Responses of growth and photosynthesis to alkaline stress in three willow species

Investigating differences in resistance to alkaline stress among three willow species can provide a theoretical basis for planting willow in saline soils. Therefore we tested three willow species (Salix matsudana, Salix gordejevii and Salix linearistipularis), already known for their high stress tolerance, to alkaline stress environment at different pH values under hydroponics. Root and leaf dry weight, root water content, leaf water content, chlorophyll content, photosynthesis and chlorophyll fluorescence of three willow cuttings were monitored six times over 15 days under alkaline stress. With the increase in alkaline stress, the water retention capacity of leaves of the three species of willow cuttings was as follows: S. matsudana > S. gordejevii > S. linearistipularis and the water retention capacity of the root system was as follows: S. gordejevii > S. linearistipularis > S. matsudana. The chlorophyll content was significantly reduced, damage symptoms were apparent. The net photosynthetic rate (Pn), rate of transpiration (E), and stomatal conductance (Gs) of the leaves showed a general trend of decreasing, and the intercellular CO2 concentration (Ci) of S. matsudana and S. gordejevii first declined and then tended to level off, while the intercellular CO2 concentration of S. linearistipularis first declined and then increased. The quantum yield and energy allocation ratio of the leaf photosystem II (PSII) reaction centre changed significantly (φPo, Ψo and φEo were obviously suppressed and φDo was promoted). The photosystem II (PSII) reaction centre quantum performance index and driving force showed a clear downwards trend. Based on the results it can be concluded that alkaline stress tolerance of three willow was as follows: S. matsudana > S. gordejevii > S. linearistipularis. However, since the experiment was done on young seedlings, further study at saplings stage is required to revalidate the results.


Experimental materials and design
The experimental site was located at the West Campus of Hebei Agricultural University, Baoding City, Hebei Province.The temperature of the climate chamber was set at 28 °C/25 °C (light/dark), light intensity was controlled by an LED cold light source at 1000 μmol m −2 s −1 , photoperiod was 14 h/10 h (light/dark), relative humidity was 60%.
The test materials were collected from the germplasm resource nursery of Jinsha Beach Forestry Farm, Huai'an County, Hebei Province.The branches of three willow species, S. matsudana, S. gordejevii and S. linearistipularis, which were the same in length and strong without pests and diseases, were collected in the early spring before bud break and then stored in a freezer at 0 °C.The hydroponic test was started in the artificial climate chamber of Hebei Agricultural University, Baoding City, Hebei Province.Before the experiment, The middle two-thirds of the selected branches were.
cut into 20 cm-long cuttings.The uppermost bud was 0.5-1 cm from the top of the cuttings.Adequate number of willow cuttings were grown in fresh water and managed regularly.When their growth reached the treatment requirements (average root length of approximately 5 cm), cuttings with uniform growth and root systems were selected to start the alkaline stress treatment experiment.
The cuttings were placed in 55 cm × 38 cm × 15 cm (L × W × H) plastic boxes for hydroponics with five treatments.Hydroponic solutions with pH values of 8.0, 8.5, 9.0, and 9.5 (mNa 2 CO 3 : mNaHCO 3 = 1:1) were prepared using 1/2 Hoagland complete nutrient solution as the background solution and 1/2 Hoagland complete nutrient solution as a control (CK).The pH of CK was 7.2.Each treatment was replicated three times, and 20 cuttings were placed in each replication and directly immersed in the solution, approximately half the cuttings' height (Fig. 1).The nutrient solution was changed every 5 days during the growth process.Before the nutrient solution was changed, the cuttings were removed and the roots were rinsed with water to wash away the last residual salt and prevent excessive salt accumulation.We randomly selected three seedlings with even growth vigor on the 1st,

