, Logan, UT, USA) with the cut end exposed to air, and then the pressure in the chamber was gradually increased; when water drops emerged at the cut end, the value of the pressure in the pressure chamber was recorded to calculate the leaf water potential (Ψw). The leaf blade samples were transiently frozen; after thawing, the leaf saps were collected, and the osmolality concentration in the leaf saps was determined using the Osmomat-070 (Gonotec GmbH, Berlin, Germany) to calculate the osmotic potential (ΨS). Then the contribution of each osmolyte to leaf ΨS was calculated according to Ma et al. [39]. The leaf turgor pressure (Ψt) was calculated as Ψw − ΨS [40].

2.8. Real-Time Quantitative PCR Analysis

Our previous transcriptome study identified several differentially expressed genes (DEGs) probably involved in ion transport under NaCl treatment [13]. In this study, we selected key DEGs associated with Na+ (NHX2, HKT1;4, and HKT1;5), K+ (AKT1, HAK5, and SKOR), Cl− (CLCc, CLCg, and NPF6.4), and NO3− (NPF3.1, NPF6.3, and NPF7.3) transports to compare their expression levels in “Lvjuren” and “Fengtian” under NaCl treatment using the qRT-PCR method.
The total RNA in the tissue samples was extracted using the Trizol reagent (Tiangen, Beijing, China). Then, RNA samples were converted into cDNA. The gene-specific primers were designed based on our RNA-sequencing data [13]. Finally, qRT-PCR was performed with the QuantStudio7 Flex PCR Thermocycler (ThermoFisher Scientific, Waltham, MA, USA) [41].

2.9. Data Analysis

Six replicate seedlings were used for all physiological parameter measurements (n = 6), and three replicate seedlings were used for qRT-PCR analysis (n = 3). The data were subjected to one-way analysis of variance (Tukey’s HSD, p < 0.05) using SPSS 19.0.

3. Results

3.1. Effects of NaCl Treatments on the Growth of Sweet Sorghum Cultivars

Under the control condition, “Fengtian” showed significantly higher plant height (PH), tissue fresh weight (FW), and dry weight (DW) than “Lvjuren” (Figure 1E,F,H). Under 50 mM NaCl treatment, the PH, tissue biomass, and leaf malondialdehyde (MDA) content in both cultivars remained at the control levels, while the shoot relative water content (RWC) in “Lvjuren” was significantly reduced (Figure 1 and Figure S1).
Compared with the control, 100 mM NaCl treatment significantly decreased the PH, tissue FW, and shoot RWC but increased the leaf MDA content of both cultivars (Figure 1 and Figure S1), indicating that this treatment inhibited the growth of sweet sorghum. However, no significant difference in the growth between “Lvjuren” and “Fengtian” was observed under this treatment (Figure 1 and Figure S1).
After 200 mM NaCl treatment, the shoot growth of seedlings was visually inhibited, while the leaf blades of “Fengtian” were wider and sturdier than those of “Lvjuren”; correspondingly, the PH, tissue FW, and DW, as well as the shoot RWC, in the former were significantly higher than those in the latter (Figure 1). In contrast, the leaf MDA content in “Fengtian” was significantly lower than that in “Lvjuren” under 200 mM NaCl treatment (Figure S1).

3.2. Effects of NaCl Treatments on the Photosynthesis of Sweet Sorghum Cultivars

As shown in Figure 2A, in comparison with the control, 50 and 100 mM NaCl significantly decreased the net photosynthesis rate (Pn) in “Lvjuren”, while they had no effect on this parameter in “Fengtian” (Figure 2A). Under 200 mM NaCl treatment, the Pn and stomatal conductance (Gs) in both cultivars showed decreasing trends, but both parameters in “Fengtian” were significantly higher than in “Lvjuren” (Figure 2A,B). Compared with the control, 50 mM NaCl treatment did not affect the chlorophyll (Chl) a and b contents, 100 mM NaCl treatment significantly reduced the Chl a content in “Lvjuren”, and 200 mM NaCl treatment significantly decreased the Chl a and b contents in both cultivars (Figure 2C,D).

