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Abstract [Objectives] This study was conducted to clarify the saline-alkali tolerance in seedlings of Lonicera caerulea L.
[Methods] The L. caerulea seedling variety， Lanjingling， was used as the test material， and alkaline solution （NaHCO3） with different concentration gradients was used for stress treatment to observe physiological effects on L. caerulea seedlings.
[Results] L. caerulea seedlings were most affected by alkaline stress at a treatment concentration of 100 mmol/L， and their osmotic substances （proline， soluble sugar， soluble protein） and antioxidant enzymes （CAT， SOD and POD） were higher in content at a concentration of 100 mmol/L compared with the alkaline stresses in this range. The contents were generally higher， and even in the detection of proline， soluble sugar， SOD and POD， the contents of these substances and enzymes reached a peak at 100 mmol/L. The contents of soluble sugar， CAT， SOD， POD and malondialdehyde were generally higher than that of the control check （CK）， and the contents of proline and soluble protein in each treatment concentration were generally higher than that of the CK.
[Conclusions] The metabolic physiology of L. caerulea seedlings has a certain adaptability to alkaline stress.
Key words Lonicera caerulea L.; Alkaline stress; Physiological response; Stress resistance
Lonicera caerulea L.， also known as Shanqiezi， Yangnaizi and Heixiaziguo， is a rare and natural wild edible berry in the world. It can be eaten raw， can be brewed into wine and prepared into beverages and jams， and has a very high edible value. In addition， L. caerulea also has good medicinal value， as it has the functions of clearing away heat and toxic materials， resisting inflammation and viral diseases， preventing capillary rupture， lowering blood pressure， improving liver detoxification function， resisting tumors， relieving discomfort symptoms after radiotherapy， and slowing down the reduction of white blood cells after chemotherapy[1].
The salt-alkalization of soil is a major environmental problem which China faces. The soil contains alkaline salts HCO-3， CO-3， etc.， which will cause the soil pH to increase， affecting plant photosynthesis， biofilm permeability and antioxidant enzyme activity changes[2]. Due to the high concentration of salt ions in saline-alkali soil， it will slow down the absorption of water by the plants， destroy the plants growth function， make plants unable to grow normally， and even cause large-scale death[3]. Tianjin Binhai and its surrounding areas are one of the main occurrence areas of coastal saline-alkali soils. The L. caerulea variety， Lanjingling， is an excellent line bred with Beilei and Lanniao as its parents. It has good fruit taste， no bitter taste， higher nutritional value and medicinal value than wild varieties， and has broad development prospects. Studying the alkali-tolerant characteristics of L. caerulea can provide theoretical guidance for the cultivation and popularization of L. caerulea in Tianjin and North China.
Materials and Methods
Materials
The tree specie to be tested was the L. caerulea variety， Lanjingling， belonging to Lonicera Linn in Caprifoliaceae. The material was 2-year-old cutting seedlings introduced from Heilongjiang Province.
Experimental methods
Planting
This experiment was carried out in the Garden Plant Experiment Base of Tianjin Agricultural University. The cultivation substrate was peat∶vermiculite∶perlite=2∶1∶1. The size of the flowerpots was 22.5 cm in diameter and 21.5 cm in height. A total of 30 pots of seedlings were planted in the experiment， with 3 plants per pot. Eighteen pot plants with normal leaf color and normal growth status were selected for the experiment.
Alkaline stress treatment
Alkaline solution was poured on October 9， October 16， October 25， and November 1， 2019.Five concentration gradients （50， 100， 150， 200， 300 mmol/L） were set ， with no application of alkali as the control （CK）. Each treatment was repeated 3 times. In order to avoid the death of plants caused by excessive alkali addition at one time， in the high concentration alkali concentration group， the alkali solution concentration was increased by 50 mmol/L every 7 d gradually to the preset alkali concentration in the pot.
Physiological index measurement methods
After 20 d of alkaline stress， sampling was performed according to the principle of sampling at the same position at the same height. The physiological indexes such as proline， soluble sugar， soluble protein， CAT， SOD， POD， malondialdehyde and chlorophyll were measured[4-7]， respectively， and the measurement of each physiological index was repeated 3 times.
Data statistics and analysis
Microsoft Office Excel 2010 and SPSS24.0 were used for data processing and plotting， and the significance of differences was tested by the single factor analysis of variance （ANOVA） method.
