Cycas Shiwandashanica, also known as Qingcuitou and Laoshutie in Chinese, is a wild plant species with extremely small populations in Guangxi, which is a very important plant germplasm resource of Cycas family in China. It is only distributed under the seasonal rain forest in low mountains and hills on the south slope of Shiwandashan, with an altitude of 100~750 m and the soil being latosolic red soil and mountain red soil developed from sand shale, conglomerate and granite, The number of existing Cycas Shiwandashanica is less than 500, so the protection research is urgently needed.

Since Cycas Shiwandashanica was discovered and named in 1994 (Zhang and Zhong, 1997), scholars at home and abroad have done very little research on it, only in morphological description and taxonomic research (Nong et al., 2011). The resources of Cycas Shiwandashanica are very scarce. At present, the research on Cycas Shiwandashanica is still at a very simple level, and is lack of basic research. The growth characteristics of seedlings, especially the photosynthetic characteristics, are in a blank state, which makes it impossible to introduce and cultivate Cycas Shiwandashanica on a large scale according to its habitat characteristics. The growth indexes of seedlings, including plant height, ground diameter and crown width, were observed to comprehensively reflect the growth and adaptability of seedlings in a specific environment. Photosynthesis is a process in which plants use light energy to produce organic matter and release oxygen in nature, which is one of the most important physiological processes in the process of plant growth, and is also the basis of plant growth and the decisive factor of productivity (Luo et al., 2020; Wang et al., 2013). The increase of plant yield potential will largely depend on the impact of photosynthesis (Molero and Reynolds, 2020). For example, high temperature, drought, low altitude and low light will affect plant photosynthesis (Maihemuti et al., 2020). At the same time, plant photosynthesis is also affected by its physiological factors. Changes in environmental factors such as leaf temperature, air humidity and photosynthetic active radiation can cause changes in physiological factors such as transpiration rate, net photosynthetic rate, intercellular carbon dioxide concentration and stomatal conductance, thus affecting plant photosynthesis, for example, the net photosynthetic rate of Acer saccharum in Canada is affected by photosynthetic active radiation and stomatal conductance, resulting in changes in photosynthesis (Li et al., 2013).

By studying the correlation between photosynthetic characteristics and growth characteristics of Cycas Shiwandashanica, this study revealed the interaction and adaptation mechanism between Cycas Shiwandashanica growth and the environmental system, so as to provide a photosynthetic theoretical basis for more scientific and effective breeding and protection of Cycas Shiwandashanica.

1 Results and Analysis

1.1 The growth index of Cycas Shiwandashanica seedlings showed that the biomass accumulated rapidly

Biomass is a direct indicator to evaluate plant growth and productivity. The determination of morphological indicators can reflect the ability of plants to adapt to the environment, which has important reference significance for the protection of plant species with extremely small populations and endangered plants. The results showed that (Table 1) the biomass of biennial seedlings of Cycas Shiwandashanica was significantly greater than that of annual seedlings. The seedling height, diameter and crown of biennial seedlings was 30.20%, 56.83% and 144.93% higher than that of annual seedlings respectively, indicating that the biomass accumulation and growth speed of Cycas Shiwandashanica seedlings were fast (Table 1).

1.2 Analysis of diurnal variation characteristics of photosynthetic physiology of seedlings

Under natural conditions, Cycas Shiwandashanica grows under the broad-leaved forest in low-altitude valleys, which is mainly affected by scattered light, and the light intensity often does not reach the light saturation point level. It is found that the diurnal variation of net photosynthetic rate P_n of Cycas Shiwandashanica presents a bimodal curve (Figure 1A). Combined with the diurnal variation trend of T_r and G_s, it was found that there was no photosynthetic “lunch break” phenomenon of Cycas Shiwandashanica. The two peaks of P_n appeared at 10:30 a.m. and 14:30 p.m., which were 1.73 μmol·m^-2·s^-1 and 1.76 μmol·m^-2·s^-1 respectively; From 8:30 a.m. to 10:30 a.m., the P_n increased with the increase of light intensity. From 10:30 to 12:30, the P_n decreased due to strong light inhibition. After 12:30, P_n began to rise due to the release of strong light inhibition. At 14:30, P_n reached the peak again. At this time, P_n was slightly higher than the peak at 10:30 a.m. After 14:30, the photosynthetic active radiation in the environment began to decrease, P_n began to decrease.