3.3. Ion Contents in Tissues of Sweet Sorghum Cultivars under NaCl Treatments

As shown in Figure 3A, compared with the control, the Na+ content in the roots, leaf sheaths, and leaf blades of both cultivars was drastically increased under NaCl treatments, and the content of Na+ in tissues followed the pattern of roots > leaf sheaths > leaf blades. Notably, “Fengtian” showed significantly higher root and leaf sheath Na+ contents but lower leaf blade Na+ content compared with “Lvjuren” (Figure 3A). After 100 and 200 mM NaCl treatments, the tissue K+ content in both cultivars was significantly decreased compared with that under control conditions (Figure 3B). However, the leaf K+ content in “Fengtian” was significantly higher than that in “Lvjuren” under all salt treatments (Figure 3B).
In comparison with the control, the Cl− content in both cultivars was also significantly increased under salt treatments, but the content of Cl− in tissues followed the pattern of leaf sheaths > leaf blades > roots (Figure 3C). Additionally, under salt treatments, “Fengtian” exhibited significantly higher leaf sheath Cl− content but lower leaf blade Cl− content than “Lvjuren” (Figure 3C). Compared with the control, all NaCl treatments significantly decreased the NO3− content in the leaf sheaths of both cultivars (Figure 3D). It is noteworthy that the NO3− content in the leaf blades of “Lvjuren” was gradually reduced with the increase in the external NaCl concentrations, while this parameter in the leaf blades of “Fengtian” under NaCl treatments was maintained at the control levels (Figure 3D).

3.4. Effect of NaCl Treatments on the Leaf Free Proline, Soluble Sugar, and Betaine Contents of Sweet Sorghum Cultivars

The contents of free proline, soluble sugar, and betaine in the leaf blades of both cultivars showed significantly increasing trends under NaCl treatments (except for the betaine content under 50 mM NaCl treatment; Figure 4). Notably, under 100 and 200 mM NaCl treatment, the free proline content in “Lvjuren” was significantly higher than that in “Fengtian”, while the soluble sugar and betaine contents in the former were significantly lower than those in the latter (Figure 4).

3.5. Effects of NaCl Treatments on the Leaf OA Capacity of Sweet Sorghum Cultivars

Although the leaf water potential (Ψw) in both cultivars significantly lowered under NaCl treatments, this parameter in “Fengtian” was much higher than in “Lvjuren” (Table 1), suggesting that the leaf hydration status in the former was better than in the latter. In comparison with the control, the leaf osmotic potential (Ψs) in both cultivars was gradually decreased after NaCl treatments, and this parameter in “Fengtian” was significantly lower than that in “Lvjuren” (Table 1). Additionally, although the leaf turgor pressure (Ψt) in both cultivars was significantly decreased after NaCl treatment (except for the leaf Ψt in “Fengtian” under 50 mM NaCl treatment; Table 1), this parameter in “Fengtian” was much higher than that in “Lvjuren” under all salt conditions (Table 1).
As shown in Table 2, the contributions of K+ and NO3− to leaf Ψs in both cultivars were significantly decreased after salt treatments; however, both parameters in “Fengtian” were significantly higher than those in “Lvjuren” under 100 and 200 mM NaCl treatments. The contribution of free proline to Ψs was negligible (<0.2%) under all growth conditions (Table 2). Interestingly, the contributions of soluble sugar and betaine to leaf Ψs showed declining trends in “Lvjuren” but increasing trends in “Fengtian” under salt treatments (Table 2).

3.6. The Expressions of Genes Related to Na+ and K+ Transport in Sweet Sorghum under NaCl Treatment

NHXs and HKTs are key proteins mediating Na+ transport in plants [19,42]. As shown in Figure 5A, SbNHX2 in the roots of “Lvjuren” and “Fengtian” showed an increasing trend after NaCl treatment. Moreover, its expression in the roots of “Fengtian” was much higher than that of “Lvjuren” (Figure 5A). SbHKT1;4 was dramatically upregulated in the leaf sheaths of both cultivars after 6 h of treatment, and the increase in “Fengtian” was higher than that in “Lvjuren” (Figure 5B). SbHKT1;5 in the roots of both cultivars (especially in “Fengtian”) was also significantly upregulated after NaCl treatment (Figure 5C).
For K+ transport, we selected AKT1, HAK5, and SKOR from our transcriptome data to investigate their expressions in “Lvjuren” and “Fengtian” after NaCl treatment. As shown in Figure 5D–F, the expressions of these genes in the roots of both cultivars were significantly increased after 6 h of treatment, and their expressions in “Fengtian” were clearly higher than those in “Lvjuren”. In addition, the expression of SbHAK5 in the leaf blades of both cultivars was also significantly induced by 48 h of treatment (Figure 5E).

