Influence of abiotic factors on CO 2 -gas exchange of Pinus pallasiana , Juniperus excelsa and Arbutus andrachne

. The complex interactions among environmental factors as incident light, temperature and soil water content create the need for used physiology-based models which describe plants performance under current and changing climatic conditions. In the present work the net photosynthetic rate of Pinus pallasiana D. Don, Juniperus excelsa M.Bieb. and Arbutus andrachne L. was modeled as a function of light irradiance using the modified rectangular hyperbola model, which is capable of describing the photoinhibition by the non-rectangular hyperbola function. A comparative assessment of the adaptive response of the photosynthetic apparatus plants on the effect of abiotic factors and their strategies in maintaining an optimal water balance in accordance with environmental conditions has been performed. The parameters of light curves of photosynthesis under conditions of full sunlight, moderate shading and drought are determined. In relation to light, Pinus pallasiana is characterized by wider ecological amplitude compared to Juniperus excelsa and Arbutus andrachne . Inefficient use of low-intensity of photosynthetically active radiation by immature plants Pinus pallasiana and Juniperus excelsa indicates poor shade tolerance and inability to resume in shade-type forests. Due to the low plasticity to changes in the light regime, Arbutus andrachne L. may experience a significant lack of light in strong shading. Arbutus andrachne has the highest ability to actively rearrange water regime in accordance with its external moisture supply, which causes the highest drought resistance, and Juniperus excelsa has a slightly lower capacity. Tolerance to hydrothermal stress in Pinus pallasiana is significantly lower than in Arbutus andrachne and Juniperus excelsa .


Introduction
Plants in the ecosystems of the southern Russia in areas of insufficient moisture in summer are often subjected to the stress of severe and prolonged drought [1]. Such conditions are excelsa is characterized as a light-loving and very drought-tolerant species, but its immature individuals are sensitive to moisture, light, and temperature conditions [24].
Much attention in the scientific literature when studying Pinus pallasiana, Juniperus excelsa and Arbutus andrachne is given to those factors that limit such processes as seed production [19][20] or germination [18], age dynamics [16]. However, their physiological response to environmental influences and climate factors limiting photosynthesis remains the least studied to the present day, and only a few papers have been devoted to it [25][26].
These studies are a necessary basis for obtaining an objective assessment of the stability of stands in warmer and drier environmental conditions predicted by global warming scenarios for the Crimea [9][10], and developing an environmentally sound system for protecting and maintaining bioecological potential.
The objective of this study was to compare the features of the adaptive response of the photosynthetic apparatus of Pinus pallasiana, Juniperus excelsa, and Arbutus andrachne to the effects of abiotic stressors: drought, high temperatures, and changes in the light regime.

Materials and methods
The study was carried out in the Nikitsky Botanical Gardens -National Scientific Center on the Southern coast of the Crimea (44°31′ N, 34°15′ E). The climate of the Southern Coast of Crimea (SCC) is subtropical Mediterranean type, characterized by hot, dry summers and mild wet winters. The average annual air temperature is 12.6°C and the mean annual rainfall amounts to 592 mm [17].
Studies were conducted during several periods of active vegetation in 2017, 2018 (April-October) and 2020 (June-July) on seedlings (8-12 years old) of Pinus pallasiana, Juniperus excelsa and Arbutus andrachne growing on the experimental site and in the conditions of the growing experience (in 15-liter vessels filled with the soil of the experimental site).
The soil of the experimental site is dark brown, medium-loamy, medium-gravelly on a gravelly-stony limestone eluvium. The lowest moisture content (LMC) ranges from 21.6 to 25.2%, the wilting humidity -9.9-11.1%.