Results and Analysis
Effects of alkaline stress on osmotic substances in Lanjingling seedlings
Effect of alkaline stress on proline content in Lanjingling seedlings With the increase of alkali concentration， the content of proline increased first and then decreased slightly with the increase of alkali concentration， and the peak appeared at the alkali concentration of 100 mmol/L. At each alkali treatment concentration， there was an extremely significant difference from the CK. It showed that in the alkaline solution treatment in this range， the content of proline increased with the increase of alkali concentration. Lanjingling was greatly affected by alkaline stress， and its proline content significantly increased than normal， but when the alkaline stress environment reached a certain level， it would adjust itself to resist the effects of adverse environments and adapt to the environment of alkaline stress.
Effect of alkaline stress on soluble sugar content in Lanjingling seedlings
With the increase of alkali concentration， the soluble sugar content of Lanjingling generally showed a trend of increasing first and then decreasing. It increased with the increase of the alkali concentration in the range of 50-100 mmol/L， and the peak was reached at the treatment concentration of 100 mmol/L. Among the various concentrations in this range， the treatment concentration of 100 mmol/L showed the highest soluble sugar content， which was significantly higher than that of the CK， while there were no significant differences between other treatment groups and the CK. It indicated that the synthesis of soluble sugar in Lanjingling seedlings was greatly affected by alkaline stress at 100 mmol/L， and the feedback of osmotic adjustment in the cells was strong.
Effect of alkaline stress on soluble protein content in Lanjingling seedlings
Under different alkali concentrations， the soluble protein content of Lanjingling seedlings was higher at each concentration than in the CK， showing a trend of first increasing and then decreasing， and the peak concentration was reached at the treatment concentration of 100 mmol/L. Among all treatment concentrations， only the 100 mmol/L treatment group increased significantly by 13.85% compared with the CK， and the remaining treatment concentrations were not significantly different from the CK， indicating that Lanjingling seedlings were more resistant to stress under 100 mmol/L alkaline stress.
Effects of alkaline stress on antioxidant enzymes in Lanjingling seedlings
Effect of alkaline stress on CAT activity in Lanjingling seedlings
The activity of CAT in Lanjinling seedlings increased first and then decreased under the treatment of alkaline solution， peaked at 100 mmol/L treatment concentration， and gradually decreased with the increase of alkali concentration after exceeding 100 mmol/L. It showed that the Lanjingling seedlings significantly improved its alkali tolerance by increasing the activity of CAT at 100 mmol/L， while under high concentration alkali stress， the plants cell activity would be affected due to persecution. Effect of alkaline stress on SOD activity in Lanjingling seedlings
With the increase of alkali concentration， the SOD activity in Lanjingling seedlings showed a trend of increasing first and then decreasing， and reached a peak at the treatment concentration of 100 mmol/L， and compared with the CK， the SOD activity of each group was higher than that of the CK. However， there were no significant differences in SOD activity between the treatment groups and the CK.
Effect of alkaline stress on POD activity in Lanjingling seedlings
With the increase of alkaline stress concentration， the POD activity of Lanjingling seedlings increased gradually before 100 mmol/L treatment concentration， and then gradually decreased after 100 mmol/L treatment concentration. The contents in various groups generally increased first and then decreased under alkaline stress overall， and were all higher than the CK. However， the POD activity at each treatment concentration was not significantly different from the CK.
Effect of alkaline stress on MDA activity in Lanjingling seedlings
The MDA content of Lanjingling seedlings increased before the treatment concentration of 100 mmol/L， and gradually decreased after the treatment concentration of 100 mmol/L. At 100 mmol/L treatment concentration， the content of MDA was significantly higher than that of the CK. The contents of malondialdehyde in the remaining treatment concentrations were not significantly different from that of the CK.
Songyan HE et al. Physiological Effects of Alkaline Stress on Seedlings of Lonicera caerulea L.
Effect of alkaline stress on chlorophyll of Lanjinling seedlings
The chlorophyll content of Lanjingling increased fluctuatingly and peaked at 50 mmol/L. The chlorophyll content generally showed an upward trend. The chlorophyll contents were higher than that of the CK. There were no significant differences between various treatments and the CK. It showed that the alkaline stress in this range had little effect on the photosynthesis of L. caerulea seedlings.