3.7. The Expressions of Key Genes Related to Cl− and NO3− Transport in Sweet Sorghum under NaCl Treatment

CLCc and CLCg mediate the sequestration of Cl− into vacuoles [23,24]. As shown in Figure 6A,B, SbCLCc and SbCLCg in all tissues (especially in the roots) of “Lvjuren” and “Fengtian” were significantly increased after 6 h of treatment; moreover, both genes in the leaf sheaths were also drastically upregulated when the treatment time was prolonged to 48 h (Figure 6A,B). NPF6.4 mediates the Cl− uptake into cells [25]. As shown in Figure 6C, SbNPF6.4 in the roots of both cultivars was downregulated after 6 h of treatment, and its expression in “Fengtian” was significantly lower than that in “Lvjuren”. In addition, SbNPF6.4 in the roots of both cultivars was downregulated to extremely low levels after 48 h of treatment (Figure 6C).
NPF3.1 is involved in NO3− transport in plants [43]. It was obvious that SbNPF3.1 in all tissues of “Lvjuren” and “Fengtian” showed a substantially increasing expression under NaCl treatment (Figure 6D). NPF6.3 and NPF7.3 are involved in root NO3− uptake and translocation of NO3− into shoots, respectively [28,29]. Under NaCl treatment, the expressions of SbNPF6.3 and SbNPF7.3 in the roots of “Lvjuren” and “Fengtian” were greatly increased, and their expressions in “Fengtian” were clearly higher than those in “Lvjuren” (Figure 6E,F).

4. Discussion

4.1. Sweet Sorghum Is a Typical Na+ Exclusion Species That Can Be Used as a Model to Elucidate the Mechanisms of Poaceae Plants to Alleviate Na+ Toxicity

Although the sweet sorghum cultivar “Lvjuren” grew faster than “Fengtian” under normal conditions, the detrimental effects of NaCl treatments on the plant height, tissue biomass, and net photosynthesis rate of “Lvjuren” were much more severe than those of “Fengtian” (Figure 1 and Figure 2). Malondialdehyde (MDA) is a product formed during the peroxidation of lipids, which is used to reflect the damages of cell membranes under various abiotic stresses [18,44]. In this study, although the leaf MDA content in both sweet sorghum cultivars was substantially increased under 100 and 200 mM NaCl treatments, this parameter in “Lvjuren” was much higher than that in “Fengtian” (Figure S1), indicating that salt treatments exerted more significant membrane damages to “Lvjuren” than to “Fengtian”. All these results suggested that “Fengtian” possessed a relatively stronger salt tolerance compared with “Lvjuren”.
The exclusion of Na+ from photosynthetic leaves is essential for Poaceae plants to deal with Na+ toxicity [19,20]. We have previously analyzed the ion accumulation traits in “Lvjuren” under NaCl treatments and found that this cultivar could accumulate much higher Na+ in roots than in shoots [13]. However, the correlation of this observation with the salt tolerance of sweet sorghum has not been well documented. In this study, the root Na+ content in the salt-tolerant cultivar “Fengtian” was significantly higher than that in “Lvjuren”, while the leaf blade Na+ content in the former was much lower than that in the latter, suggesting that the large accumulation of Na+ in roots to restrict Na+ overaccumulation in leaf blades is an effective strategy of sweet sorghum to alleviate the Na+ toxicity. It has been reported that durum wheat (Triticum turgidum) can accumulate a great quantity of Na+ in the leaf sheaths under salt stresses [45]. Similarly, the Na+ content in the leaf sheaths was much higher than that in the leaf blades of sweet sorghum under NaCl treatments (Figure 3A). Moreover, “Fengtian” displayed a significantly higher leaf sheath Na+ content compared with “Lvjuren” (Figure 3A). Therefore, the retention of Na+ in leaf sheaths should also contribute to excluding Na+ from leaf blades under saline conditions.
We have previously investigated the transcriptome of “Lvjuren” under salt stresses and identified several differentially expressed genes associated with ion transports after salt treatments [13]. Nevertheless, the expression patterns of these genes and their function in the adaptation of sweet sorghum to salinity are still unclear. Therefore, we compared the expressions of key genes under NaCl treatment in “Lvjuren” and “Fengtian” in this study. The vacuolar Na+ sequestration helps to accumulate Na+ in specific tissues, and NHX1 is thought to mediate this process [42,46]. However, in the transcriptome data of sweet sorghum, no transcript of NHX1 is identified; instead, a transcript of SbNHX2 undergoes significant changes after NaCl treatment [13]. In this study, SbNHX2 in the roots of “Lvjuren” and “Fengtian” was significantly upregulated under NaCl treatment (Figure 5A). Moreover, its expression in “Fengtian” (showing a relatively higher root Na+ content) was clearly higher than that in “Lvjuren” (showing a relatively lower root Na+ content) (Figure 5A). Considering that NHX2 also localizes on the tonoplast [47], we speculate that SbNHX2 probably is a candidate gene for regulating Na+ accumulation in the roots of sweet sorghum.
HKT1;4 and HKT1;5 have been proven to mediate the root xylem Na+ unloading to restrict Na+ transport into the shoots [2,48]. In this study, SbHKT1;4 and SbHKT1;5 in roots were significantly induced by NaCl treatment, and their expressions in “Fengtian” were much higher than in “Lvjuren” (Figure 5B,C), suggesting that SbHKT1;4 and SbHKT1;5 should be indispensable components for restricting Na+ translocation into shoots of sweet sorghum. It has been found that HKT1;4 in durum wheat is also expressed in the leaf sheaths [48]. Notably, SbHKT1;4 in the leaf sheaths of sweet sorghum cultivars (especially in “Fengtian”) was substantially upregulated after NaCl treatment (Figure 5B), indicative of a possible role of SbHKT1;4 in the Na+ accumulation in leaf sheaths. It has been reported that grain sorghum and rice (Oryza sativa) accumulate more Na+ in leaves than in roots under salt stresses [49,50]. Therefore, the identified genes associated with Na+ transport in sweet sorghum such as NHX2, HKT1;4, and HKT1;5 should be important gene resources for the improvement of Na+ exclusion abilities in traditional crops through genetic approaches. Taken together, sweet sorghum could effectively restrict the overaccumulation of Na+ in leaf blades under saline conditions, which could be regarded as a promising model for clarifying Na+ exclusion mechanisms of Poaceae plants.