Effects of alkaline stress on the water content of three willow cuttings
As in from Fig. 3, the root and leaf water content of the three willow cuttings was affected to different degrees under different concentrations of alkaline stress, and root and leaf water content showed an overall decreasing Table 1.Effects of alkali stress on changes in leaf and root dry weight of three willow cuttings.(In statistical terms, the letters a, b, c, and d represent significant differences among the respective data at the P ≤ 0.05 level).trend with increasing pH.When the pH of the alkaline solution was 8.0 and 8.5, the leaf water content of the three willow cuttings decreased, but there was no significant difference compared with that of the control group (Fig. 3a, b, c).When the pH of the alkaline solution was 9.0 and 9.5, the leaf water content of the three willow cuttings decreased significantly, with the most significant decrease occurring in S. linearistipularis (Fig. 3c).This result indicated that the water retention capacity of S. linearistipularis leaves was the worst among the three willow cuttings under high concentrations of alkaline stress.At alkaline stress pH values of 8.0 and 8.5, there was no significant difference in the root water content of S. matsudana and S. gordejevii compared with the control (Fig. 3d, e).When the pH was 9.0 and 9.5, the root water content of S. matsudana and S. gordejevii decreased significantly compared with that of the control.In contrast, the difference in root water content of S. linearistipularis was only significant at pH 9.5 compared with the control (Fig. 3f).The water retention capacity of three willow cuttings' roots were in the order of S. linearistipularis > S. gordejevii > S. matsudana.

Effects of alkaline stress on the chlorophyll content of three willow cuttings
As seen in Table 2, the changes in chlorophyll a, chlorophyll b and total chlorophyll content of the leaves of the three willow cuttings were consistent with a decreasing trend.S. linearistipularis showed the most significant decrease under alkaline stress at different pH values, while S. matsudana showed the smallest.At pH 8.0, the total chlorophyll contents of the three willow cuttings decreased by 1.40%, 10.30% and 17.70%, respectively, compared with the control.

Effects of alkaline stress on photosynthetic gas exchange parameters in three willow cuttings
Photosynthesis is an essential pathway for biosynthesis and physiological metabolism during the growth and development of plant bodies and is more sensitive to changes in the external environment.Photosynthesis is an important indicator of the level of alkalinity resistance in plants.Figure 4 shows that S. linearistipularis had the lowest level of photosynthetic gas exchange among the three willow cuttings.The alkaline stress treatment concentration increased, and the leaf Pn, Gs and E of the three willow cuttings decreased significantly.Intercellular CO 2 concentration of S. matsudanau leaves and S. gordejevii leaves showed a decreasing trend under alkali stress, and that of S. linearistipularis leaves showed a decreasing and then increasing trend (Fig. 4j, k, l).
Changes in the rate of decline of Pn in the leaves of the three willow cuttings differed with increasing pH.The rate of Pn decline in S. gordejevii and S. linearistipularis peaked between pH CK-8.0.They slowed down significantly between pH 8.0-9.5, whereas the rate of Pn decline in S. matsudana leaves was more stable throughout alkaline stress (Fig. 4a, b, c).www.nature.com/scientificreports/

Effects of alkaline stress quantum yield and energy partition ratio of three willow cuttings
As shown in Fig. 5, the trends of the quantum yield and energy allocation ratios of the leaves of the three willow cuttings under alkaline stress were generally consistent, all of which showed an overall gradual decrease in φPo, Ψo, and φEo and an overall gradual increase in φDo with the aggravation of the stress.There were differences in tolerance to alkaline stress among different species.S. matsudana showed better index stability under alkaline stress than the other two willows (Fig. 5a, d, g, j).Notably, S. gordejevii was able to better regulate its adaptive capacity to the adverse environment by changing the energy absorbed, converted, used for electron transfer and dissipated by heat radiation in the leaves at lower pH (pH < 9) under alkaline stress, during which its indices were less inhibited than those of the other two willows.From the point of view of energy utilisation, S. gordejevii had a more vital short-term alkaline stress tolerance ability.However, with further aggravation of the alkaline stress, the indices changed abruptly.The speed and magnitude of the changes were greater than those of S. matsudana and S. linearistipularis, and the degree of damage was significant (Fig. 5b, e, h, k).

Effects of alkaline stress on leaf performance indices and propulsion of three willow cuttings
As shown in Table 3, the changes in the PI ABS and PI CSm of S. matsudana leaves were the same, and both showed an overall decreasing trend with increasing pH.The DF CSm of S. matsudana leaves showed an overall decreasing trend with increasing pH, and the differences were significant when the pH was 8.5, 9.0 and 9.5 compared with the control group.S. gordejevii leaf PI ABS , PI CSm and DF CSm all showed a trend of increasing and then decreasing with increasing pH.The three indices peaked when the pH was 8.0 and then decreased significantly with increasing pH in all treatments compared with the control group.The PI ABS, PI CSm and DF CSm of S. linearistipularis leaves showed an overall decreasing trend with increasing pH, and each treatment group decreased significantly compared with the control group.