Conclusions and Discussion
During plant growth， plant photosynthesis and physiological metabolism are affected by salt-alkali stress， which can lead to metabolic disorders in the growth phase of the plant， resulting in plant wilting or death[8]. Soluble sugar is the energy source for the synthesis of the organic carbon framework by plants， and the level of sugar metabolism in plants can be reflected through the measurement of soluble sugar content. In this study， the soluble sugar content of Lanjinling seedlings increased first and then decreased. When the concentration of alkali increased， the water potential inside the cells is lower than that outside the cells， causing the cells to absorb water. As a result， the osmosis of plant cells is enhanced， and the plant sugar metabolism level also increases. The plants are more stressed under alkaline stress due to the influence of high pH， and the content of proline and organic acids and other osmotic substances increase[9-10]， which can effectively resist the adversity environment. Therefore， even if the Lanjinling seedlings maintain a low or higher level of sugar metabolism， they could still maintain the normal growth of the plant. This balance allows the Lanjinling seedlings to have a certain adaptability to the metabolic physiology of alkali stress. When plants are subjected to alkaline stress， a large amount of oxygen free radicals will be produced in the body， which will cause oxidative damage to membrane lipids. Meanwhile， alkaline stress will also induce plants to produce antioxidant enzymes to scavenge too much activated oxygen by increasing their own resistance oxidase activity， to thereby improve their alkali resistance[11]. The activity of SOD， POD and CAT in L. caerulea seedlings were higher than that in the CK， indicating that Lanjingling was affected by alkaline stress at this range of alkali concentration， but due to the enhanced activity of the Lanjingling plants in the body， their own alkali tolerance was improved. Therefore， Lanjingling had resistance to alkaline stress environment.
The contents of MDA in Lanjingling at different concentrations were higher than the CK mostly. With the increase of the alkali concentration， the content of MDA in the cells first increased and then decreased， indicating that the membrane structure and tissue of Lanjingling might be injured under the high concentration of alkaline stress， which affected its stress resistance. The chlorophyll content of Lanjingling did not change significantly under different treatment concentrations， which indicated that the photosynthesis of the Lanjingling plants in this range of alkaline environment was not affected.
References
[1] HUO JW. RAPD study on biological characteristics and germplasm resources of Lonicera L. subsect. Caeruleae[D]. Harbin： Northeast Agricultural University， 2004. （in Chinese）
[2] ZHANG RR. The harm and improvement of soil salinization[J]. Modern Agricultural Science and Technology， 2019（21）： 178-179. （in Chinese）
[3] GUO ZY. Analysis on the characteristics of soil salinization in Tianjin Binhai and surrounding areas[D]. Tianjin： Tianjin University， 2018. （in Chinese）
[4] ZOU Q. Plant physiology experiment guidance[M]. Beijing： China Agriculture Press， 2000. （in Chinese）
[5] BATES LS， WALDREN RP， TEARE ID. Rapid determination of free proline for water stress studies[J].Plant Soil， 1973（39）： 205-207.
[6] YOSHIDA S， FORNO DA， COCK JH， et al. Laboratory manual for physiological studies of rice[M] . Los Banos， Philippines： The International Rice Research Institute， 1973.
[7] LI HS. Principles and techniques of plant physiology and biochemistry experiments[M]. Beijing： Higher Education Press， 2000. （in Chinese）
[8] TAO J， CHEN SG， QIN CY， et al. Influences of salt alkali on MDA and protective enzyme activity of Popular varieties[J]. Journal of Northeast Forestry University， 2005（3）： 13-15， 37. （in Chinese）
[9] ZHANG XY， ZHANG R， JIA XM， et al. Physiological characteristics of two apple plantlets in response to saline alkali stress[J]. Gansu Agricultural Science and Technology， 2019（7）： 33-40. （in Chinese）
[10] LIU B， JIA XM， ZHU ZL， et al. Effect of Saline-alkali on Photosynthesis and Osmotic Regulation Substances of Malus halliana Koehne[J]. Acta Botanica Boreali-Occidentalia Sinica，2019，39（9）：1618-1626.（in Chinese）
[11] GAO ZW. Research on the salt-alkali resistance mechanism of alfalfa and oat[D]. Changchun： Northeast Normal University， 2011. （in Chinese）
[Methods] The L. caerulea seedling variety， Lanjingling， was used as the test material， and alkaline solution （NaHCO3） with different concentration gradients was used for stress treatment to observe physiological effects on L. caerulea seedlings.