The diurnal variation of stomatal conductance G_s of Cycas Shiwandashanica showed a single peak trend (Figure 1B), and when P_n appeared the minimum value of 0.47 μmol·m^-2·s^-1, G_s did not decrease and was still open, indicating that the photosynthetic inhibition was caused by non-stomatal factors, mainly due to the excess light energy absorbed by the photosynthetic structure of leaves under strong light system, resulting in the decrease of photosynthetic function and the decrease of P_n. At 14:30, with the peak value of P_n of Cycas Shiwandashanica, G_s also reached the maximum value. With the gradual decrease of the degree of stomatal opening, its P_n also gradually decreased, and the lowest value was -0.62 μmol·m^-2·s^-1 at 18:30, indicating that P_n of Cycas Shiwandashanica was restricted by leaf structure and stomatal conductance.

The change trend of transpiration rate T_r of Cycas Shiwandashanica was basically consistent with the daily change of stomatal conductance (Figure 1C), showing a single peak change trend. In the morning, with the advance of time and the continuous rise of atmospheric temperature, the pressure deficit of Cycas Shiwandashanica leaves and atmospheric water vapor was increasing; The transpiration rate continued to rise, resulting in the continuous decline of leaf water potential and leaf water use efficiency, and finally resulting in the weakening of leaf photosynthesis. At 12:30 a.m., the transpiration rate of leaves was 1.05 mmol·m^-2·s^-1, which basically reached the peak. T_r increased, water consumption accelerated, and leaf water use efficiency decreased, resulting in the weakening of leaf photosynthesis; At about 14:30, T_r reached the peak and then began to decrease. The G_s and T_r values at 18:30 were lower than those at 8:30 a.m., mainly because the light intensity at 18:30 was weaker than that at 8:30 a.m., and the photosynthetic capacity of leaves was weakened, resulting in the decline of G_s and T_r.

The change trend of intercellular carbon dioxide concentration C_i of Cycas Shiwandashanica was opposite to the net photosynthetic rate. In the process of diurnal change, C_i increased first and then decreased (Figure 1D). The concentration of CO₂ entering the cells increased at 10:30 a.m., but its net photosynthetic rate did not increase. Again, it showed that the main factor for the decrease of net photosynthetic rate in the morning is non-stomatal factors. In the afternoon, the strong light, high temperature and relative humidity of the air decreased. In order to avoid excessive evaporation of water, the stomata contracted, and the decrease of transpiration rate caused the temperature of the internal structure of the leaves to rise, resulting in the decrease of the activity of ribulose diphosphate carboxylase in the dark reaction of photosynthesis, the decrease of affinity for CO₂ and the decrease of net photosynthetic efficiency.

1.3 Correlation analysis between environmental factors and diurnal variation index of photosynthesis

The results of correlation analysis between net photosynthetic rate P_n, stomatal conductance G_s, intercellular carbon dioxide concentration C_i and transpiration rate T_r and environmental factors (atmospheric temperature T_a, air relative humidity RH, photosynthetic active radiation PAR, atmospheric carbon dioxide concentration C_a) showed that (Table 2): P_n of Cycas Shiwandashanica was very significantly correlated with photosynthetic indexes G_s, C_i and T_r, but not correlated with environmental factors; G_s was very significantly correlated with photosynthetic indexes P_n, C_i and T_r, and significantly correlated with environmental factor PAR; C_i was very significantly correlated with photosynthetic indexes P_n and T_r, and significantly correlated with environmental factors PAR and C_a; T_r was very significantly correlated with other photosynthetic indexes and environmental factors. It can be seen that P_n has no significant correlation with environmental factors such as T_a, RH, PAR and C_a, but has a very significant correlation with photosynthetic indexes G_s, C_i and Tr, while photosynthetic indexes G_s, C_i, T_r have a significant or very significant correlation with different environmental factors. It can be seen that the impact of environmental factors on the photosynthesis of Cycas Shiwandashanica was mainly through affecting the photosynthetic rate by affecting its stomatal conductance, intercellular carbon dioxide concentration and transpiration rate.

1.4 Photoresponse curve (P_n-PAR)

The relationship between plant photosynthetic rate and light intensity can be objectively presented by measuring its light response curve (Du et al., 2018). The light response curve of Cycas Shiwandashanica leaves was measured under stable temperature and CO₂ concentration. The light response curve of Cycas Shiwandashanica was fitted through the right angle hyperbola correction model, and the degree of fitting was good (Figure 2, R²=0.99). When photosynthetic active radiation was in the range of 0~200 μmol·m^-2·s^-1, the net photosynthetic rate (P_n) increased linearly with the increase of photosynthetic active radiation (PAR); When the photosynthetic active radiation was 200~600 μmol·m^-2·s^-1, the growth rate of net photosynthetic rate slowed down, and when the photosynthetic active radiation was greater than 600 μmol·m^-2·s^-1, the net photosynthetic rate increased slowly and finally tended to reach the light saturation point. According to the hyperbolic fitting model, the relevant physiological parameters of Cycas Shiwandashanica such asP_max, LSP, LCP, R_d and apparent quantum efficiency AQY were obtained. The maximum photosynthetic rate (P_nmax) of Cycas Shiwandashanica was only 3.56 μmol·m^-2·s^-1, the maximum net photosynthetic rate was at a low value, indicating that the ability of Cycas Shiwandashanica to use light energy to produce organic matter was weak as a whole. Light saturation point and light compensation point represent the utilization ability of plants to strong light and weak light respectively. The light saturation point of Cycas Shiwandashanica was 1023.67 μmol·m^-2·s^-1 and light compensation point (LCP) were 3.68 μmol·m^-2·s^-1, with low light compensation point and high light saturation point, belonging to mesophyte.

