Effects of salt stress on the photosynthetic physiology and mineral ion absorption and distribution in white willow (Salix alba L.)

Objective The purpose of this study was to explore the adaptive mechanism underlying the photosynthetic characteristics and the ion absorption and distribution of white willow (Salix alba L.) in a salt stress environment in cutting seedlings. The results lay a foundation for further understanding the distribution of sodium chloride and its effect on the photosynthetic system. Method A salt stress environment was simulated in a hydroponics system with different NaCl concentrations in one-year-old Salix alba L.branches as the test materials. Their growth, ion absorption, transport and distribution in the roots and leaves, and the changes in the photosynthetic fluorescence parameters were studied after 20 days under hydroponics. Results The results show that The germination and elongation of roots are promoted in the presence of 171mM NaCl, but root growth is comprehensively inhibited under increasing salt stress. Under salt stress, Na+ accumulates significantly in the roots and leaves, and the Na+ content and the Na+/K+ and Na+/Ca2+ root ratios are significantly greater than those in the leaves. When the NaCl concentration is ≤ 342mM, Salix alba can maintain relatively stable K+ and Ca2+ contents in its leaves by improving the selective absorption and accumulation of K+ and Ca2+ and adjusting the transport capacity of mineral ions to aboveground parts, while K+ and Ca2+ levels are clearly decreased under high salt stress. With increasing salt concentrations, the net photosynthetic rate (Pn), transpiration rate (E) and stomatal conductance (gs) of leaves decrease gradually overall, and the intercellular CO2 concentration (Ci) first decreases and then increases. When the NaCl concentration is < 342mM, the decrease in leaf Pn is primarily restricted by the stomata. When the NaCl concentration is > 342mM, the decrease in the Pn is largely inhibited by non-stomatal factors. Due to the salt stress environment, the OJIP curve (Rapid chlorophyll fluorescence) of Salix alba turns into an OKJIP curve. When the NaCl concentration is > 171mM, the fluorescence values of points I and P decrease significantly, which is accompanied by a clear inflection point (K). The quantum yield and energy distribution ratio of the PSⅡ reaction center change significantly (φPo, Ψo and φEo show an overall downward trend while φDo is promoted). The performance index and driving force (PIABS, PICSm and DFCSm) decrease significantly when the NaCl concentration is > 171mM, indicating that salt stress causes a partial inactivation of the PSII reaction center, and the functions of the donor side and the recipient side are damaged. Conclusion The above results indicate that Salix alba can respond to salt stress by intercepting Na+ in the roots, improving the selective absorption of K+ and Ca2+ and the transport capacity to the above ground parts of the plant, and increasing φDo, thus shows an ability to self-regulate and adapt.

-Do not repeat words of the title in the Keywords.
-Please for the first use Salix alba L., then please follow the only Salix alba in the manuscript.
[Reply] We have rewrote the name throughout the manuscript. Please look at the example from line 15+18.
-Add economic and other importance of plant in introduction to make it more valuable.
[Reply] We have added economic and other importance of plant in introduction. See Line 64-69 .
-It is better to NaCl concentration in mM instead of %.
[Reply] We've changed % to mM. Please see line 91.
-Objective of the study needs to be refined.
[Reply] We have refined our research objectives. Please see Line 14-17.
-Line: 98. Which instrument (its name model, company) used for the determination of ions? [Reply] We have added information about this instrument. We used the atomic absorption spectrometer of Analytikjena in Germany for atomic absorption determination.Please see line 116-117.
-In my opinion, chlorophyll content (a, b, and total) and carotenoids needs to be measured. It have direct link with salt stress and adaptability. NaCl and RWC have direct link when talking about stress and adaptability. So, I will suggest to study RWC (relative water content) under salt stress.
[Reply] This study mainly focuses on the effects of salt ions in plants on photosynthetic performance, without considering chlorophyⅡ and water. Thank you for your valuable suggestions, which will be added and improved in our future studies.
-References need to be revised. Many articles on salinity response are published in high impact factor journals. So try to cite them, so everyone can access the references as well. Also, try to cite the latest articles.
[Reply] We have deleted some references. We have added some influential and recent articles. Please see line 472-495.

COMMENTS FOR THE AUTHOR:
Reviewer 2# -However, this mechanism seems to be general in many plants, and also, as lack of data, the conclusions can not be rich and for further discussion.
[Reply] Thank you very much for your suggestions. We have revised the manuscript. Most of the researches on White Willow mainly focus on the medicinal value of substances such as salicin contained in the bark, or the value of studying the enrichment of heavy metals in white willow, which is mainly used to purify water resources and realize agricultural irrigation and fishery breeding. Habitat stress is mainly drought and flooding, but there are few literatures on salt stress, most of which focus on the responses of physiological indexes and photosynthetic indexes of plants to ion absorption and transport under salt stress. This is also one of the reasons for our research. We hope to use white willow as experimental material to observe the various effects of salt stress on plants from this perspective.

COMMENTS FOR THE AUTHOR: Reviewer 3#
The manuscript titled "Effects of salt stress on the photosynthetic physiology and mineral ion absorption and distribution in white willow (Salix alba L.)" one of classic example of tree species response to salt stress. The manuscript is well organized and language easy to follow although there were few grammatical and Syntex errors besides SI units.
