Physiological and proteomics insights into salt tolerance of two Jerusalem artichoke cultivars

Jerusalem artichoke (Helianthus tuberosus L.) is an insulin-containing crop, which has been paid an intensive attention in recent decades. Although, some progress has been made in the biochemistry of Jerusalem artichokes (JA), the inner adaptive mechanism of salt tolerance among JA varieties is still unclear. Elucidating salt-tolerant differences by integrated stress physiology and proteomics approach will provide comprehensive insights into their adaptive mechanism for various JA varieties, therefore serving for the large-scare cultivation in salt-affected marginal lands. JA seedlings were initially grown in half-strength Hoagland solution, and then exposed to 100 and 200 mM NaCl for 30 days. We found that salt stress decreased the plant height, root length, fresh and dry weight in both varieties, and the decreasing extents of N1 (Helianthus tuberosus var. N1) was greater than M1 (Helianthus tuberosus var. M1). Chloroplast ultrastructure in N1 was severely damaged, but appeared unaltered in M1. Also, N1 remained lower selective for K+ over Na+, exhibiting more Na+ accumulation in plant tissues compared to M1. Penetrating cutting-edge elementary proteomic results showed the regulation of protein expression in M1 was much more positive than in N1. Taken together, these results illustrated the considerable differences in adaption to saline environment between varieties.


Introduction
Salinity is a major problem affecting crop production all over the world: 20% of cultivated land in the world, and 33% of irrigated land, are salt-affected and degraded (Machado and Serralheiro 2017). Salt-affected soils occur in more than 100 countries worldwide, although varying from their extent of different places (Bui 2013). Recently, many plants have been intensely studied on their salt-tolerant processes, including physiological or molecular responses to salinity, aiming at well utilizing saline-alkali soil for crop production (Singh et al. 2012;Roy et al. 2014;Munns and Gilliham 2015).
JA is widely regarded with the relevant literature as moderate salt-tolerant plant, and it is rich in carbohydrates, of which 70-90% is inulin (Newton et al. 1991;Zhao et al. 2010). Many studies documented that JA species had stronger ecological adaptability to cold, drought and salinity, which just complied with China government's concept of exploiting coastal saline soils for agricultural production (Zhao et al. 2010;Bhagia et al. 2018).
It has been reported that JA growth is generally inhibited by ionic toxicity, which can result in a little bit tube yield losses (Xue and Liu 2008), and some salt-tolerant mechanisms might play a vital role in compensating this adverse effects, including ion regionalization, fluctuations in antioxidant enzyme activity and osmotic adjustment under saline conditions (Yang et al. 2016;Long et al. 2009;). Additionally, salt resistance to plants is mainly controlled by polygenes, and this helps form newly synthesized or enhanced proteins, so-called salt-stress proteins which might be closely correlated to plant regulatory mechanisms (Duché et al. 2002). Recently, proteomics were widely used to analyze salt tolerance of many conventional plants (Eldakak et al. 2013;Aslam et al. 2017). However, it has not been applied to the study on the JA plant by far. Proteomics methodologies achieve rapid development within several years, and shift from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gelfree workflows in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry, which provides a reliable analysis for salt-tolerant mechanism of plants for special purpose. The hypothesis of this paper is that the salt tolerance of JA plant has significant intraspecific differences and salt stress results in a series of adaptive responses in physiological and biochemical bases.
The objective of this study was to elucidate the salttolerant mechanisms of two JAs at the physiological and proteomics level, thereby provided comprehensive insights into differences in adaptation to saline environments between the two JAs. This study was of great significance on the JA variety breeding and cultivation in salt-affected region for food industrial purpose.