4.2. The Large Accumulation of Cl− in Leaf Sheaths Play a Vital Role in Sweet Sorghum to Cope with Cl− Toxicity

We have previously found that the Cl− content in shoots of “Lvjuren” was significantly higher than that in roots under NaCl treatment [13]. However, the strategies in sweet sorghum, even in Poaceae plants, are still unclear. In the present study, the sweet sorghum cultivar “Fengtian” also accumulated much more Cl− in shoots under NaCl treatments (Figure 3C), suggesting that different from Na+ transport, sweet sorghum somehow lacks abilities to restrict Cl− transport into shoots. Interestingly, in the shoots of sweet sorghum, Cl− was mainly accumulated in the leaf sheaths rather than in the leaf blades (from the quantitative perspective, the Cl− and Na+ contents in leaf blades were at the same levels; Figure 3A,C). Furthermore, “Fengtian” showed significantly higher leaf sheath Cl− content but lower leaf blade Cl− content compared with “Lvjuren” (Figure 3C). Given that “Fengtian” displayed a stronger tolerance to salinity than “Lvjuren” (Figure 1 and Figure 2), the reservation of Cl− in the leaf sheaths should be a vital strategy of sweet sorghum to avoid Cl− overaccumulation in leaf blades under saline conditions.
It has been proposed that the nitrate transporter 1/peptide transporter NPF6.4 in maize (Zea mays) possibly mediates Cl− uptake by roots [25]. However, the expression pattern of this gene under salt stresses has not been reported. In this study, SbNPF6.4 in the roots of “Lvjuren” and “Fengtian” showed a significantly decreasing trend after salt treatment, and the decrease in “Fengtian” was larger than that in “Lvjuren” (Figure 6C), suggesting that the downregulated expression of SbNPF6.4 might play an essential role in reducing Cl− uptake by the roots. In addition, NPF6.4 in maize is found to also express in multiple cell types of shoots [25]. In this study, SbNPF6.4 in the leaf sheaths and leaf blades was increased significantly after NaCl treatment (Figure 6C), indicating that SbNPF6.4 might also regulate Cl− transport in shoots of sweet sorghum.
The sequestration of Cl− into vacuoles can not only reduce the disturbance of Cl− on cellular metabolisms but also accumulate this ion in specific tissues, which is mediated by chloride channels CLCc and CLCg [51]. Although the expressions of CLCc and CLCg in the roots and leaves of several plants are substantially increased under saline conditions [23,24,52], the expression patterns of homologous genes of CLCc and CLCg in Poaceae plants have not been well documented. In the present study, SbCLCc and SbCLCg in all tissues of both sweet sorghum cultivars were significantly upregulated after short-term NaCl treatment (Figure 6A,B). Notably, after long-term salt treatment, these two genes were highly expressed in the leaf sheaths (Figure 6A,B). Therefore, SbCLCc and SbCLCg play indispensable roles in the large accumulation of Cl− in the leaf sheaths.