The intensity of CO 2 -gas exchange of leaves was determined with 3-fold repetition on well-developed intact leaves of the upper part of the shoot with an interval of 15-20 minutes. An automatic 4-channel open-type system for monitoring CO 2 gas exchange and leaf transpiration " РТМ-48A photosynthesis monitor" (Bioinstruments S.R.L., Moldova) was used for measurements [27]. The measurements were carried out at a natural CO 2 concentration in the air of about 0.04 %. The leaf chamber was oriented so that its elements did not obscure the leaf. Temperature (°C) and humidity (%), photosynthetically active radiation (PAR) were recorded using sensors of the RTH-48 Weather module, soil moisture (%) and leaf temperature (°C) were measured by sensors of the РТМ-48A system and the PM-11z phytomonitor (Bioinstruments S.R.L., Moldova) [14].
The values of net photosynthetic rate (P N , μmol СО 2 m -2 s -1 ), total respiration rate (R total , μmol СО 2 m -2 s -1 ), dark respiration rate (R D , μmol СО 2 m -2 s -1 ) and photorespiration rate (R PR , μmol СО 2 m -2 s -1 ) were used to characterize the CO₂-gas exchange of the leaf in the PAR range from 0 to 2000 μmol photons m -2 s -1 . When studying the dependence of P N on light intensity, experimental measurements were made on sunny, mostly clear days. The studied plants grew in favorable hydrothermal conditions: the daytime air temperature varied within 18-31°C, relative humidity -45-70%, soil moisture -60-100% Field Capacity (FC). The maximum measured value of the PAR in full light conditions varied in the range from 1350 to 1900 μmol photons m -2 s -1 , and in moderate shading conditions in the greenhouse it varied in the range of 400-800 μmol photons m -2 s -1 . In conditions of moderate drought, soil moisture was from 35 to 55 % FC.
A modified model of a rectangular hyperbola describing photoinhibition of the process by a non-rectangular hyperbola (1) was chosen for a comparative assessment of the physiological differences of plants in relation to the light factor [28]: (1) where P N is the net photosynthetic rate, μmol СО 2 m -2 s -1 ; I is photosynthetically active radiation (PAR), μmol photons m -2 s -1 ; I comp is the light compensation point -the light intensity at which the total CO 2 -gas exchange (P N /I (x,t) ) is zero, μmol photons m -2 s -1 ; φ (Io-Icomp) -the quantum yield of photosynthesis (the tangent of the angle of inclination of the light curve was calculated as a derivative of P N at point I) I = I o -I comp , μmolCO 2 μmol photons -1 ; β and γ are correction coefficients that do not depend on the intensity of solar radiation [28], m² s μmol photons -1 , and β is a correction coefficient for the tendency to decrease P N when the PAR exceed the light saturation point due to photoinhibition and is similar to the convexity parameter [28].
The maximum gross photosynthetic rate (P gmax ), dark respiration rate (R D ), light saturation point (I sat ) and a number of other additional parameters were calculated from equation (1) [29].
When studying the influence of abiotic factors on the processes of leaf gas exchange, plants were exposed to environmental conditions that mimic the hot summer of the Southern Coast of the Crimea and conditions of water scarcity. Soil moisture in the vessels with control plants was maintained at a level corresponding to the moisture content of 60-90% FC.
For a comparative assessment of photosynthetic activity of plants under optimum conditions and under the influence of abiotic stressors we calculated the rate of use of PAR during photosynthesis -the number μmol СО 2 mmol -1 photons (Kr) and the economic ratio of photosynthetic ability, or the coefficient of photosynthetic efficiency (Keff), which is the ratio of gross photosynthetic rate to dark respiration rate (Keff ≈ (P N + R D )/R D ) and describes the maximum possible efficiency of gas exchange [30].
The resulting data array was analyzed using information and mathematical criteria in the MS Excel2010 and Statistica10 software packages. The least squares and robust locally weighted regression methods were used to model and smooth the data (Statistica10). All calculations were performed at a given significance level p < 0.05.

Results and discussion
Photosynthetic light response curves describe the dependence of the net photosynthetic rate on the intensity of the PAR. They reflect the photosynthetic phenotype of plants: they provide information about the maximum photosynthetic capacity, quantum yield, light compensation point, and the efficiency of using solar radiation by leaves. Analysis of the photosynthetic light response curves provides an important potential ecological and physiological characteristic of this species, which allows us to obtain valuable information about the physiological differences of plants in relation to the light factor, their adaptation to the light environment. Table 1 shows the average values of the measured maximum values of PAR, photosynthesis and leaf respiration of the studied plant species, obtained during the registration of the light curve.