[Reply] Thank you very much for your suggestions. We have corrected grammatical and syntactic errors .Please see the manuscript.
-1. Adapting a hydroponic system to research salt stress may be the most recent best strategy, but there is no way of knowing how the experimental setting was done. Authors may include a photo of the same in order to make any relevant comments.
[Reply] We have added a picture to the manuscript. Please see the Figure 1.
-2.Similarly for rooting length, kindly provide some photo to understand plant response.
[Reply] We have added a photo. Please see the figure 2.
-3. Throughout the manuscript I can see that salt stress has a significant impact on above-ground biomass, such as leaf area index, shoot length, and so on, but the fact that it was not included in this study is a significant disadvantage. In the meantime, various physiological characteristics have been subjected leaves.
[Reply] This study mainly focuses on the effects of salt ions in plants on photosynthetic performance, without considering the aspects of leaves and stems. Thank you for your valuable suggestions, which will be added and improved in future research.
-4. When it comes to hydroponic systems, CK medium may be the best option, however, the reaction of Salix alba seedlings is substantially higher than 0.1% NaCl, but no justification from authors how or why although the salt concentration less than 0.1% NaCl.
[Reply] we have made an explanation, please see line 263-269.Thank you very much for your valuable suggestions.
-5. A statistical analysis was performed, however it was not up to scientific merit. For example, the manuscript frequently mentions significance of attributes, but there are no ANOVA tables (except LSD rank), at least as a supplemental to support. I request the authors to include F=xxx, df=xxx, and sig.=>0.001 wherever statistical significance was indicated.
[Reply] We added some these information throughout the manuscript. Please see the example form line 173-175+179+181.
6.Throughout the article, there are discussions about the trends of the morphological and physiological response of plant to the treatments, but the presented table does not provide viewpoints. Authors profusely mentioned the response trends widely without statistical analysis about trends which containing the scatter plot and best fit regression models (r2 and P). I advise authors to use graphs instead of tables, and I've included a sample presentation for ready reference.
[Reply] Thank you very much for your valuable suggestions. We have changed some tables into charts. Please see figure 7. As shown in Fig 7, with increasing NaCl stress, the SA k, Na, ST k, Na, SA Ca, Na, and ST Ca, Na all showed a trend 168 of first increasing and then decreasing. When the NaCl concentration was less than or equal to 342mM/L, the selective 169 absorption capacity of the roots for K + (F=998.922, df=4，Sig.<0.001)and Ca2+ (F=1018.689, df=4，Sig.<0.001) 170 and the selective transport capacity of the leaves for K + (F=168.047, df=4，Sig.<0.001) and Ca 2+ (F=29.925, df=4， 171 Sig.<0.001) were enhanced and reached a significant level. The selective absorption capacity of roots for K + is greater 172 than that of Ca 2+ , but the selective transport capacity of the leaves to Ca 2+ is greater than that of K + . These results 173 indicated that Salix willow could adjust the upward transport capacity of K + and Ca 2+ via the selective absorption and 174 accumulation of mineral ions to compensate for the change in concentration under salt stress, to prevent the impacts 175 of nutrient deficiency and ion toxicity on the shoot growth. there was no significant difference from the control. Later, as the salt stress intensified, the photosynthetic carbon 180 assimilation ability of Salix alba. leaves was significantly inhibited (F=95.66, df=4，Sig.<0.001); when the NaCl 181 concentration was greater than 171mM/L, both the E (F=100.091, df=4，Sig.<0.001)and gs (F=69.346, df=4， 182 Sig.<0.001) were significantly lower than the control and became stronger; but at a low salt concentration (171mM/L 183 NaCl), there is no significant difference from the control. With the increased salt concentration, the leaf Ci showed a 184 trend of first decreasing and then increasing, reaching the lowest when the salt concentration was 342mM/L, which 185 was significantly lower than the control by 10.4%, and then it gradually increased. The Ci of leaves (F=20.50, df=4， 186 Sig.<0.001) under 513mM/L and 684mM/L NaCl treatments were not significantly different from that of the control, 187 but they were significantly higher than the lowest value by 12.4% and 14.6%, respectively. increasing NaCl concentration, the maximum photochemical efficiency (φPo) of Salix alba. leaves after dark 206 adaptation gradually decreased. Under the 342mM/L NaCl treatment, the φPo was significantly lower than that of 207 the control. At that time, salt stress triggered photoinhibition, and the photosynthetic capacity of the leaves was 208 reduced. 209 The excitons captured by the reaction center transfer electrons to the electron transport chain, and the ratio of 210 excitons that exceed QA's other electron acceptors to promote QA reduction excitons (Ψo) and the light energy 211 absorbed by the reaction center are used for electron transfer. The quantum yields (φEo) all increased first and then 212 decreased with the increasing salt stress. At 171mM/L NaCl, although the Ψo and φEo increased, they were not 213 significantly different from the control. Later, as the stress intensified, both the Ψo and φEo were significantly lower 214 than those of the control. When the NaCl concentration was 342mM/L, the Ψo and φEo were significantly lower than 215 the 11.1% and 11.9% of the control group, respectively. Compared with the control group, salt stress increased the 216 quantum ratio (φDo) of Salix alba. leaves for heat dissipation. When the NaCl concentration was 513mM/L, φDo 217 was significantly higher than that of the control. Sig.<0.001) , indicating that the Salix alba. leaves experienced photoinhibition, the PSⅡ was damaged, and the 226 measurement at the 684mM/L NaCl concentration was significantly lower than that of the control, by 60.2%. When 227 the NaCl concentrations were 342mM/L, 513mM/L and 684mM/L, the PICSm values were significantly lower 228 (F=202.821, df=4，Sig.<0.001) than that of the control by 20.1%, 43.9% and 66.4%; when the NaCl concentrations 229 were 513mM/L and 684mM/L, the DFCSm values were significantly lower (F=40.755, df=4，Sig.<0.001) than the 230 control by 6.3% and 11.2%. Salt stress seriously affects the absorption of light energy by plants and leads to a decline 231 in the basic driving force. 232