Correlation analysis of indicators of three willow cuttings under alkaline stress
The relationship between 16 indicators of S. matsudana root and leaf water content, chlorophyll content, and photosynthesis can be determined by analysing a heatmap of the correlation matrix.From Fig. 6a, it is evident that in S. matsudana, there are 92 pairs of indicators with a highly significant correlation level (P ≦ 0.01) , 5 pairs with a significant correlation level (P ≦ 0.05) , and 22 pairs with no correlation or insignificant correlation.The correlation between root water content and other indicators was insignificant except for the highly significant correlation between root and leaf water content.The intercellular CO 2 concentration in S. matsudana leaves had weak or no correlation with other indicators except for a highly significant correlation with stomatal conductance and significant correlations with chlorophyll b, net photosynthetic rate, transpiration rate, PI ABS and PI CSm .There were 105 pairs of highly significant correlations (P ≦ 0.01) , 4 pairs of significant correlations (P ≦ 0.05) , and 10 pairs of weak or nonexistent correlations among the indicators in S. gordejevii (Fig. 6).The intercellular CO 2 concentration of S. gordejevii showed weak or no correlation with most indicators, except for a negative correlation with root and leaf water content and significant correlations with φPo, φDo, and DF CSm .The correlation between Pn and leaf water content did not reach the level of significance, which was different from that of S. matsudana and S. linearistipularis.Table 2. Effects of alkaline stress on chlorophyll a, chlorophyll b and total chlorophyll contents of three willow cuttings.(In statistical terms, the letters a, b, c, and d represent significant differences among the respective data at the P ≤ 0.05 level).correlation with root and leaf water content, Ψo, φEo and DF CSm , a significant negative correlation with φPo, a significant positive correlation with φDo, and a weak or nonexistent correlation with the other indicators.Except for the intercellular CO 2 concentration, there were highly significant correlations between the other indicators.As one of the main organs for material exchange in plants, the growth of the root system is closely related to the growth and development of the above-ground parts, whether the root system can function properly, and the efficiency of water and nutrient utilisation by plants 15,16 .However, because of its direct contact with adversity stress, it can be the earliest to feel the signals of adversity stress and become the first and foremost site of damage 17 .It has been shown that the metabolic regulation of roots is the first barrier for plants to cope with saline and alkaline stress 18,19 , and in particular, the alkaline resistance of plants mainly depends on roots 20 .This study found that the dry weights of roots and leaves of three willow cuttings generally tended to decrease during alkaline stress.The dry weight of the root system of S. matsudana, which is highly alkaline tolerant was always maintained at a high level.S. gordejevii root dry weight also maintained a stable state at higher levels, although it changed significantly under low pH stress.S. linearistipularis root system dry weight remained low and decreased significantly at pH 8.5.Therefore, the alkaline tolerance of the three willow cuttings was thus S. matsudana > S. gordejevii > S. linearistipularis.www.nature.com/scientificreports/

Analysis of changes in root and leaf water content of three willow cuttings under alkaline stress
Salinity stress can lead to osmotic pressure imbalance in the plant, preventing water uptake and, in severe cases, leading to water exudation or even plant death 21 .In this experiment, the water content of the root systems of S. gordejevii and S. linearistipularis decreased by approximately the same amount.In contrast, the water content of the root system of S. matsudana decreased by the most significant amount.However, the water content of S. matsudana leaves was able to remain stable.S. matsudana reduced root water uptake as much as possible without affecting the growth of the root system, which is an essential mechanism for plant adaptation to saline and alkaline stress.This supports Jia's study on the impact of alkaline stress on hickory's photosynthetic properties 22 .Yang et al. pointed out in their study on alfalfa that plant water loss can be used as a rapid and economical osmotic adjustment to cope with osmotic imbalance brought about by saline and alkaline stress 23 .The results of this study showed that the stability of the leaf water content of the three willow cuttings was a vital reflection of the strength of their resistance.S. matsudana leaf water content remained stable throughout the alkaline stress period, S. gordejevii leaves retained water better under low-concentration stress, and S. linearistipularis leaves retained water the least.This difference in water uptake among willow species may be due to variations in their ability to accumulate osmoregulatory substances, which affect osmotic pressure.