Juniperus excelsa was characterized by the highest photosynthetic ability under comfortable external growing conditions (favorable temperature, absence of water deficit and shading). The intensity of net photosynthetic rate in this species is on average 11-15% higher than the needles of Pinus pallasiana and Arbutus andrachne leaves (table 1). The light saturation curve of photosynthesis reached a plateau in Juniperus excelsa at a PAR of more than 1000 μmol photons m -2 s -1 (more than 50-60% of total light intensity). Light saturation of photosynthesis in Pinus pallasiana and Arbutus andrachne was observed at 850-900 μmol photons m -2 s -1 . Dark respiration rate in full light conditions in Pinus pallasiana and Juniperus excelsa leaves was almost twice higher than in Arbutus andrachne, indicating their need for more light to compensate for CO 2 .
Under moderate shading conditions, net photosynthetic rates decreased on average by 24% in Pinus pallasiana, 19% in Juniperus excelsa, 16% in Arbutus andrachne, and dark respiration rates by 6-11%, which can be regarded as a direct reaction to a decrease in assimilate formation due to a reduction in the arrival of PAR.
With a moderate water deficit, the decrease in net photosynthetic rate did not exceed 3-5%, and the increase in respiration intensity associated with an increase in energy costs for the synthesis of osmotic that protect proteins from dehydration was 11-13% in Pinus pallasiana and Arbutus andrachne and 22% in Juniperus excelsa, which is one of the ways plants adapt to drought.
The use of PAR during photosynthesis in the formed leaves of all the studied species has significantly increased with the deterioration of lighting conditions. The Kr values for moderate shading increased by 89% in Juniperus excelsa, 64% in Pinus pallasiana, and 55% in Arbutus andrachne. Compared to optimal conditions, there were different trends in the use of PAR and when exposed to moderate droughts. Kr values for Pinus pallasiana decreased by 15%, for Juniperus excelsa, on the contrary, increased by 21%, and for Arbutus andrachne they remained unchanged. Keff -photosynthetic efficiency coefficient, dimensionless; ±standard deviation; afull sunlight, bmoderate shading, ccombined treatment of full sunlight and moderate drought.
The variation in the values of the photosynthesis efficiency coefficients (Keff), which characterize the maximum possible efficiency of gas exchange and reflect the physiological state of this species in these habitat conditions, also significantly differed both in size and dynamics. The Keff indicator is species-specific and very sensitive to environmental factors [30]. Analysis of the calculations showed that the highest photosynthesis efficiency from the analyzed plant species is characterized by Arbutus andrachne, which has this value almost twice higher than that of Pinus pallasiana and Juniperus excelsa. In comparison with full sunlight, with moderate shading, Крe decreased in all species by 11-15%. Under moderate drought conditions, photosynthesis efficiency decreased by 20% in Pinus pallasiana, 14% in Juniperus excelsa, and 7% in Arbutus andrachne. Differences in Kr and Keff indicators under different lighting conditions and moderate droughts were statistically significant with a 95% probability.