Influence of salt stress on the root growth status of Salix alba 234
As the primary organ responsible for plant material exchange, the root system and its growth status are closely 235 related to the growth and development of the aboveground plant parts, whether the root system can function normally, 236 and the plant's water and nutrient utilization efficiency [33] . Under salt stress, the root system is the first to feel the 237 adversity stress signal, and it is also the most directly affected part [34] . Its ring-stripe inhibition is primarily manifested 238 in the low levels of the root length, surface area and other parameters, and the root system grows slowly. A high-salt 239 environment will cause plants to experience osmotic stress and ion toxicity, which will lead to changes in membrane 240 permeability, which will in turn affect the absorption of water and nutrient elements by the roots, causing the plants 241 to lose a large amount of water; the ions near the roots will be unbalanced, the physiological functions of the roots 242 will eventually be lowered, and even the structure will be destroyed. Some of the aboveground leaves wilt, and 243 photosynthetic production cannot be performed normally, which causes plant growth and metabolic disorders until 244 the loss of physiological functions. 245 The change in root growth and the time of the root sprouting period can directly reflect the degree of damage to 246 plants by salt stress and represent the strength of plant salt tolerance [35] . This study showed that the 171mM/L NaCl 247 concentration significantly promoted the increase in the average number of roots and the elongation of the average 248 root length of Salix alba. cuttings, and it can promote the rooting of the root system in advance, to a certain extent, 249 which is consistent with Wang Shufeng et al. [36] and Ci Dun. The research results of Wei et al. [35] were basically the 250 same. This growth response may be due to the decrease in water potential outside the roots under salt stress, which 251 stimulates the growth of the roots instead of moderate osmotic stress to ensure the normal absorption of water and 252 nutrients to meet the physiological and metabolic needs of the aboveground parts. 253 Some plants do have the phenomenon that low salt promotes the increase of some indicators, such as: promoting 254 the germination of sorghum seeds [37] , the roots of the seedlings of wolfberry [38] and rice [39] , and the growth indicators 255 of corn [40,41] . Both ChorophyⅡin chrysanthemum [42] and proline content of cherry seedlings [43] are increased, while 256 the net photosynthetic rate of wild chrysanthemum [44] and hazel trees increased [45] . The reason for the low salt 257 concentration may be that the salt stress has a dual effect of stimulus and inhibition on plants. The strong and weak 258 relationship between stimulus and inhibition triggers changes in various plant indicators, resulting in the same low 259 salt. It can promote growth, and it will be inhibited after high salt.This finding shows that Salix alba. has some ability 260 12 to adjust and adapt to salt stress, and this adaptability is of great significance to the survival and continuation of the 261 plant itself under adversity. However, as the salt stress intensifies, the ability of plants to coordinate their own growth 262 is destroyed, root germination and elongation are significantly inhibited and become more intense, the root functions 263 are destroyed, and the plants cannot maintain their normal growth and development. In this study, when the salt concentration was low, the growth of Salix alba. was basically normal, the symptoms 272 of salt damage were not significant, and the damage was obvious under severe stress. Na + accumulates significantly 273 in the roots and leaves of Salix alba. under salt stress, but the Na + content in different organs is significantly different, 274 and it is primarily concentrated in the roots. This result shows that the willow root system has a compensation 275 mechanism that can reduce the transportation of salt to aboveground parts by enriching Na + in the root, thereby 276 effectively reducing or delaying the occurrence of salt damage in the aboveground parts. This conclusion is consistent 277 with the study by Hao Han et al. [47] . When the salt stress is too high, this balance is broken, and growth is blocked. 278 As an important inorganic solute, K + is essential for reducing the cell osmotic potential and maintaining the 279 water balance. Generally, plants have an antagonistic effect on the absorption of Na + and K + [48] , and the competition 280 between the two usually leads to a decrease in the K + content. The loss of K + will cause changes in the physical 281 structure of the stomata, frustrating photosynthesis [49] . In addition, K + participates in the metabolism of various 282 enzymes in plants [50] . As salt stress increases, an excessive loss of K + will lead to K + dependent enzymes in Salix 283 alba. The enzyme activity decreases, which affects the metabolic reactions in which it participates. Therefore, if 284 13 plants are to grow in a salty environment, the selective absorption of K + by the root system and the transportation of 285 K + to the ground are particularly important. This study showed that the K + content in the roots of Salix alba. 286 significantly decreased with increasing stress, but the K + in the