Two JAs (Helianthus tuberosus var. N1 and Helianthus tuberosus var. M1) were long-term preserved in Jiangsu Provincial Key Lab of Marine Biology. N1 is tested as a moderate salt-tolerant variety which was widely cultivated in coastal saline land, while M1 is introduced from Russia in 2009. Prior to cultivation, JA tubers were cut in many nubbles with buds, then sterilized with 1.0 g L -1 HgCl 2 for 10 min, rinsed thoroughly with distilled water and germinated into moist sand in an incubator at 25°C. Emergening with 4th leaf, uniform JA seedlings were transplanted into plastic kegs and hold up by polyethylene lid through which plants were supported over the nutrient solution. There are three treatments of this experiment, including non salttreated (0 mM), 100 mM and 200 mM NaCl addition treatments. Among treatments, a factorial setup was arranged on the basis of a completely randomised design with three replications. JA seedlings were exposed to salt solution when they were four-week old, and harvested after 30 days. While harvesting, plant samples were collected in two separate sets, one was used for determining the growth status and analysing ion composition of plant tissues, the other was for physiological and biochemical analysis, such photosynthetic pigments, antioxidant enzymes and proteome.
The plant height, root length and fresh weight were measured to according to Lu (2000), plant dry weight (DW) and the relative water content (RWC) were measured to according to Smart and Bingham (1974). The chlorophyll a (Chl a), chlorophyll b (Chl b) and total chlorophyll (tChl) concentrations were calculated according to Litchenthaler (1987), and the total carotenoids were analysed as described by Mencarelli and Saltveit (1988).CAT and POD activity was assayed according to the method described by Bradford (1976), Bergmeyer and Bernt (1974) and Kwak et al. (1995). Total proteins were extracted from JA leaves by a conventional trichloroacetic acid (TCA)/ acetone method with some embellishment (Chen et al. 2011;Thiede et al. 2013).The data and comparative analysis between samples were performed by the soft of PDQuest7.2 (GE Healthcare Life Science). The mean values were calculated according to three replicates of plant samples by Microsoft Excel for Windows, and the standard error of means was generated through SPSS15.0 software. Two-way ANOVA was further applied to determine their significance of treatments. Also, the Duncan's new multiple range test (P \ 0.05) was adopted for data statistics in this research.
There were phenotypic differences between the two JAs immersed in the different concentrations of salt solution (Fig. 1). Salt stress exerted a negative impact on the plant fresh weight compared to the control, decreased by approximately 23.7% and 16.5% in 100 mM NaCl treatment, 42.5% and 34.2% in 200 mM NaCl treatment for N1 and M1, respectively (Table 1). And there was the same trend which was clearly observed for the plant dry weight. The plant height and root length were also influenced by salt stress, whilst their values of N1 were considerably lower than M1. Moisture contents of N1 showed a significant reduction in 200 mM NaCl treatment; however those of M1 remained unchanged. Those results indicated that M1 had a stronger resistance to salt stress compared to N1. Figure 2 shows for M1, Chl a decreased to some extent merely under 200 mM NaCl stress, whilst Chl b and T chl remained unchanged under the tested salinities, and Car, however, increased with increasing salinities. For N1, the Fig. 1 Representative photographs of the two s showed a growth reduction N1 and M1 with increasing salt stress levels. Plants were grown in hydroponic solution and treated with 0 (control), 100 mM NaCl, and 200 mM NaCl, respectively for 30 days result almost reversed, showing a significant reduction in Chl a, Chl b and T chl under successively strengthened salt stresses; however, the maximum value of Car contents was observed in the 100 mM other than 200 mM NaCl treatments. Additionally, the levels of chlorophyll pigments were remarkably higher in M1 than in N1, no matter if JAs were under salt stress or not, suggesting M1 displayed stronger photosynthetic capacity than N1 against salinity stress.
To further elucidate the differences of photosynthetic capacities between the two JAs subjected to salt stress, the ultrastructure changes were determined by transmission electron microscopy (TEM) (Fig. 3). The chloroplasts in control were located in mesophyll and parenchyma cells, containing large starch grains (SG), whereas in the salt-treated plants, chloroplasts showed somewhat degradation. Overall, N1 suffered severe alteration of the chloroplast ultrastructure by salt stress, but M1 remained relatively stable, showing that the thylakoidal membranes in N1 disorganized with more swelling and curling than M1 at the same salt concentration. The cell plasmolysis occurred in two JAs, which became increasingly rigorous in conjunction with increasing salinity. And for the two JAs, the ultrastructural response to salinity was that salinity scarcely influenced the thylakoids and perturbed the granum lamella (GL) in M1, whereas GL numbers in N1 were reduced and the thylakoid membrane was also decreased markedly by salt stress.