Analysis of changes in photosynthetic pigments and the photosynthetic system of three willow cuttings under alkaline stress
Photosynthesis in plant leaves depends mainly on the absorption, transfer and conversion of light energy in Thylakoid Membrane 24 .Studies have shown that photosystem II (PSII) is the most sensitive to salinity stress, and chloroplasts are one of the most essential organelles in plant PS II that respond to salinity stress 25 .Chlorophyll a and chlorophyll b are concentrating pigments that are not photochemically active.They are mainly responsible for absorbing light energy and transferring it to the unique chlorophyll pair in the reflection centre during photosynthesis 26 .Changes in the external environment will directly affect the chlorophyll content of leaves, which in turn will impact the photosynthetic function of plants.Therefore, chlorophyll content has become an important reference index for plant salinity tolerance 27 .In addition, Mg 2+ and Fe 2+ , as essential elements for chlorophyll synthesis and chloroplast structure, precipitate at high pH, which is one of the reasons for the decrease in plant chlorophyll content due to alkaline stress 28 .In this study, we found that during alkaline stress, the photosynthetic pigment content of the three willow species varied in a gradual decline with increasing pH during alkaline stress.S. matsudana was able to ensure the relative stability of its photosynthetic pigment content during alkaline stress and was less affected by alkaline stress, followed by S. gordejevii.Furthermore, S. linearistipularis showed the most significant decrease in photosynthetic pigment content.The total chlorophyll content of the three willow cuttings showed significant differences in their sensitivity to low concentrations of alkaline stress, with S. linearistipularis > S. gordejevii > S. matsudana.This indicates that alkaline stress destroys the plant environment, affects the physiological functions of the plant, reduces the stability of the cyst-like membrane, significantly inhibits the synthesis of chlorophyll, and accelerates the decomposition of chlorophyll by increasing chlorophyll hydrolase, thus weakening the absorption of light energy by chlorophyll 29 .However, there were significant differences in the magnitude and rate of decline in chlorophyll content of the three willow cuttings under alkaline stress, which may be related to the resistance of the tree species to saline and alkaline stress.Photosynthesis, as a critical anabolic process of plant material energy sources, not only provides energy for the average growth, development and physiological metabolism of plants but is also more sensitive to adverse environments and is one of the critical indicators for determining the damage status of plants 30 .Alkaline stress is one of the critical factors for the inhibition of plant photosynthesis compared to salt stress.In addition to ionic toxicity and osmotic stress from high concentrations of salt ions in the plant body, high pH stress in the interroot is also one of the critical factors for inhibiting plant photosynthesis 31 .Studies have demonstrated that plants can recover from the effects of salt stress.The degree of recovery from damage to the acceptor side of the PS II reaction centre under salt stress was close to 100%.The degree of recovery from damage to the donor side was less than 85%.
In contrast, the effects of alkaline stress on the photosynthetic system of phytomass were not reversible, which may be related to the effect of high pH of alkaline stress on the plant 32 .In addition, alkaline stress inhibits chlorogenesis and reduces the photosynthetic area and carbon assimilation in monocots, which results in physiological metabolic disorders and the accumulation of toxic substances 33 .Salinity stress inhibits energy-related processes, such as photosynthesis, carbohydrate metabolism, and the TCA cycle 34 .
The net photosynthetic rate, as a critical indicator of photosynthesis, can straightforwardly reflect the material production capacity of plants per unit leaf area 35 .Based on previous studies, limiting factors affecting photosynthesis under adverse stress can be categorised into two main groups: stomatal limitation and nonstomatal limitation 36 .In this study, the leaf Pn, Gs and E of all three willow cuttings decreased significantly with increasing pH.Among them, S. matsudana was better able to maintain the stability of its own Pn under more substantial stress concentrations.Furthermore, S. gordejevii and S. linearistipularis were significantly inhibited at the beginning of alkaline stress.The reduction in transpiration rate and stomatal conductance may be an adaptive response to the reduction in water content, which is consistent with the reduction in willow leaf water content in the results of this experiment 37 .Reducing the transpiration rate as much as possible without affecting the photosynthetic rate is essential for plant adaptation to adversity 38 .Leaf Ci decreased with decreasing Gs at lower pH in three willow cuttings, showing typical stomatal factor limitation 39 .At higher pH for the three willow cuttings, S. matsudana and S. gordejevii leaf Ci did not change significantly with decreasing Gs. S. linearistipularis leaf Ci increased with decreasing Gs, showing typical nonstomatal factor limitation.The changes in Ci of S. matsudana and S. gordejevii leaves indicated that the two types of willows were less affected by nonstomatal limiting factors www.nature.com/scientificreports/under high alkaline stress, which differed from the results of previous studies and might be highly related to the unique salinity resistance mechanism of the two types of willows.