leaves could be maintained at a high level at a 287 342mM/L NaCl concentration and below and even increased significantly when the NaCl concentration was 288 171mM/L, according to Zhou Qi et al. [51] A study on Carpinus chinensis also confirmed this result. At this time, the 289 value and increase of Na + /K + in the roots of the Salix alba. were greater than that of the leaves, and the SA k, Na and 290 ST k, Na all increased significantly. Studies have shown that under salt stress, the Na + /K + value can represent the 291 degree of salt damage to the plant, and the lower Na + /K + value of the leaves can help the plant better maintain its 292 growth and photosynthetic function [52] , and the SA k, Na and ST k, Na indicates that the plants can better tolerate salt 293 stress [53] . This result shows that at that time, Salix alba. could maintain a relatively stable leaf K + content and the 294 normal progress of photosynthesis by restricting the transportation of Na + from the root to the leaves, increasing the 295 selective absorption of K + through the plant roots and the ability to transport K + to the ground. The accumulation of 296 Na + causes damage to plants, which may be an important mechanism by which Salix alba. copes with salt stress. 297 Later, with the increase in salt stress, the K + in the roots and leaves clearly flowed out. A high concentration of Na + 298 will replace the Ca 2+ bound to the membrane system, which will damage the integrity of the membrane structure and 299 membrane function, thereby destroying the ion balance in the plant body and causing a large amount of organic solute 300 extravasation [54] . The establishment of Ca 2+ homeostasis in the cytoplasm is a key condition for salt adaptation [55] . 301 This experiment showed that as the salt stress intensified, the Ca 2+ content in the Salix alba. roots continued to 302 decrease, but it could accumulate in the leaves when the NaCl concentration was ≤342mM/L. The results of Jia Yin 303 et al. [56] were similar; the Na + /Ca 2+ value of white Salix roots was higher than that of the leaves, and the Sa Ca, Na and 304 ST ca, Na were all significantly increased. This result may be due to the large influx of Na + into the root system under 305 salt stress, activating Ca 2+ signal transduction, triggering the sodium elimination system to reduce the damage of Na + , 306 14 and enhancing the selective absorption of Ca 2+ in leaves, thereby enhancing the selective transport of Ca 2+ from root 307 to shoot to maintain the low cell osmotic potential and the stability of the cell membrane. In addition, studies have 308 shown that the increase in intracellular Ca 2+ contents under salt stress can inhibit the outflow of K + , thereby alleviating 309 the damage of salt stress to plants [57] . Therefore, the upward transportation of Ca 2+ in the roots of Salix alba. may be 310 an important mechanism for it to maintain the balance of K + and Na + in the aerial part, establish ion homeostasis in 311 the aerial part, and adapt to salt stress. However, due to the limited ability of the roots of Salix alba. to absorb Ca 2+ , 312 under high salt stress, the absorption of the roots will not be able to offset the loss of nutrient elements caused by ion 313 poisoning. 314

Effects of salt stress on photosynthetic parameters of Salix alba 315
Photosynthesis is a key metabolic process that provides material energy for plants. High salt stress will 316 comprehensively affect the photosynthesis of plants through osmotic stress, ion toxicity, and feedback inhibition 317 caused by the accumulation of photosynthetic products [58] . These effects will cause the destruction of the membrane 318 structure and the imbalance of ions in tissue cells, affecting the absorption of light energy by plants and the process 319 of carbon assimilation [59] . This change inhibits the formation of leaf primordia and reduces the photosynthetic area 320 and carbon assimilation of individual plants, resulting in physiological metabolic disorders and the accumulation of 321 toxic substances. In fact, the energy supply related to photosynthesis, carbohydrate metabolism, and the TCA cycle 322 are all inhibited by salt stress [60] . 323 Because stomata are directly connected to the external environment, their coordinated response under stress 324 determines whether the photosynthetic capacity of the plant is normal [61] . In this experiment, the Pn, E, and gs did not 325 change significantly when the NaCl concentration was 171mM/L. As the salt concentration further increased, each 326 index decreased significantly, which is basically consistent with the results of previous studies [62,63] . When the NaCl 327 concentration was less than 342mM/L, the Ci of the Salix alba. leaves decreased with decreasing gs. Thus, the 328 diffusion resistance of CO2 in the leaves increases, and the carbon sequestration ability weakens. The stoma factor is 329 the dominant factor restricting the decline in Salix alba. leaf photosynthesis. Later, as the degree of salt stress further 330 intensified, the Ci increased with the decreasing gs, and the photosynthetic system activity of the mesophyll cells 331 15 decreased, resulting in a decrease in the assimilation capacity, which is a typical non-stomatal limiting factor. Previous 332 studies have shown that under adverse stress, stomatal restriction and non-stomatal restriction and the interaction of 333 the two will reduce the photosynthetic rate of plants; under mild stress, stomatal restriction is dominant; and under 334 severe stress, stomatal restriction leads to non-stomatal restriction [64,65] . Our experiment also supports this view. 335

Effects of salt stress on chlorophyll fluorescence kinetics of Salix alba 336