4.3. K+, NO3−, Soluble Sugars, and Betaine Should Be Important for the Osmotic Adjustment of Sweet Sorghum under NaCl Treatments

It was observed that “Fengtian” possessed significantly higher shoot water content and leaf water potential than “Lvjuren” under NaCl treatments (Figure 1G and Table 1), suggesting that the hydration status in the former was better than in the latter. Moreover, the leaf osmotic potential in “Fengtian” was significantly lower than that in “Lvjuren”, while the leaf turgor pressure in the former was significantly higher than that in the latter under NaCl treatments (Table 1), suggesting that “Fengtian” displayed a stronger OA ability than “Lvjuren” under saline conditions.
K+ and NO3− are two important inorganic osmolytes in plants, while their accumulations in plant tissues are generally inhibited by salt stresses as a consequence of the well-known competition of Na+ and Cl− for ion binding sites of K+ and NO3− transporters [1,53]. Notably, K+ and NO3− contents in the leaf blades of “Fengtian” were maintained at relatively high levels, while they were significantly reduced in “Lvjuren” under NaCl treatment (Figure 3B,D). Furthermore, the contributions of K+ and NO3− to the leaf osmotic potential of “Fengtian” were significantly higher than those of “Lvjuren” under salt treatments (Table 2), which should be an important reason why the former displayed better hydration status and stronger OA ability than the latter.
AKT1 and HAK5 constitute important pathways for K+ acquisition by plant roots, and SKOR mediates the transport of K+ from roots into shoots [26,54]. SbAKT1, SbHKA9, and SbSKOR in the roots of both sweet sorghum cultivars were drastically upregulated after NaCl treatment, and moreover, all these genes in “Fengtian” showed significantly higher expressions than in “Lvjuren” (Figure 5D,F), suggesting the upregulation of SbAKT1, SbHAK5, and SbSKOR should be closely associated with maintaining the K+ homeostasis in sweet sorghum. In addition, SbHAK5 in the leaf blades of both cultivars was also significantly upregulated after NaCl treatment (Figure 5E), indicating that SbHAK5 is possibly also involved in K+ transport in the leaf blades.
NPF6.3 and NPF7.3 control root NO3− uptake and translocation of NO3− into shoots, respectively [29,30]. It was noticed that after NaCl treatment, the expressions of SbNPF6.3 and SbNPF6.4 in the roots of sweet sorghum were substantially upregulated (Figure 6E,F), which should play a vital role in maintaining shoot NO3− homeostasis. NPF3.1 has been proven to also mediate NO3− uptake into cells [43]. Interestingly, the expression of SbNPF3.1 in all tissues of both sweet sorghum cultivars was drastically increased after NaCl treatment (Figure 6D). Therefore, we speculate that the upregulated expression of SbNPF3.1 also contributed to maintaining NO3− homeostasis under saline conditions.
Although the free proline content in the leaf blades of both cultivars under NaCl treatments was significantly increased, its contribution to the leaf osmotic potential was negligible (Figure 4A and Table 2), suggesting that the OA of sweet sorghum should not mainly rely on the biosynthesis of free proline. Except for serving as an organic osmolyte, proline is also an antioxidant in plants [55]. The cell membranes of “Lvjuren” were more severely damaged than those of “Fengtian” under 100 and 200 mM NaCl treatments (Figure S1). Correspondingly, the leaf free proline content in “Lvjuren” was significantly higher than that in “Fengtian” (Figure 4A), suggesting that free proline should play an important role in alleviating the oxidative stress of sweet sorghum under saline conditions. It was observed that the contents of soluble sugars and betaine in “Fengtian” were also increased under NaCl treatment, and the contributions of these metabolites to leaf osmotic potential were substantially increased in “Fengtian” after salt treatment (Figure 4B,C and Table 2), indicating that the increased biosynthesis of soluble sugars and betaine should be essential for leaf osmotic adjustment of sweet sorghum under saline conditions.