Analysis of changes in chlorophyll fluorescence kinetic parameters in three willow cuttings under alkaline stress
φPo, Ψo, φEo and φDo reflect the energy allocation ratio of plants.In this study, at lower pH at the beginning of alkaline stress, all fluorescence indices of the three willow cuttings showed no significant difference compared with the control group, indicating that the low concentration of alkaline stress had less effect on the energy allocation in the PS II reaction centre of the three willow cuttings, which was the same as the results of Zhang et al. 40 .With stress intensification, φPo, Ψo, and φEo significantly decreased while φDo significantly increased in S. gordejevii and S. linearistipularis, and the magnitude of changes was positively correlated with stress intensity.S. matsudana φPo, Ψo, φEo and φDo had the same trend of change as those of the other two willows, but the magnitude of change was smaller, and φPo differed from the control only at pH 9.5.The three willow cuttings adjusted the energy allocation ratios of their own PS II reaction centres under alkaline stress, i.e., the quantum ratio of energy absorbed by antenna pigment cells for heat dissipation increased and the energy share of photochemical reactions decreased 41 .Plants rely on light energy through photosynthesis to synthesise organic matter.However, too much light energy can harm their photosynthetic apparatus.To prevent damage, excess energy is dissipated through the lutein cycle for photoprotection, according to Barbara et al. 42 .To reduce the damage to the photosynthetic apparatus caused by excess light energy, plants will also appropriately reduce chlorophyll content to reduce the capture of excess light energy without affecting photosynthesis, which is also a self-regulatory mechanism for plants to cope with stress 43,44 .In conclusion, the three willow cuttings showed different degrees of decreases in φPo, Ψo and φEo, indicating that the photosynthetic apparatus was significantly damaged and that the ability of the PSII receptor side Q A to transfer electrons and the ability of Q B and PQ to be reduced were reduced, as evidenced by the reduced opening of the active reaction centres and the inhibition of the electron transfer process 45 .Compared with the three willow cuttings, S. matsudana had the most vital ability to maintain its stability under alkaline stress; S. gordejevii was more tolerant at lower alkaline stress concentrations, but the degree of damage increased rapidly with a further increase in alkaline stress; and S. linearistipularis was the least resistant throughout the alkaline stress process.
Plants are prone to photoinhibition or exacerbation in adverse environments, and PI ABS can comprehensively reflect the activity of photosystems through the three aspects of absorption, capture and electron transfer of light energy under adverse conditions 46 .In this study, the overall trend of decreasing PI ABS , PI CSm and DF CSm in the leaves of the three willow cuttings showed a more obvious decreasing trend with the increasing degree of alkaline stress, among which S. matsudana showed a minor change, S. gordejevii showed a more vital ability to tolerate the low concentration of stress, and S. linearistipularis showed the most apparent decreasing trend.This indicates that alkaline stress seriously affects the absorption and utilisation of light energy and leads to a decrease in the basal driving force, and the degree of reduction in PSII activity varies among different species of willow leaves.The stress triggered photoinhibition, reversible inactivation or irreversible degradation of the PSII reaction centre, reduced light energy conversion efficiency and impaired functioning of the photosynthetic apparatus in willow leaves, thus limiting the normal conduct of photosynthesis.