AQY is the apparent quantum efficiency, which reflects the transformation and utilization ability of plants to light energy in the weak light stage. The AQY of Cycas Shiwandashanica was only 0.02, which was lower than the apparent quantum efficiency of most plants (Teng et al., 2019), and its utilization ability of weak light was poor. R_d is the dark respiration rate, and R_d of Cycas Shiwandashanica was 0.18 μmol·m^-2·s^-1, the R_d was low, indicating that the dark respiration rate of Cycas Shiwandashanica seedlings was small and the metabolic efficiency of organic matter was high, which corresponds to the need to continuously accumulate organic matter to promote growth in the seedling stage. In conclusion, the maximum net photosynthetic rate and light compensation point of Cycas Shiwandashanica were low, but its light saturation point was high. It can be seen that it has poor ability to use weak light and can use some strong light. Therefore, it is necessary to choose a habitat with certain light intensity to facilitate its growth.

2 Discussion

Photosynthesis is the basis of organic matter accumulation during plant growth, which is very important for plant growth and stress resistance (Robert et al., 2004). The plant height, diameter, crown and other biomass of the same plant in different habitats will be affected to varying degrees (Ma et al., 2012). After the introduction and cultivation of Cycas Shiwandashanica in this study, it was found that the seedling height, diameter and crown of Cycas Shiwandashanica biennial seedlings were significantly higher than those of annual seedlings, indicating that the growth environment of Cycas Shiwandashanica seedlings was suitable and the growth of Cycas Shiwandashanica seedlings was in good condition, and the hydrothermal environment of the introduction and cultivation site can meet the growth needs of Cycas Shiwandashanica.

Under natural conditions, the diurnal variation curve of photosynthesis reflected the physiological and ecological rhythm of various parameters and the different adaptive characteristics of plants to environmental changes. The diurnal variation of net photosynthetic rate (P_n) of Cycas Shiwandashanica was a bimodal curve, but when the net photosynthetic rate decreased, the stomatal conductance and intercellular carbon dioxide concentration did not decrease, indicating that it had no obvious photosynthetic “lunch break phenomenon”. The first peak of net photosynthetic rate was due to the high intensity of solar radiation in the afternoon. The decrease of net photosynthetic rate caused by photoinhibition of Cycas Shiwandashanica seedlings. The second peak was due to the closure of stomata and the decrease of stomatal conductance after 14:30 pm.

The simple correlation between physiological indexes such as net photosynthetic rate and environmental factors such as ambient temperature showed that in the diurnal variation process of Cycas Shiwandashanica, stomatal conductance, intercellular carbon dioxide concentration and transpiration rate directly affected its net photosynthetic rate, and intercellular carbon dioxide concentration inhibited the net photosynthetic rate. Although environmental factors in the process of diurnal variation did not directly affect the net photosynthetic rate of plants, they affect the photosynthetic capacity of plants by affecting intercellular carbon dioxide concentration, stomatal conductance and transpiration rate, so as to increase or decrease the net photosynthetic rate of Cycas Shiwandashanica. The research results were similar to those of Acer saccharum in Canada (Li et al., 2013).

Light intensity is the main environmental factor affecting plant growth and survival. The ability of photosynthesis in leaves with different light intensity is related to CO₂ intensity and biochemical characteristics of plants. At the same time, the change of light intensity is of great significance to the formation of flowering morphology of plants (Chai et al., 2013; Bos et al., 2000). The light saturation point and light compensation point of plant leaves reflect the requirements of plants for light intensity (Luo et al., 2019, Seed, 38(9): 52-56). The light saturation point and light compensation point of Cycas Shiwandashanica showed that the optimum light intensity range was 3.68~1 023.67 μmol·m^-2·s^-1, with obvious mesophyte characteristics. In the results of photoresponse curve, the maximum net photosynthetic rate of Cycas Shiwandashanica was 3.56 μmol·m^-2·s^-1, the ability of photosynthesis was weak, mainly because the light in its environment was scattered light, which can not meet its demand for light intensity for a long time, which was consistent with the original habitat conditions of Cycas Shiwandashanica. This study found that Cycas Shiwandashanica was only distributed under the arbor forest on the south slope of Shiwandashan and the slopes on both sides of the valley. The canopy density of the upper forest is very high and the light intensity of the whole day is very low. Therefore, selecting roadsides with certain vegetation coverage or forests with certain direct sunlight is a link that must be paid attention to in the process of introduction, cultivation and ex-situ protection of Cycas Shiwandashanica.