[Results] L. caerulea seedlings were most affected by alkaline stress at a treatment concentration of 100 mmol/L， and their osmotic substances （proline， soluble sugar， soluble protein） and antioxidant enzymes （CAT， SOD and POD） were higher in content at a concentration of 100 mmol/L compared with the alkaline stresses in this range. The contents were generally higher， and even in the detection of proline， soluble sugar， SOD and POD， the contents of these substances and enzymes reached a peak at 100 mmol/L. The contents of soluble sugar， CAT， SOD， POD and malondialdehyde were generally higher than that of the control check （CK）， and the contents of proline and soluble protein in each treatment concentration were generally higher than that of the CK.
[Conclusions] The metabolic physiology of L. caerulea seedlings has a certain adaptability to alkaline stress.
Key words Lonicera caerulea L.; Alkaline stress; Physiological response; Stress resistance
Lonicera caerulea L.， also known as Shanqiezi， Yangnaizi and Heixiaziguo， is a rare and natural wild edible berry in the world. It can be eaten raw， can be brewed into wine and prepared into beverages and jams， and has a very high edible value. In addition， L. caerulea also has good medicinal value， as it has the functions of clearing away heat and toxic materials， resisting inflammation and viral diseases， preventing capillary rupture， lowering blood pressure， improving liver detoxification function， resisting tumors， relieving discomfort symptoms after radiotherapy， and slowing down the reduction of white blood cells after chemotherapy[1].
The salt-alkalization of soil is a major environmental problem which China faces. The soil contains alkaline salts HCO-3， CO-3， etc.， which will cause the soil pH to increase， affecting plant photosynthesis， biofilm permeability and antioxidant enzyme activity changes[2]. Due to the high concentration of salt ions in saline-alkali soil， it will slow down the absorption of water by the plants， destroy the plants growth function， make plants unable to grow normally， and even cause large-scale death[3]. Tianjin Binhai and its surrounding areas are one of the main occurrence areas of coastal saline-alkali soils. The L. caerulea variety， Lanjingling， is an excellent line bred with Beilei and Lanniao as its parents. It has good fruit taste， no bitter taste， higher nutritional value and medicinal value than wild varieties， and has broad development prospects. Studying the alkali-tolerant characteristics of L. caerulea can provide theoretical guidance for the cultivation and popularization of L. caerulea in Tianjin and North China.
Materials and Methods
Materials
The tree specie to be tested was the L. caerulea variety， Lanjingling， belonging to Lonicera Linn in Caprifoliaceae. The material was 2-year-old cutting seedlings introduced from Heilongjiang Province.
Experimental methods
Planting
This experiment was carried out in the Garden Plant Experiment Base of Tianjin Agricultural University. The cultivation substrate was peat∶vermiculite∶perlite=2∶1∶1. The size of the flowerpots was 22.5 cm in diameter and 21.5 cm in height. A total of 30 pots of seedlings were planted in the experiment， with 3 plants per pot. Eighteen pot plants with normal leaf color and normal growth status were selected for the experiment.
Alkaline stress treatment
Alkaline solution was poured on October 9， October 16， October 25， and November 1， 2019.Five concentration gradients （50， 100， 150， 200， 300 mmol/L） were set ， with no application of alkali as the control （CK）. Each treatment was repeated 3 times. In order to avoid the death of plants caused by excessive alkali addition at one time， in the high concentration alkali concentration group， the alkali solution concentration was increased by 50 mmol/L every 7 d gradually to the preset alkali concentration in the pot.
Physiological index measurement methods
After 20 d of alkaline stress， sampling was performed according to the principle of sampling at the same position at the same height. The physiological indexes such as proline， soluble sugar， soluble protein， CAT， SOD， POD， malondialdehyde and chlorophyll were measured[4-7]， respectively， and the measurement of each physiological index was repeated 3 times.
Data statistics and analysis
Microsoft Office Excel 2010 and SPSS24.0 were used for data processing and plotting， and the significance of differences was tested by the single factor analysis of variance （ANOVA） method.