Generally, superfluous Na ? uptake by JA plants caused retardant plant growth, which was shown in Table 2. For Significant differences (P B 0.05) between the NaCl treatments are indicated by different letters. Data are presented as the means ± SD (n = 3)

Fig. 2
Effect of NaCl stress on chlorophyll a, chlorophyll b, total chlorophyll and carotenoid contents in leaves of JA s. The values are presented as the means ± standard error (SE); n = 3 for all groups.
The bars represent the SE. Bars with the same letter are not significantly different, denoted by P \ 0.05 according to Duncan's multiple range tests the two JAs, Na ? was markedly increased with increasing salinity, and it was mostly accumulated in the roots, followed by the stems and then in the leaves. Instead, Mg 2? , Ca 2? , K ? concentration in plant tissues declined with increasing salinity. Superfluous Na ? entering into JA tissues had a negative effect on K ? uptake by plant roots, resulted in a relatively lower K ? /Na ? ratio. There was no significant difference for K ? /Na ? ratio in the plant tissues at low-concentration of salt stress (100 mM), nevertheless M1 possessed better capacity to regulate K ? /Na ? homeostasis than N1 at high-concentration of salt stress (200 mM). Figure 4 shows 100 mM NaCl significantly elevated the SOD, POD and CAT activities of JA plant, but 200 mM NaCl had exactly the reverse effect. The SOD activity in M1 was found to be considerably higher than that in N1 in 200 mM NaCl treatment, whereas no differences were observed in 100 mM NaCl treatment. The POD activity response to salt stress in both JA s had the same trend with the SOD activity. The CAT activity, however, was obviously activated in 100 mM NaCl treatment, but was severely inhibited in 200 mM NaCl treatment for the two JA s. Malondialdehyde (MDA), measured as TBARS, increased consistently with increasing level of salinity in two s, therefore indicating a sharp increase in lipid peroxidation. High salinity conditions resulted in increases in MDA by 72.9% and 44.3% in N1 and M1, respectively, from which we can surmise that more severe lesions occurred in the biological membranes of N1.
Variant protein spots of double varieties were shown in Fig. 5. The protein spots detected in all three replicates of silver-stained gels were analysed with an image scanner and the average response content of every spot protein was then calculated. In the protein analysis of N1, the expression of greater than 39 proteins was markedly reduced and 9 proteins were significantly increased. In M1, approximately 30 proteins decreased and 18 proteins increased. Further analysis revealed that plots 5306, 6409, 4306, 3308 increased with increasing salinity, whereas plots 2609, 5607, 5510 as well as ten other plots consistently decreased. Of those, plot 6309 showed the greatest negative correlation with salinity. Previously described protein plots likely had similar effects on the regulation of salt stress responses. Comparatively, the regulation of protein expression in M1 was much more positive than in N1. Therefore, analogous response mechanisms of JA to salt stress disclosed some divergent procedures.
Salinity did adversely affect the tested plant growth, including height, root length and fresh/dry weight (Table 1), although JA plant was generally considered as a moderate salt-tolerant species (Zhao et al. 2010). This phenomenon had also been found in many other crop species like wheat (Datta et al. 2009), soybean (Dolatabadian et al. 2011), canola (Tuncturk et al. 2011) and some halophytes (Akhzari et al. 2012). In this experiment, JA plant growth exhibited varying responses to NaCl stress among s. Overall, M1 grew relatively well than N1 under the salt-stressed conditions, demonstrating M1 had stronger salt-resistant capacity. This was mainly because, JA s suffered from salt stress were under moisture lose, resulting in the retardant plant growth rate. Therefore, plant moisture content could be regarded as an indicator characterizing salt tolerant capacity of JA plant.
The depression of photosynthesis due to salt stress is mainly ascribed to a reduction in chlorophyll content (Ashraf and Harris 2004). In plants, the chlorophyll content of leaves is not only a straightforward, reflective and revealing index of plant photosynthetic capacity but also an important physiological index to measure the ability of salt resistance (Willekens et al. 1994). Salt stress also resulted in a reduction in chlorophyll of JA plant, which agreed with previous studies of salt-treated rice, sorghum and maize (Yilmaz and Kina 2008). But between JA s, M1 showed higher photosynthetic pigment contents in its leaves in comparison with N1.