The analysis of the light dependences of the gas exchange of the leaves of the studied plants confirmed the conclusions made on the basis of the measurements. Juniperus excelsa had the highest rates of photochemical reactions. Its values were slightly lower in Pinus pallasiana and Arbutus andrachne. The response to the effects of moderate shading and drought in all species was manifested in a steady trend of decreasing the intensity of gas exchange (table 2, figure 1). The values of the obtained determination coefficients indicate that the variation of net photosynthetic rate by 98-99% in the P N /I dependence model is explained by a change in the intensity of the PAR (table 2). The parameter of the substrate light constant I K equal to the value of the light intensity at the intersection of the maximum photosynthetic rate with the line of the initial slope of the light curve (I K = P gmax /φ (I 0 )), is one of the oldest and most used in the description of light dependencies. The value of the I K parameter can be used to assess the adaptive properties of the species, since it characterizes light conditions when photosynthesis is limited by dark reactions, under which protective mechanisms begin to act. The adaptation of photophysical and photochemical stages to changes in the light regime determines the nature of dark reactions of photosynthesis. A low I K value often indicates inefficient use of high PAR, rather than efficient use of low ones, and vice versa [31]. Studies have shown that under favorable growing conditions with saturating light intensity, the average net photosynthetic rate in light leaves of Pinus pallasiana was 12.3 μmol CO 2 m -2 s -1 , in Juniperus excels -14.7 μmol CO 2 m -2 s -1 and in Arbutus andrachne -13.8 μmol CO 2 m -2 s -1 (table 3). Under the influence of moderate drought, the intensity of photosynthetic CO 2 -gas exchange in Pinus pallasiana decreased by 13%, while the functional activity of the photosynthetic apparatus in Arbutus andrachne did not change, which is obviously due to the degree of stomatal openness. 10.4±4.9 20.9±1.1 15.2±1.6 PARphotosynthetically active radiation; P N , P g , R PR , R D and R total (R total = R PR + R D )mean of net photosynthetic rate, gross photosynthetic rate, photo respiration rate, dark respiration rate and total respiration rate; ±standard deviation; afull sunlight, bcombined treatment of full sunlight and moderate drought.
Total light respiration includes photorespiration, Moeler reactions, and dark or mitochondrial respiration [32]. The data obtained by us suggest that all the plants presented in the study have a low intensity of photorespiration and a low intensity of dark respiration. The highest intensity of total respiration was observed in Juniperus excelsa with the highest level of light saturation of photosynthesis. An average level of respiration intensity was observed in Arbutus andrachne.
The ratio of respiration and photosynthesis (R total /Pg) is an integral indicator of the energy balance of the whole plant, the consistency of the main physiological processes, and characterizes the share of respiratory costs from the total number of photoassimilates during plant functioning [33]. From the results obtained, we can conclude that Juniperus excelsa and Arbutus andrachne spend a fairly high amount of photoassimilates on respiration. Evaluation of the efficiency of carbon use for plant growth processes showed that the share of fixed carbon, which is realized in net productivity, was 92% in Pinus pallasiana, 81% in Juniperus excelsa, and 86% in Arbutus andrachne. The obtained ratios of photosynthesis and respiration processes reflect the regularities of quantitative organization of the whole plant under optimal conditions [33][34]. Under conditions of moderate water deficit, the ratio of respiration and photosynthesis in the studied plant species increased by 1-2%. This ratio under stress, as a rule, increases due to an increase in total respiration by the value of Ra (adaptive component of respiration), and this value is more significant in less resistant to this particular stress plant species. The new R total /Pg ratio has the lowest possible value under these conditions [34].
The study of the temperature dependence of photosynthesis in the absence of water stress in conditions of sufficient light intensity showed a variation of the temperature optima P N in the considered species within a fairly wide range. The analysis of experimental data revealed that the rate of assimilation of carbon dioxide per unit leaf surface of Juniperus excelsa accelerated with increasing leaf temperature up to 28-32°C, Arbutus andrachne -up to 37-39°C. Under optimal conditions of soil moisture with nonlimiting light, the maximum net photosynthetic rate in Pinus pallasiana was observed at a leaf temperature of 16-22°C ( fig. 2). When the leaf temperature increased above the threshold values, there was a violation of the assimilation balance, increased respiration, and as a result, a decrease in photosynthesis. Under the influence of moderate drought, the temperature optimum zones changed insignificantly (within 2-3°C). However, under conditions of severe hydrothermal stress with soil moisture below 25% of FC, photosynthesis inhibition in the studied plant species was observed at temperatures of 8-10°C below the threshold.
In conditions of insufficient water supply, the ability of plants to regulate the water regime of aboveground parts plays a primary role. A promising approach to study the mechanisms that ensure plant adaptation to water scarcity is to compare species that differ in drought tolerance.
Determination of the ecological and physiological characteristics of net photosynthetic rate in a wide range of changes in soil moisture allowed us to determine the dependence of the intensity of gas exchange of the studied plant species on soil moisture ( fig. 3).