5. Conclusions

The reservation of Na+ in the roots or Cl− in the leaf sheaths could effectively restrict the overaccumulation of these salt ions in the leaf blades and, therefore, plays a vital role in alleviating ion toxicity to sweet sorghum under salt stresses. The maintenance of high K+ and NO3− contents and large biosynthesis of soluble sugars and betaine in leaf blades should be important for osmotic adjustment in sweet sorghum under saline conditions. The upregulated expressions of SbNHX1, SbHKT1;4, SbHKT1;5, SbNPF6.4, SbCLCc, and SbCLCg and the downregulated expression of SbNPF6.4 should play key roles in alleviating Na+ and Cl− toxicities in sweet sorghum under salt stresses (Figure 7). Moreover, SbAKT1, SbHAK5, SbSKOR, SbNPF3.1, SbNPF6.3, and SbNPF7.3 are closely involved in maintaining K+ and NO3− homeostasis in sweet sorghum (Figure 7).

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14081849/s1, Figure S1: Effects of 50–200 mM NaCl treatments on MDA content in leaf blades of sweet sorghum cultivars “Lvjuren” and “Fengtian”. Data are means (±SD), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05); Figure S2: The photographs of 12 sweet sorghum cultivars under control conditions or treated with 100 mM NaCl. Seedlings were grown in silica sands and cultivated with Hoagland solution for 3 weeks, then seedlings were transferred to Hoagland solution containing 0 (control) or 100 mM NaCl for 10 d; Figure S3: The shoot fresh weigh of 12 sweet sorghum cultivars under 100 mM NaCl treatment. FT, Fengtian; YG, Yanggan; HG, Hunnigreen, NMW, Niumowang; BZ, Baizhan; DK, Daka; JJ, Juejia; PK, Paka; HN, Hainiu; LJR, Lvjuren; KL, Keller; DL, Dale; Table S1: The primers for qRT-PCR analysis.