The OJIP curve contains a great deal of information about the original photochemical reaction of the PSII 337 reaction center [66] . When environmental conditions change, chlorophyll fluorescence can directly or indirectly affect 338 the photosystem performance of plants [67] . The changes in the PSII can reflect the impact of changes in the stress 339 environment on the photosynthetic capacity of plants and the adaptation mechanism of photosynthetic machinery to 340 environmental changes. High salt stress can inhibit or destroy parts of the functions of PSⅡ, hinder the original 341 photochemical reaction and electron transfer process of PSⅡ, and reduce the photosynthetic capacity of Salix alba. 342 leaves. This consequence may be the result of the accumulation of Na + . The typical fast fluorescence kinetics curve 343 generally has O, J, I, and P phases during the rising phase of fluorescence [68] . This study shows that when the 344 concentration of NaCl is ≥ 342mM/L, the OJIP curve of Salix alba. will be deformed to OKJIP, the fluorescence 345 values of points I and P will decrease significantly, and obvious inflection point K will appear. The occurrence of the 346 K point is caused by damage to the PSII donor side oxygen release complex (OEC) due to the inhibition of the water 347 lysis system and the receptor-side part before QA, and the relatively variable fluorescence of the K point can represent 348 the degree of OEC damage [69,70] . In addition, the high salt treatment greatly shortened the time required to reach the 349 P point (the maximum fluorescence value). This result indicates that the higher the degree of salt stress, the greater 350 the damage to the stability of the PSⅡ reaction center and the OEC on the PSⅡ donor side of Salix alba. leaves, the 351 weaker the ability to provide electrons downstream and the stronger the reduction of the PSⅡ acceptor side is 352 hindered. 353 The φPo, Ψo, φEo, φDo reflect the energy distribution ratio of plants. In this study, when the NaCl concentration 354 was 171mM/L, there was no significant difference among the indicators. As the stress intensified, the φPo, Ψo and 355 φEo decreased significantly while the φDo increased significantly, which is different from the results of Huang 356 Qinqin et al. [71] . This finding shows that Salix alba. adjusted the energy distribution ratio of the PSII reaction center 357 under different degrees of stress. This adjustment occurs to increase the quantum ratio used for heat dissipation and 358 16 reduce the proportion of energy in photochemical reactions, which is an adaptive regulation mechanism of Salix alba. 359 under salt stress. The decrease in the φPo, Ψo and φEo indicates that the photosynthetic machinery is clearly damaged, 360 the ability to reduce the QB and PQ on the PSII receptor side is diminished, and the electron transfer process is 361 inhibited. Plants are prone to occur or aggravate photoinhibition in adverse environments [69] . In this study, when the 362 concentration of NaCl was greater than 171mM/L, the PIABS, PICSm and DFCSm all showed a significant downward 363 trend. This trend shows that Salix alba. leaves exhibit photoinhibition, the PSII reaction center is reversibly 364 inactivated or irreversibly degraded, the conversion efficiency of light energy is reduced, and the function of the 365 photosynthetic apparatus is impaired, which restricts the normal progress of photosynthesis. 366 In this study, 171mM/L NaCl stress had no significant effect on the growth status of the Salix alba. root system, 367 ion distribution or photosynthetic fluorescence characteristics and even increased these parameters to a certain extent. 368 As the salt treatment concentration gradually increased, the average root number, average root length, and rooting 369 index decreased significantly; Na + accumulated in the root system, K + and Ca 2+ were significantly lost; the 370 photosynthetic rate decreased significantly, the PSⅡ reaction center was partially inactivated, and the donor side OEC 371 and the electron acceptor on the acceptor side were damaged. Salix alba can respond to salt stress by intercepting 372 Na + in the root system, improving the selective absorption of K + and Ca 2+ and the ground transportation capacity, and 373 increasing the quantum ratio used for heat dissipation, indicating that Salix willow has some tolerance to salt stress 374 environments. 375 Supporting Information 376 S1  show that root germination and elongation are promoted in the presence of 0.1% 171mM/L NaCl, but root growth is 21 comprehensively inhibited under increasing salt stress. Under salt stress, Na + accumulates significantly in the roots 22 and leaves, and the Na + content and the Na + /K + and Na + /Ca 2+ root ratios are significantly greater than those in the 23 leaves. When the NaCl concentration is ≤ 0.2% 342mM/L, Salix alba. can maintain relatively stable K + and Ca 2+ 24 contents in its leaves by improving the selective absorption and accumulation of K + and Ca 2+ and adjusting the 25 transport capacity of mineral ions to aboveground parts, while K + and Ca 2+ levels are clearly decreased under high 26 salt stress. With increasing salt concentrations, the net photosynthetic rate (Pn), transpiration rate (E) and stomatal 27 conductance (gs) of leaves decrease gradually overall, and the intercellular CO2 concentration (Ci) first decreases and 28 then increases. When the NaCl concentration is < 342mM/L, the decrease in leaf Pn is primarily restricted by the 29 stomata. When the NaCl concentration is > 342mM/L, the decrease in the Pn is largely inhibited by non-stomatal 30 factors. Due to the salt stress environment, the OJIP curve (Rapid chlorophyll fluorescence) of Salix alba. turns into 31 an OKJIP curve. When the NaCl concentration is > 171mM/L, the fluorescence values of points I and P decrease 32 significantly, which is accompanied by a clear inflection point (K). The quantum yield and energy distribution ratio 33 of the PSⅡ reaction center change significantly (φPo, Ψo and φEo show an overall downward trend while φDo is 34 promoted). The performance index and driving force (PIABS, PICSm and DFCSm) decrease significantly when the NaCl 35 concentration is > 171mM/L, indicating that salt stress causes a partial inactivation of the PSII reaction center, and 36 the functions of the donor side and the recipient side are damaged.