3 Materials and Methods

3.1 Materials

The test seeds were collected from Jinhuacha Nature Reserve, Nasuo Town, Fangcheng District, Fangcheng Port, Guangxi (108°12′33″E, 21°46′16″N) in October 2017. The collected seeds of Cycas Shiwandashanica were peeled off, washed and dried, and placed under the ginkgo forest with a shading of 75% for sand storage; The photosynthetic materials were the leaves of annual seedlings of Cycas Shiwandashanica; The seeds of Cycas Shiwandashanica were identified by researcher Xiong Zhongchen from Guangxi Institute of Botany.

3.2 Overview of test site

The experimental site was located in the Endangered Plant Protection Park of Guangxi Institute of Botany, Yanshan District, Guilin City (110°18ʹ01ʺE, 25°05ʹ23ʺN). Guilin is a mid subtropical monsoon climate zone, with an average annual temperature of 19.2℃. The monthly average temperature is higher than 20℃ for 6~7 months, the annual relative humidity is 78.0%, and it has an obvious dry and wet season.

3.3 Measurement of growth potential

The seed coat of Cycas Shiwandashanica collected in the experiment was hard, the structure was dense and the water permeability was poor, resulting in the low germination rate of seeds in the field habitat; The seeds were soaked in warm water for 24 hours to promote the expansion of the seeds, and then stored in sand, so as to improve the germination rate of the seeds. The experiment began with the sand storage at the end of October. After breaking dormancy for 2~3 months, the seeds germinated continuously from January to February of the second year. After the seed germinated and two true leaves grew, they were transplanted into the nutrition bag. The seedling height, diameter and crown of the same 10 annual and biennial seedlings were measured on August 12, 2018-2019. The seedling height and crown were measured with a tape measure, and the diameter was measured with a vernier caliper.

3.4 Determination of diurnal variation of photosynthesis

From 8:30 to 18:30 in the sunny weather in September, the net photosynthetic rate (P_n) of annual seedlings of Cycas Shiwandashanica was measured every 2 hours. The transparent leaf chamber was used to measure diurnal variation under complete natural light. Because the leaves of Cycas Shiwandashanica were small, the two leaves were superimposed for measurement. The maximum light intensity in the environment on that day could reach 2 000 μmol·m-²·s^-1, the concentration of carbon dioxide in the air was about 400 μmol/mol, the maximum temperature was about 34℃, and the maximum humidity was 40%.

3.5 Measurement of photoresponse curve

The photoresponse curve of annual seedlings of Cycas Shiwandashanica was measured by Li-6400 portable photosynthetic instrument with LED red and blue light source from 09:00 to 12:00. The leaves with mature middle section and no diseases and pests of each plant were selected. The light intensity was set to 0, 20, 50, 100, 200, 400, 800, 1000, 1200, 1400 and 1500 μmol·m-²·s^-1 in turn, and the three repeated results were averaged. The carbon dioxide cylinders were used to control the carbon dioxide concentration to 400 μmol·m^-2s^-1. Before measurement, the leaves were placed under the light intensity of 1 500 μmol·m-²·s^-1 for induction.

3.6 Data processing

The diurnal variation of photosynthetic rate and photoresponse curve were preliminarily analyzed by Excel 2016, the significance was analyzed by SPSS, the photoresponse curve was fitted by the right angle hyperbola correction model in photosynthetic calculation software (Ye, 2010), and Origin8.5 was used to make relevant charts.

Authors’ contributions

TJM was the experimental designer and executor of this study; TJM and QHZ completed the data analysis and wrote of the first draft of the manuscript; ZR, ZCH, WX and JYS participated in the experimental design, experiment and result analysis; XZC was the conceiver and person in charge of the project, guiding experimental design, data analysis, manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by Guangxi Fund of Natural Science (2020GXNSFAA259029), Special Funds for Guiding Local Scientific and Technological Development by the Central Government in China (Guike ZY1949013) and Basic Business Expenses of Guangxi Institute of Botany (Guizhiye 18013; 18014 and 19002).

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