Author Contributions

Conceptualization, H.G. and Y.-N.C.; Methodology, J.K., X.-L.W. and S.-J.Y.; Software, X.-L.W.; Validation, X.-L.W.; Formal analysis, X.-L.W.; Investigation, J.K. and S.-J.Y.; Data curation, S.-J.Y.; Writing—original draft, J.K.; Writing—review & editing, H.G. and Y.-N.C.; Supervision, Y.-N.C.; Funding acquisition, H.G. and Y.-N.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (32301505 and 32101253), the Shaanxi Postdoctoral Science Foundation (2023BSHEDZZ120), the Natural Science Basic Research Program of Shaanxi (2023-JC-QN-0255), and the Shenzhen Science and Technology Program (JCYJ20220530161404010).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of 50–200 mM NaCl treatments on the growth-related parameters of sweet sorghum cultivars “Lvjuren” and “Fengtian”. (A–D) Growth photographs of seedlings grown under the control condition or treated with 50, 100, and 200 mM NaCl, respectively. (E) Plant height. (F) Tissue fresh weight. (G) Shoot relative water content. (H) Tissue dry weight. The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
Figure 1. Effects of 50–200 mM NaCl treatments on the growth-related parameters of sweet sorghum cultivars “Lvjuren” and “Fengtian”. (A–D) Growth photographs of seedlings grown under the control condition or treated with 50, 100, and 200 mM NaCl, respectively. (E) Plant height. (F) Tissue fresh weight. (G) Shoot relative water content. (H) Tissue dry weight. The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
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Figure 2. Effects of 50–200 mM NaCl treatments on the photosynthesis-related parameters of sweet sorghum cultivars “Lvjuren” and “Fengtian”. (A) Net photosynthesis rate. (B) Stomatal conductance. (C) Chlorophyll a content. (D) Chlorophyll b content. The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
Figure 2. Effects of 50–200 mM NaCl treatments on the photosynthesis-related parameters of sweet sorghum cultivars “Lvjuren” and “Fengtian”. (A) Net photosynthesis rate. (B) Stomatal conductance. (C) Chlorophyll a content. (D) Chlorophyll b content. The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
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Figure 3. Effects of 50–200 mM NaCl treatments on the tissue ion contents of sweet sorghum cultivars “Lvjuren” and “Fengtian”. (A) Na+ content. (B) K+ content. (C) Cl− content. (D) NO3− content. The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
Figure 3. Effects of 50–200 mM NaCl treatments on the tissue ion contents of sweet sorghum cultivars “Lvjuren” and “Fengtian”. (A) Na+ content. (B) K+ content. (C) Cl− content. (D) NO3− content. The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
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Figure 4. Effects of 50–200 mM NaCl treatments on the free proline, soluble sugar, and betaine contents in the leaf blades of sweet sorghum cultivars “Lvjuren” and “Fengtian”. (A) Free proline content. (B) Soluble sugar content. (C) Betaine content. The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
Figure 4. Effects of 50–200 mM NaCl treatments on the free proline, soluble sugar, and betaine contents in the leaf blades of sweet sorghum cultivars “Lvjuren” and “Fengtian”. (A) Free proline content. (B) Soluble sugar content. (C) Betaine content. The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
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Figure 5. The relative expression levels of genes related to Na+ and K+ transport in the tissues of the sweet sorghum cultivars “Lvjuren” and “Fengtian” after 200 mM NaCl treatment. (A) Relative expression of SbNHX2. (B) Relative expression of SbHKT1;4. (C) Relative expression of SbHKT1;5. (D) Relative expression of SbAKT1. (E) Relative expression of SbHAK5. (F) Relative expression of SbSKOR. The data are means (±SDs), n = 3. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
Figure 5. The relative expression levels of genes related to Na+ and K+ transport in the tissues of the sweet sorghum cultivars “Lvjuren” and “Fengtian” after 200 mM NaCl treatment. (A) Relative expression of SbNHX2. (B) Relative expression of SbHKT1;4. (C) Relative expression of SbHKT1;5. (D) Relative expression of SbAKT1. (E) Relative expression of SbHAK5. (F) Relative expression of SbSKOR. The data are means (±SDs), n = 3. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
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Figure 6. The relative expression levels of genes related to Cl− and NO3− transport in the tissues of the sweet sorghum cultivars “Lvjuren” and “Fengtian” after 200 mM NaCl treatment. (A) Relative expression of SbCLCc. (B) Relative expression of SbCLCg. (C) Relative expression of SbNPF6.4. (D) Relative expression of SbNPF3.1. (E) Relative expression of SbNPF6.3. (F) Relative expression of SbNPF7.3. The data are means (±SDs), n = 3. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