[Conclusion] The above results indicate that Salix 37 alba. can respond to salt stress by intercepting Na + in the roots, improving the selective absorption of K + and Ca 2+ 38 and the transport capacity to the above ground parts of the plant, and increasing φDo, thus showing an ability to self-39 regulate and adapt. 40

Introduction 43
With economic and social development, the problem of soil salinization has become increasingly prominent, 44 resulting in approximately 1 billion hectares of saline-alkali land in the world [1] . The total area of China's saline-45 alkali land is believed to reach more than 100 million hectares [2] . Among the areas of concern, the coastal area, one 46 of the primary types of saline-alkali land, has frequent water-salt interactions and secondary salinization because it is close to the sea [3] . Because the ecological environment of the salinization area is fragile and natural conditions are 48 limited by many factors, it is highly significant to develop and use saline-alkali land scientifically and rationally while 49 under pressure from a rapid population increase and a sharp decline in land resources; the goal is to advance towards 50 the sustainable and healthy development of China's forestry and ecological environment [4] . 51 Choosing and cultivating excellent salt-tolerant tree species through biotechnology is currently one of the most 52 economical, effective, ecological and environmentally friendly biological measures to solve the soil salinization 53 problem [5] . Salix is a deciduous tree or shrub belonging to the genus Salix in the Salicaceae. It has strong ecological 54 adaptability and can grow well under saline-alkali, drought and barren soil conditions [6] . Previous studies on the salt 55 tolerance of Salix plants were mostly focused on the physiological responses of seedlings to salt stress [7,8,9] , but there 56 are few studies on the adaptability of plants to salt stress specific to seedlings. High salt stress will cause plant water 57 loss, ion imbalance and nutrient element deficiency through osmotic stress and ion poisoning [10] , which will affect 58 the normal growth and morphology of plants. A series of physiological growth changes in plants under salt stress are 59 the comprehensive embodiment of their salt tolerance ability, among which the growth status of plant roots, the ion 60 accumulation in different organs and the change in photosynthetic fluorescence parameters are important factors 61 affecting the salt tolerance ability of plants [11,12,13] . These indicators can not only represent the extent of the effects 62 of stress factors on plants, but they can also reflect the growth of plants under salt stress, the selective absorption and 63 transport of ions, and the photosynthesis ability. Willow has the characteristics of antipyretic, analgesic, anti-64 inflammatory, anti-rheumatism, astringent, drought resistance [14] and anti-corrosion [15] , among which the bark of 65 White Willow contains salicin [16,17] with antibacterial, bactericidal, antioxidant, antipyretic, analgesic and other 66 functions, and is a good natural food additive and food resource of health care products [18] . Its roots can also enrich 67 harmful elements, reduce the impact of harmful elements on the surrounding soil [19,20] , and play a role in purifying 68 polluted water [21] .Salix alba.has strong adaptability to adversity [22] , so it has great potential for use and promotion in 69

Effects of salt stress on ion content, absorption and transport in the roots and leaves of Salix alba 153
The ion content measurements (Table Fig 4) showed that under different concentrations of NaCl, the Na + 154 contents in the roots and leaves of Salix alba. were significantly higher than that in the control group, and the range 155 of Na + change was positively correlated with the stress concentration. The comparison of Na + contents in the roots 156 and leaves shows that the Na + content of the roots is much higher than that in the leaves. Under 684mM/L NaCl 157 stress, the Na + content in the roots could reach twice that in the leaves. With increasing stress concentration, the K + 158 content in the leaves first increased and then decreased, reaching a peak at a concentration of 171mM/L NaCl, which 159 was a significant increase of 14.0% compared to the control group. However, after the NaCl concentration was greater 160 than 342mM/L, the concentration was significantly lower than that of the control. As the stress concentration 161 increased, the K + contents in the roots of each treatment group showed a gradual decrease, which were all significantly 162 lower than that of the control. The Ca 2+ content in the leaves of Salix alba. increased first and then decreased with 163 increasing salt concentration. At 342mM/L NaCl, compared with the control group, the concentration significantly 164 increased by 13.6% and then showed a significant downward trend. The Ca 2+ content in the roots decreased 165 continuously with increasing stress, and when the NaCl concentration was 684mM/L, the Ca 2+ content dropped to 166 35.6% of the control. 