Results and Analysis
Effects of alkaline stress on osmotic substances in Lanjingling seedlings
Effect of alkaline stress on proline content in Lanjingling seedlings With the increase of alkali concentration， the content of proline increased first and then decreased slightly with the increase of alkali concentration， and the peak appeared at the alkali concentration of 100 mmol/L. At each alkali treatment concentration， there was an extremely significant difference from the CK. It showed that in the alkaline solution treatment in this range， the content of proline increased with the increase of alkali concentration. Lanjingling was greatly affected by alkaline stress， and its proline content significantly increased than normal， but when the alkaline stress environment reached a certain level， it would adjust itself to resist the effects of adverse environments and adapt to the environment of alkaline stress.
Effect of alkaline stress on soluble sugar content in Lanjingling seedlings
With the increase of alkali concentration， the soluble sugar content of Lanjingling generally showed a trend of increasing first and then decreasing. It increased with the increase of the alkali concentration in the range of 50-100 mmol/L， and the peak was reached at the treatment concentration of 100 mmol/L. Among the various concentrations in this range， the treatment concentration of 100 mmol/L showed the highest soluble sugar content， which was significantly higher than that of the CK， while there were no significant differences between other treatment groups and the CK. It indicated that the synthesis of soluble sugar in Lanjingling seedlings was greatly affected by alkaline stress at 100 mmol/L， and the feedback of osmotic adjustment in the cells was strong.
Effect of alkaline stress on soluble protein content in Lanjingling seedlings
Under different alkali concentrations， the soluble protein content of Lanjingling seedlings was higher at each concentration than in the CK， showing a trend of first increasing and then decreasing， and the peak concentration was reached at the treatment concentration of 100 mmol/L. Among all treatment concentrations， only the 100 mmol/L treatment group increased significantly by 13.85% compared with the CK， and the remaining treatment concentrations were not significantly different from the CK， indicating that Lanjingling seedlings were more resistant to stress under 100 mmol/L alkaline stress.
Effects of alkaline stress on antioxidant enzymes in Lanjingling seedlings
Effect of alkaline stress on CAT activity in Lanjingling seedlings
The activity of CAT in Lanjinling seedlings increased first and then decreased under the treatment of alkaline solution， peaked at 100 mmol/L treatment concentration， and gradually decreased with the increase of alkali concentration after exceeding 100 mmol/L. It showed that the Lanjingling seedlings significantly improved its alkali tolerance by increasing the activity of CAT at 100 mmol/L， while under high concentration alkali stress， the plants cell activity would be affected due to persecution. Effect of alkaline stress on SOD activity in Lanjingling seedlings
With the increase of alkali concentration， the SOD activity in Lanjingling seedlings showed a trend of increasing first and then decreasing， and reached a peak at the treatment concentration of 100 mmol/L， and compared with the CK， the SOD activity of each group was higher than that of the CK. However， there were no significant differences in SOD activity between the treatment groups and the CK.
Effect of alkaline stress on POD activity in Lanjingling seedlings
With the increase of alkaline stress concentration， the POD activity of Lanjingling seedlings increased gradually before 100 mmol/L treatment concentration， and then gradually decreased after 100 mmol/L treatment concentration. The contents in various groups generally increased first and then decreased under alkaline stress overall， and were all higher than the CK. However， the POD activity at each treatment concentration was not significantly different from the CK.
Effect of alkaline stress on MDA activity in Lanjingling seedlings
The MDA content of Lanjingling seedlings increased before the treatment concentration of 100 mmol/L， and gradually decreased after the treatment concentration of 100 mmol/L. At 100 mmol/L treatment concentration， the content of MDA was significantly higher than that of the CK. The contents of malondialdehyde in the remaining treatment concentrations were not significantly different from that of the CK.
Songyan HE et al. Physiological Effects of Alkaline Stress on Seedlings of Lonicera caerulea L.
Effect of alkaline stress on chlorophyll of Lanjinling seedlings
The chlorophyll content of Lanjingling increased fluctuatingly and peaked at 50 mmol/L. The chlorophyll content generally showed an upward trend. The chlorophyll contents were higher than that of the CK. There were no significant differences between various treatments and the CK. It showed that the alkaline stress in this range had little effect on the photosynthesis of L. caerulea seedlings.