Figure 6. The relative expression levels of genes related to Cl− and NO3− transport in the tissues of the sweet sorghum cultivars “Lvjuren” and “Fengtian” after 200 mM NaCl treatment. (A) Relative expression of SbCLCc. (B) Relative expression of SbCLCg. (C) Relative expression of SbNPF6.4. (D) Relative expression of SbNPF3.1. (E) Relative expression of SbNPF6.3. (F) Relative expression of SbNPF7.3. The data are means (±SDs), n = 3. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
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Figure 7. The schematic diagram showing the gene regulatory network underlying ion transports of sweet sorghum under NaCl stress. The upregulated expression of NHX2 in root cells contributes to withholding Na+ in the roots, and the upregulated expression of HKT1;4 and HKT1;5 enhance the xylem unloading of Na+. The downregulation of NPF6.4 could restrict Cl− uptake by root epidermal cells, and the upregulations of CLCc and CLCg in roots, leaf sheaths, and leaf blades help to relieve the disturbance of Cl− on cellular metabolic processes. Sweet sorghum maintains the K+ and NO3− homeostasis in the leaf blades by upregulating the expression of AKT1, HAK5, NPF3.1, and NPF6.3 in root epidermal cells, as well as SKOR and NPF7.3 in root stelar cells. The upregulated expression of HAK5 and NPF3.1 should also contribute to the uptake of K+ and NO3− into leaf blade cells.
Figure 7. The schematic diagram showing the gene regulatory network underlying ion transports of sweet sorghum under NaCl stress. The upregulated expression of NHX2 in root cells contributes to withholding Na+ in the roots, and the upregulated expression of HKT1;4 and HKT1;5 enhance the xylem unloading of Na+. The downregulation of NPF6.4 could restrict Cl− uptake by root epidermal cells, and the upregulations of CLCc and CLCg in roots, leaf sheaths, and leaf blades help to relieve the disturbance of Cl− on cellular metabolic processes. Sweet sorghum maintains the K+ and NO3− homeostasis in the leaf blades by upregulating the expression of AKT1, HAK5, NPF3.1, and NPF6.3 in root epidermal cells, as well as SKOR and NPF7.3 in root stelar cells. The upregulated expression of HAK5 and NPF3.1 should also contribute to the uptake of K+ and NO3− into leaf blade cells.
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Table 1. The water potential, osmotic potential, and turgor pressure in leaf blades of sweet cultivars “Lvjuren” and “Fengtian” under 50–200 mM NaCl treatments.
Table 1. The water potential, osmotic potential, and turgor pressure in leaf blades of sweet cultivars “Lvjuren” and “Fengtian” under 50–200 mM NaCl treatments.
CultivarsTreatmentsWater Potential
(Ψw, MPa)		Osmotic Potential (Ψs, MPa)Turgor Pressure
(Ψt, MPa)
LvjurenControl−0.18 ± 0.04 a−0.63 ± 0.02 a0.45 ± 0.01 a
50−0.46 ± 0.03 c−0.75 ± 0.03 b0.29 ± 0.03 b
100−0.81 ± 0.05 e−1.02 ± 0.05 d0.21 ± 0.01 c
200−1.12 ± 0.06 g−1.25 ± 0.05 f0.13 ± 0.02 d
FengtianControl−0.19 ± 0.03 a−0.64 ± 0.01 a0.45 ± 0.03 a
50−0.39 ± 0.02 b−0.83 ± 0.03 c0.44 ± 0.04 a
100−0.70 ± 0.03 d−1.18 ± 0.06 e0.30 ± 0.01 b
200−0.96 ± 0.04 f−1.35 ± 0.08 g0.29 ± 0.01 b
The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
Table 2. The contribution of each osmolyte to the leaf osmotic potential of the sweet cultivars “Lvjuren” and “Fengtian” under 50–200 mM NaCl treatments.
Table 2. The contribution of each osmolyte to the leaf osmotic potential of the sweet cultivars “Lvjuren” and “Fengtian” under 50–200 mM NaCl treatments.
CultivarsTreatmentsK+NO3−ProlineSugarsBetaine
LvjurenControl39.44 ± 2.15 a42.03 ± 2.38 a0.11 ± 0.01 b5.33 ± 0.23 c6.86 ± 0.38 b
5031.81 ± 1.74 b33.88 ± 2.01 b0.13 ± 0.01 b5.30 ± 0.26 c5.86 ± 0.29 c
10018.01 ± 1.05 d21.79 ± 1.00 d0.14 ± 0.01 b4.61 ± 0.21 d5.90 ± 0.30 c
20015.07 ± 0.69 e16.31 ± 0.57 e0.18 ± 0.01 a4.40 ± 0.21 d6.01 ± 0.35 c
FengtianControl38.63 ± 1.77 a42.00 ± 1.90 a0.11 ± 0.01 b5.16 ± 0.31 c6.78 ± 0.37 b
5031.39 ± 1.60 b33.90 ± 1.47 b0.11 ± 0.01 b5.93 ± 0.33 b6.28 ± 0.28 bc
10022.02 ± 1.10 c25.00 ± 1.11 c0.10 ± 0.01 b6.08 ± 0.33 ab7.44 ± 0.39 a
20020.00 ± 0.87 cd23.40 ± 1.12 c0.12 ± 0.01 b6.43 ± 0.5 a7.80 ± 0.40 a
The data are means (±SDs), n = 6. Different letters indicate significant differences as determined using Tukey’s HSD test (p < 0.05).
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Kang, J.; Wang, X.-L.; Yan, S.-J.; Guo, H.; Cui, Y.-N. Comparative Physiological and Gene Expression Analyses Provide Insights into Ion Transports and Osmotic Adjustment of Sweet Sorghum under Salt Stress. Agronomy 2024, 14, 1849. https://doi.org/10.3390/agronomy14081849

AMA Style

Kang J, Wang X-L, Yan S-J, Guo H, Cui Y-N. Comparative Physiological and Gene Expression Analyses Provide Insights into Ion Transports and Osmotic Adjustment of Sweet Sorghum under Salt Stress. Agronomy. 2024; 14(8):1849. https://doi.org/10.3390/agronomy14081849

Chicago/Turabian Style

Kang, Jie, Xiao-Long Wang, Shi-Jie Yan, Huan Guo, and Yan-Nong Cui. 2024. "Comparative Physiological and Gene Expression Analyses Provide Insights into Ion Transports and Osmotic Adjustment of Sweet Sorghum under Salt Stress" Agronomy 14, no. 8: 1849. https://doi.org/10.3390/agronomy14081849

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