167 Table Figs 5 and 6 shows that both the Na + /K + and Na + /Ca 2+ in the roots and leaves increased significantly with 168 increasing NaCl stress concentration. This finding shows that as the stress intensifies, the relative absorption of Na + 169 by Salix alba. increases greatly, but the absorption of K + and Ca 2+ decreases. The Na + /K + and Na + /Ca 2+ contents of 170 all the treatments gradually decreased from root to leaf, and the rising Na + /K + (F=1263.766, df=4，Sig.<0.001) and 9 Na + /Ca 2+ (F=10485.256, df=4，Sig.<0.001) in the roots were significantly higher than those in leaves (F=1235.223, 172 df=4，Sig.<0.001; F=2335.783, df=4，Sig.<0.001), suggesting that Salix willow could reduce the salt stress damage 173 to young tissues by regulating ion transport. 174 As shown in Table Fig 7, with increasing NaCl stress, the SA k, Na, ST k, Na, SA Ca, Na, and ST Ca, Na all showed a 175 trend of first increasing and then decreasing. When the NaCl concentration was less than or equal to 342mM/L, the 176 selective absorption capacity of the roots for K + (F=998.922, df=4，Sig.<0.001)and Ca2+ (F=1018.689, df=4，  177 Sig.<0.001) and the selective transport capacity of the leaves for K + (F=168.047, df=4，Sig.<0.001) and Ca 2+ 178 (F=29.925, df=4，Sig.<0.001) were enhanced and reached a significant level. The selective absorption capacity of 179 roots for K + is greater than that of Ca 2+ , but the selective transport capacity of the leaves to Ca 2+ is greater than that 180 of K + . These results indicated that Salix willow could adjust the upward transport capacity of K + and Ca 2+ via the 181 selective absorption and accumulation of mineral ions to compensate for the change in concentration under salt stress, 182 to prevent the impacts of nutrient deficiency and ion toxicity on the shoot growth. 183

Effects of salt stress on photosynthetic parameters in Salix alba.Leaves 184
Table Figs 8 and 9 shows that the photosynthetic parameters of Salix alba. leaves were affected to different 185 degrees under different salt concentrations. When the NaCl concentration was 171mM/L, the Pn of the leaves 186 increased, but there was no significant difference from the control. Later, as the salt stress intensified, the 187 photosynthetic carbon assimilation ability of Salix alba. leaves was significantly inhibited (F=95.66, df=4 ， 188 Sig.<0.001); when the NaCl concentration was greater than 171mM/L, both the E (F=100.091, df=4，Sig.<0.001)and 189 gs (F=69.346, df=4，Sig.<0.001) were significantly lower than the control and became stronger; but at a low salt 190 concentration (171mM/L NaCl), there is no significant difference from the control. With the increased salt 191 concentration, the leaf Ci showed a trend of first decreasing and then increasing, reaching the lowest when the salt 192 concentration was 342mM/L, which was significantly lower than the control by 10.4%, and then it gradually 193 increased. The Ci of leaves (F=20.50, df=4，Sig.<0.001) under 513mM/L and 684mM/L NaCl treatments were not 194 10 significantly different from that of the control, but they were significantly higher than the lowest value by 12.4% and 195 14.6%, respectively. The excitons captured by the reaction center transfer electrons to the electron transport chain, and the ratio of 217 excitons that exceed QA's other electron acceptors to promote QA reduction excitons (Ψo) and the light energy 218 11 absorbed by the reaction center are used for electron transfer. The quantum yields (φEo) all increased first and then 219 decreased with the increasing salt stress. At 171mM/L NaCl, although the Ψo and φEo increased, they were not 220 significantly different from the control. Later, as the stress intensified, both the Ψo and φEo were significantly lower 221 than those of the control. When the NaCl concentration was 342mM/L, the Ψo and φEo were significantly lower than 222 the 11.1% and 11.9% of the control group, respectively. Compared with the control group, salt stress increased the 223 quantum ratio (φDo) of Salix alba. leaves for heat dissipation. When the NaCl concentration was 513mM/L, φDo 224 was significantly higher than that of the control.