Conclusions and Discussion
During plant growth， plant photosynthesis and physiological metabolism are affected by salt-alkali stress， which can lead to metabolic disorders in the growth phase of the plant， resulting in plant wilting or death[8]. Soluble sugar is the energy source for the synthesis of the organic carbon framework by plants， and the level of sugar metabolism in plants can be reflected through the measurement of soluble sugar content. In this study， the soluble sugar content of Lanjinling seedlings increased first and then decreased. When the concentration of alkali increased， the water potential inside the cells is lower than that outside the cells， causing the cells to absorb water. As a result， the osmosis of plant cells is enhanced， and the plant sugar metabolism level also increases. The plants are more stressed under alkaline stress due to the influence of high pH， and the content of proline and organic acids and other osmotic substances increase[9-10]， which can effectively resist the adversity environment. Therefore， even if the Lanjinling seedlings maintain a low or higher level of sugar metabolism， they could still maintain the normal growth of the plant. This balance allows the Lanjinling seedlings to have a certain adaptability to the metabolic physiology of alkali stress. When plants are subjected to alkaline stress， a large amount of oxygen free radicals will be produced in the body， which will cause oxidative damage to membrane lipids. Meanwhile， alkaline stress will also induce plants to produce antioxidant enzymes to scavenge too much activated oxygen by increasing their own resistance oxidase activity， to thereby improve their alkali resistance[11]. The activity of SOD， POD and CAT in L. caerulea seedlings were higher than that in the CK， indicating that Lanjingling was affected by alkaline stress at this range of alkali concentration， but due to the enhanced activity of the Lanjingling plants in the body， their own alkali tolerance was improved. Therefore， Lanjingling had resistance to alkaline stress environment.
The contents of MDA in Lanjingling at different concentrations were higher than the CK mostly. With the increase of the alkali concentration， the content of MDA in the cells first increased and then decreased， indicating that the membrane structure and tissue of Lanjingling might be injured under the high concentration of alkaline stress， which affected its stress resistance. The chlorophyll content of Lanjingling did not change significantly under different treatment concentrations， which indicated that the photosynthesis of the Lanjingling plants in this range of alkaline environment was not affected.
References
[1] HUO JW. RAPD study on biological characteristics and germplasm resources of Lonicera L. subsect. Caeruleae[D]. Harbin： Northeast Agricultural University， 2004. （in Chinese）
[2] ZHANG RR. The harm and improvement of soil salinization[J]. Modern Agricultural Science and Technology， 2019（21）： 178-179. （in Chinese）
[3] GUO ZY. Analysis on the characteristics of soil salinization in Tianjin Binhai and surrounding areas[D]. Tianjin： Tianjin University， 2018. （in Chinese）
[4] ZOU Q. Plant physiology experiment guidance[M]. Beijing： China Agriculture Press， 2000. （in Chinese）
[5] BATES LS， WALDREN RP， TEARE ID. Rapid determination of free proline for water stress studies[J].Plant Soil， 1973（39）： 205-207.
[6] YOSHIDA S， FORNO DA， COCK JH， et al. Laboratory manual for physiological studies of rice[M] . Los Banos， Philippines： The International Rice Research Institute， 1973.
[7] LI HS. Principles and techniques of plant physiology and biochemistry experiments[M]. Beijing： Higher Education Press， 2000. （in Chinese）
[8] TAO J， CHEN SG， QIN CY， et al. Influences of salt alkali on MDA and protective enzyme activity of Popular varieties[J]. Journal of Northeast Forestry University， 2005（3）： 13-15， 37. （in Chinese）
[9] ZHANG XY， ZHANG R， JIA XM， et al. Physiological characteristics of two apple plantlets in response to saline alkali stress[J]. Gansu Agricultural Science and Technology， 2019（7）： 33-40. （in Chinese）
[10] LIU B， JIA XM， ZHU ZL， et al. Effect of Saline-alkali on Photosynthesis and Osmotic Regulation Substances of Malus halliana Koehne[J]. Acta Botanica Boreali-Occidentalia Sinica，2019，39（9）：1618-1626.（in Chinese）
[11] GAO ZW. Research on the salt-alkali resistance mechanism of alfalfa and oat[D]. Changchun： Northeast Normal University， 2011. （in Chinese）