The zone of optimal soil moisture for Pinus pallasiana is in the range of 60-80% FC. When the soil moisture was below or above the specified limits, the intensity of photosynthesis decreased. The effect of soil drought on the activity of CO 2 assimilation was already evident in Pinus pallasiana when the soil moisture dropped to 55% of the FC. Below 30% FC, there was a negative carbon balance between the level of CO₂ assimilation during photosynthesis and its loss during respiration.
For young Juniperus excelsa plants, the optimal soil moisture was 65-100% FC. When humidification was less than 60% of FC, a gradual decrease in CO₂-gas exchange began. Periodic complete inhibition of photosynthesis due to stomatal closure occurred at soil moisture below 20% FC.
Arbutus andrachne was distinguished by its high tolerance to drought and ability to function effectively in a wide range of soil moisture (within 35-100% FC). A negative carbon balance under sufficient soil water content for Pinus pallasiana plants occurs when the needles overheat above 42-43°C. For Juniperus excelsa and Arbutus andrachne plants it was observed periodically at temperatures above 46-47°C. However, under the complex effect of hydrothermal stress as a result of leaf dehydration and overheating, periodic complete inhibition of photosynthesis in the studied plant species occurs already at temperatures 8-10°C below the threshold. With increasing soil drought, a critical loss of soil water content, leading to an almost complete cessation of photosynthesis due to stomata closure, occurs in Pinus pallasiana plants when relative water content reduce below 30% of FC, in Juniperus excelsabelow 20% of FC, in Arbutus andrachnebelow 10-15% of FC. Carbon assimilation by young plants of Pinus pallasiana onset only when PAR above 15-24 μmol photons m -² s -¹, Juniperus excelsaabove 17-24 μmol photons m -² s -¹ and Arbutus andrachneabove 8-10 μmol photons m -² s -¹.

Conclusion
As a result of the research, a comprehensive assessment of the features of regulation of photosynthetic activity of young plants Pinus pallasiana, Juniperus excelsa and Arbutus andrachne under various lighting, temperature and humidity conditions was carried out. The cardinal points of their light curves are determined and quantitative values of optimal and threshold values of environmental factors that limit gas exchange processes are obtained.
It was found that in the absence of stress effects of moisture deficiency and high temperatures, Juniperus excelsa has the highest intensity of CO 2 -gas exchange, and Arbutus andrachne has the highest efficiency. In relation to light, Pinus pallasiana is characterized by wider ecological amplitude compared to Juniperus excelsa and Arbutus andrachne.
Based on the results of the study of CO 2 -gas exchange, we can conclude that Pinus pallasiana and Juniperus excelsa are heliophytes with their characteristic efficient use of high-intensity of PAR. Variations in the values of the light compensation point in different lighting conditions indicate a better adaptation to shading of Pinus pallasiana than Juniperus excelsa. High values of the light compensation point indicate that the immature plants Pinus pallasiana and Juniperus excelsa do not effectively use low PAR intensities, weak shade tolerance, and inability to renew in shade-type forests.
Features of CO₂ assimilation during photosynthesis allow us to conclude that Arbutus andrachne is a light-loving plant with a weakly expressed plasticity to changes in the light regime and may experience a significant lack of light under strong shading. However, the low level of dark respiration and low values of the light compensation point indicate the ability to absorb CO₂ in low light.
Under optimal conditions, the share of fixed carbon that is realized in net productivity is 92% for Pinus pallasiana, 81% for Juniperus excelsa, and 86% for Arbutus andrachne. During the period of increasing soil moisture deficit, there is a natural decrease in the intensity of leaf gas exchange. Comparison of the results obtained between the species showed a significant variability in their sensitivity to temperature and water stress. Arbutus andrachne has the highest ability to actively rearrange the water regime in accordance with its external moisture supply, while Juniperus excelsa has a slightly lower capacity. Tolerance to hydrothermal stress in Pinus pallasiana is significantly lower than in Arbutus andrachne and Juniperus excelsa.