Influence of salt stress on the root growth status of Salix alba 241
As the primary organ responsible for plant material exchange, the root system and its growth status are closely 242 related to the growth and development of the aboveground plant parts, whether the root system can function normally, 243 and the plant's water and nutrient utilization efficiency [33] . Under salt stress, the root system is the first to feel the 244 adversity stress signal, and it is also the most directly affected part [34] . Its ring-stripe inhibition is primarily manifested 245 in the low levels of the root length, surface area and other parameters, and the root system grows slowly. A high-salt 246 environment will cause plants to experience osmotic stress and ion toxicity, which will lead to changes in membrane 247 permeability, which will in turn affect the absorption of water and nutrient elements by the roots, causing the plants 248 to lose a large amount of water; the ions near the roots will be unbalanced, the physiological functions of the roots 249 will eventually be lowered, and even the structure will be destroyed. Some of the aboveground leaves wilt, and 250 photosynthetic production cannot be performed normally, which causes plant growth and metabolic disorders until 251 the loss of physiological functions. 252 The change in root growth and the time of the root sprouting period can directly reflect the degree of damage to 253 plants by salt stress and represent the strength of plant salt tolerance [35] . This study showed that the 171mM/L NaCl 254 concentration significantly promoted the increase in the average number of roots and the elongation of the average 255 root length of Salix alba. cuttings, and it can promote the rooting of the root system in advance, to a certain extent, 256 which is consistent with Wang Shufeng et al. [36] and Ci Dun. The research results of Wei et al. [35] were basically the 257 same. This growth response may be due to the decrease in water potential outside the roots under salt stress, which 258 stimulates the growth of the roots instead of moderate osmotic stress to ensure the normal absorption of water and 259 nutrients to meet the physiological and metabolic needs of the aboveground parts. 260 Some plants do have the phenomenon that low salt promotes the increase of some indicators, such as: promoting 261 the germination of sorghum seeds [37] , the roots of the seedlings of wolfberry [38] and rice [39] , and the growth indicators 262 of corn [40,41] .Both ChorophyⅡin chrysanthemum [42] and proline content of cherry seedlings [43] are increased, while 263 the net photosynthetic rate of wild chrysanthemum [44] and hazel trees increased [45] . The reason for the low salt 264 concentration may be that the salt stress has a dual effect of stimulus and inhibition on plants. The strong and weak 265 13 relationship between stimulus and inhibition triggers changes in various plant indicators, resulting in the same low 266 salt. It can promote growth, and it will be inhibited after high salt.This finding shows that Salix alba. has some ability 267 to adjust and adapt to salt stress, and this adaptability is of great significance to the survival and continuation of the 268 plant itself under adversity. However, as the salt stress intensifies, the ability of plants to coordinate their own growth 269 is destroyed, root germination and elongation are significantly inhibited and become more intense, the root functions 270 are destroyed, and the plants cannot maintain their normal growth and development. In this study, when the salt concentration was low, the growth of Salix alba. was basically normal, the symptoms 279 of salt damage were not significant, and the damage was obvious under severe stress. Na + accumulates significantly 280 in the roots and leaves of Salix alba. under salt stress, but the Na + content in different organs is significantly different, 281 and it is primarily concentrated in the roots. This result shows that the willow root system has a compensation 282 mechanism that can reduce the transportation of salt to aboveground parts by enriching Na + in the root, thereby 283 effectively reducing or delaying the occurrence of salt damage in the aboveground parts. This conclusion is consistent 284 with the study by Hao Han et al. [47] . When the salt stress is too high, this balance is broken, and growth is blocked. 285 As an important inorganic solute, K + is essential for reducing the cell osmotic potential and maintaining the 286 water balance. Generally, plants have an antagonistic effect on the absorption of Na + and K + [48] , and the competition 287 between the two usually leads to a decrease in the K + content. The loss of K + will cause changes in the physical 288 structure of the stomata, frustrating photosynthesis [49] . In addition, K + participates in the metabolism of various 289 enzymes in plants [50] . As salt stress increases, an excessive loss of K + will lead to K + dependent enzymes in Salix 290 14 alba. The enzyme activity decreases, which affects the metabolic reactions in which it participates. Therefore, if 291 plants are to grow in a salty environment, the selective absorption of K + by the root system and the transportation of 292 K + to the ground are particularly important. This study showed that the K + content in the roots of Salix alba. 293 significantly decreased with increasing stress, but the K + in the leaves could be maintained at a high level at a 294 342mM/L NaCl concentration and below and even increased significantly when the NaCl concentration was 295 171mM/L, according to Zhou Qi et al. [51] A study on Carpinus chinensis also confirmed this result. At this time, the 296 value and increase of Na + /K + in the roots of the Salix alba. were greater than that of the leaves, and the SA k, Na and 297 ST k, Na all increased significantly. Studies have shown that under salt stress, the Na + /K + value can represent the 298 degree of salt damage to the plant, and the lower Na + /K + value of the leaves can help the plant better maintain its 299 growth and photosynthetic function [52] , and the SA k, Na and ST k, Na indicates that the plants can better tolerate salt 300 stress [53] . This result shows that at that time, Salix alba. could maintain a relatively stable leaf K + content and the 301 normal progress of photosynthesis by restricting the transportation of Na + from the root to the leaves, increasing the 302 selective absorption of K + through the plant roots and the ability to transport K + to the ground. The accumulation of 303 Na + causes damage to plants, which may be an important mechanism by which Salix alba. copes with salt stress. 304 Later, with the increase in salt stress, the K + in the roots and leaves clearly flowed out. A high concentration of Na + 305 will replace the Ca 2+ bound to the membrane system, which will damage the integrity of the membrane structure and 306 membrane function, thereby destroying the ion balance in the plant body and causing a large amount of organic solute 307 extravasation [54] . The establishment of Ca 2+ homeostasis in the cytoplasm is a key condition for salt adaptation [55] . 308 This experiment showed that as the salt stress intensified, the Ca 2+ content in the Salix alba. roots continued to 309 decrease, but it could accumulate in the leaves when the NaCl concentration was ≤342mM/L. The results of Jia Yin 310 et al. [56] were similar; the Na + /Ca 2+ value of white Salix roots was higher than that of the leaves, and the Sa Ca, Na and 311 ST ca, Na were all significantly increased. This result may be due to the large influx of Na + into the root system under 312 25 temperature-induced damage in wheat. Sensors 20 (1)

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