Analysis of correlation differences among indicators of three willow cuttings under alkaline stress
Correlation analysis among indicators of three willow cuttings under alkaline stress.This study showed no significant correlation between the root water content of S. matsudana and other indicators, except for a significant positive correlation with leaf water content (P ≦ 0.05) .Unlike S. matsudana, the correlation between root system water content and other indicators of S. gordejevii and S. linearistipularis reached a significant level (P ≦ 0.01) .The difference in correlations among the three willow cuttings may be related to the existence of unique salinity resistance mechanisms in S. matsudana.Compared with S. gordejevii and S. linearistipularis, S. matsudana has better drought resistance, which reduces the effect of alkaline stress on the plant by reducing the water content of the root system and synthesising more osmoregulatory substances to maintain the water content of the leaves without affecting the normal physiological metabolism of the plant.Some studies have shown that the decrease in the water content of alfalfa cuttings under alkaline stress is not only a result of osmotic stress but also may be due to the damage of high pH to the root system structure of the plant body and osmotic regulatory substances 23 .Suitable water content may enable salt-resistant plants to develop less energy-consuming osmotic pressure, thus increasing the resistance of saline plants to stress 47 .
It was also observed in this study that the intercellular CO 2 concentration in S. matsudana was significantly and positively correlated with the net photosynthetic rate (P ≦ 0.05) , transpiration rate (P ≦ 0.05) , and stomatal conductance (P ≦ 0.01) and was not correlated with root and leaf water contents.In contrast, the intercellular CO 2 concentrations in S. gordejevii and S. linearistipularis were not correlated with the net photosynthetic rate, transpiration rate, and stomatal conductance, root and leaf water content was significantly negatively correlated (P ≦ 0.01) .The results indicated that stomatal factors were the main limiting factors for the growth of S. matsu- dana under alkaline stress.In contrast, nonstomatal factors were the main limiting factors for the growth of S. gordejevii and S. linearistipularis under alkaline stress.

Conclusion
Alkaline stress affected root and leaf dry weight, root and leaf water content, chlorophyll content, photosynthesis and chlorophyll fluorescence kinetic parameters in all three willow cuttings.To some extent, the three willow cuttings can cope with the damage caused by alkaline stress by appropriately reducing the root water content while increasing the water-holding capacity of the leaves.Stronger alkaline stress also inhibited chlorophyll synthesis and accelerated chlorophyll decomposition.At low concentrations of alkaline stress, stomatal closure was the leading cause of decline in all three willow cuttings.In contrast, at high concentrations of alkaline stress, nonstomatal factors were the main factors limiting photosynthesis in all three willow cuttings.S. matsudana and S. linearistipularis photosynthesis were less affected by nonstomatal limiting factors, while S. linearistipularis photosynthesis was significantly nonstomatal limited.In terms of chlorophyll fluorescence kinetic parameters, alkaline stress degraded or inactivated the leaf PSII reaction centre and caused damage to both the donor and acceptor sides of PSII, triggering a series of responses, such as photoinhibition, reduction in light energy conversion efficiency and impaired functioning of the photosynthetic apparatus in the leaves of the three willow cuttings.The correlation between the root water content of S. matsudana and the indicators once again illustrated the idea that plants can cope with alkaline stress by reducing the root water content.Furthermore, a comparison of the correlations between Ci and Pn, Gs and E showed that nonstomatal limiting factors had less inhibitory effects on the growth of S. matsudana and that the growth of S. gordejevii and S. linearistipularis was significantly nonstomatal-limited.
The adaptive mechanisms of the three willow cuttings to alkaline stress differed.The performance of the three willow cuttings showed that S. matsudana could better maintain the relative stability of the indicators at higher alkaline stress concentrations, followed by S. gordejevii, which showed a stronger ability to tolerate low alkaline stress concentrations.However, the rate of change in the indicators increased rapidly with further stress intensification.S. linearistipularis was the least adaptive to the regulation of alkaline stress.

Figure 1 .
Figure 1.Design of three willows experimental treatment.

Figure 2 .
Figure 2. Effects of alkaline stress on the morphology of three willow cuttings (a: S. matsudana; b: S. gordejevii; c: S. linearistipularis).Note: From left to right are the changes of willow cuttings growth with the increase of pH on the 8th day of stress.

Table 3 .
Effects of alkaline stress on performance indices PI ABS , PI CSm and driving force DF CSm of three willow cuttings.
(In statistical terms, the letters a, b, c, and d represent significant differences among the respective data at the P ≤ 0.05 level).