It has been reported that salt stress causes membranes to swell, increases in plastoglobuli, changes in starch levels and the destruction of grana in many plants (Keiper et al. 1998;Hernandez et al. 1999). This phenomenon were analogous to those observed in chloroplasts of salt-treated N1 (Fig. 3). In contrast, M1 maintained inerratic grana with no swelling of thylakoid membranes in 100 mM and 200 mM NaCl treatments, which indicated that M1 was a relatively salt-tolerant. On average, the photosynthetic organelle ultrastructures in M1 appeared to be well maintained under salt stress. Thus, M1 possessed more beneficial defense mechanisms against salt stress than N1. When treated with high salinity, superfluous Na ? depolarizes the plasma membrane and the resultant outflow of K ? can damage the membrane structure and then result in disorders of cell metabolism, which ultimately restrains plant growth (Shabala et al. 2003). According to Tester (Tester and Davenport 2003), the effects of salt stress on photosynthesis in plants include damage caused by the unbalance of ion absorption, and the water potential of osmotic stress reduction, etc. The regulation and distribution of ions in various plants parts and within cells is an indispensable characteristic of the mechanism of salt tolerance (Mahmoodzadeh et al. 2015) because the specific accumulation of Na ? in tissues of plants was toxic and had been found to be one of the primary causes of reduced growth under salinity conditions. Such a distribution mode of inorganic cations decreased the osmotic potential of the root environment and guaranteed the natural physiological function and metabolism of the plant. Our studies showed a significant accumulation of Na ? and substantial reduction of Mg 2? , K ? , Ca 2? in two JAs under higher salinity stress (200 mM NaCl). Clearly, osmotic adjustment capacity of M1 was greater than of N1, especially at high lever salt stress.
Under normal circumstances, plants have a large armament of antioxidants to employ against the injury by active oxygen. Among these are SOD, POD and CAT, which play obligatory roles in this process. It has been reported that ROS scavenging by the enhanced activation of antioxidant enzymes can strengthen salt tolerance (Alscher et al. 2002). Our results showed that superfluous lipid peroxidation occurred progressively in JAs subjected to salinity (Fig. 4), which resulted in increasing membrane permeability as expressed by electrolyte leakage. The process of lipid peroxidation was closely related to the increasing activity of antioxidative enzymes in leaves, and the increment in the electrolyte leakage, which indicated as an elevated amount of MDA content in the plant leaves. Compared with the control, there were significantly increased levels of SOD, POD and CAT activity under 100 mM NaCl, but a prominent reduction under 200 mM NaCl. The levels of MDA in N1 were significantly higher than in M1. Therefore, these results suggested that M1 had a greater capacity of salt resistance than N1.
Protein is the executor of the physiological function as well as the direct manifestation of biological phenomena (Wilkins et al. 2013). The study of protein structure and function helps clarify changes in physiological mechanisms. Two-dimensional gel electrophoresis (2-D gels) can authenticate the complicated protein samples at the proteome-wide level. Salt tolerance of JA plant consists of complex biochemical pathways including a mass of gene expression, and the application of 2-D gels offers great Fig. 4 Effect of NaCl stress on MDA content and SOD, CAT and POD activities in leaves of two JA s. The values are presented as the means ± standard error (SE); n = 3 for all groups. The bars represent the SE. Bars with the same letter are not significantly different, denoted by P \ 0.05 according to Duncan's multiple range tests opportunities to clarify the process of protein change during salt tolerance. Comparatively, the regulation of protein expression in M1 was much more positive than in N1 (Fig. 5), which provided comprehensive insights into differences in adaptation to saline environments between the two JAs.
In the present study, it can be concluded that although the Jerusalem artichoke plant showed a similar salt-tolerant mechanism but with a much larger difference between cultivars. In general, the growth of both JA cultivars was strongly influenced by salt stress. However M1 exhibited a higher salt tolerance than N1, which mainly reflected in the differences in the water retaining capacity, chlorophyll content and stability of the chloroplast structure as well as osmotic regulatory capability. Penetrating cutting-edge elementary proteomics results further provided comprehensive insights into differences in salt-tolerant adaption for the two JA cultivars.

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Conflict of interest The authors declare that they have no conflict of interest.
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