Photosynthetic characteristics and antioxidant protective substances of two coniferous plants in karst fissures and under rainfall distribution

doi:10.21203/rs.3.rs-4966886/v1

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Research Article

Photosynthetic characteristics and antioxidant protective substances of two coniferous plants in karst fissures and under rainfall distribution

https://doi.org/10.21203/rs.3.rs-4966886/v1

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published 28 Oct, 2024

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Background The widely distributed hard limestone matrix is believed to exacerbate drought and increase the difficulty of restoring fragile karst areas. The cracks in this matrix may alleviate the negative effects of drought on plants, but their underlying mechanisms are still poorly understood.

Studying the physiological growth status of Pinus yunnanensis F. and Pinus elliottii E. seedlings under different karst fissure thicknesses and rainfall distributions is of great significance for the management, vegetation restoration, and tree species selection in karst rocky desertification areas.

In this study, we used a two-factor block experiment and set different rainfall durations, namely reduced rainfall duration (I_3d), natural rainfall duration (I_6d), and extended rainfall duration (I_9d); Different karst small habitats, i.e., stone-free soil (S₀), less stone and more soil (S_1/4), and half stone and half soil (S_1/2), are simulated at these three levels. Analyze the changes in physiological growth and photosynthetic characteristics in two types of coniferous seedlings under different treatments with different karst thicknesses.

Results The results showed that with the increase of karst thickness, the growth volumes of height and diameter of P. yunnanensis F. seedlings, the biomass of various organs, and the accumulation of K⁺, Ca²⁺, Na⁺, and Mg²⁺ showed a significant change pattern of first increasing and then decreasing (P<0.05); P. elliottii E. seedlings show a gradually decreasing trend (except for Ca²⁺). The biomass of each organ in two types of coniferous seedlings showed leaf>stem>root, while K⁺, Ca²⁺, and Mg²⁺ in each organ of P. yunnanensis F. seedlings showed leaf>root>stem, and Na⁺ showed root>leaf>stem. The accumulation of mineral elements in various organs of P. elliottii E. seedlings is as follows: roots>stems>leaves, and the accumulation of mineral elements in both types of coniferous seedlings is as follows: Ca²⁺>Mg²⁺>K⁺>Na⁺. Root length, root volume, root surface area, root diameter, SOD, POD, SP, photosynthetic pigment content, fluorescence parameters, and gas exchange parameters of P. yunnanensis F. seedlings gradually increase with the increase of karst thickness (except for the 9-day rainfall duration), while those of P. elliottii E. seedlings gradually decrease. Under different treatments, the maximum saturated light intensity and minimum light intensity of P. yunnanensis F. seedlings were 1624.530 and 21.395μmol·m^-2·s^-1, and 1081.100 and 27.148μmol·m^-2·s^-1 for P. elliottii E. seedlings, respectively. I_6dS_1/4 and I_3dS₀ treatments have the best growth effects on P. yunnanensis F. and P. elliottii E. seedlings.

Conclusions In summary, prolonging rainfall duration has an inhibitory effect on the growth of two types of coniferous seedlings. Reducing rainfall duration promotes the growth and development of P. elliottii E. seedlings, increasing karst thickness inhibits the growth of P. elliottii E. seedlings, and to some extent, promotes the growth and development of P. yunnanensis F. seedlings. Therefore, we give priority to P. yunnanensis F. as the tree species for vegetation restoration or rocky desertification management in karst areas. Our study reveals the role of limestone-filled different karst fissures in mitigating the effects of drought as "containers" for plant growth. These findings help us understand the response of plants to drought stress and provide valuable insights for vegetation restoration in karst environments affected by global climate change.Therefore, further experiments with various karst fissure sizes are necessary to test the universality of the reactions of various plants under different karst fissures. The results of this study can be used to help restore ecosystems damaged by karst rocky desertification processes.

pinus yunnanensis F. and pinus elliottii E

karst rocky desertification

karst fissures

physiological growth

enzyme activity

photosynthetic characteristics

Karst rocky desertification (KRD) is a major area with fragile ecology and environment, seriously threatening the sustainable development of society and economy (Reynolds et al., 2007; Dai et al.2018; Zhu et al., 2021). According to reports, most of the land in the Mediterranean region is threatened by karst development, and Lebanon is one of the countries severely affected by karst, with karst areas accounting for 70% of its land area (Kheir et al., 2008). With the development of karst landforms, soil erosion in western Ireland has accelerated, and the loss of water resources has become increasingly barren in the local soil (Drew 1983). Research has shown that due to the presence and development of karst, soil erosion in Cuba has exceeded a certain range, with an average variation between 12.3-13.7t ha^− 1a^− 1, and the risk of soil erosion is particularly high, which greatly hinders social and economic development (Febles-González et al., 2011). This is also the case in China, especially in the karst areas of southwestern China. The deterioration caused by land degradation and rocky desertification of the ecological environment is particularly prominent, affecting the survival and development space of residents (Wang et al., 2004; Bai et al., 2013; Pan et al., 2021. Like the three major karst concentrated distribution areas in the world, the karst area in southwestern China is the largest (about 550,000 square kilometers) and also the most typical distribution area of carbonate rocks in the world (Jiang et al.,2014). Most typical features of the region have been broken, such as land surface, exposed bedrock, poor soil, and severe decline in land productivity (Wang et al., 2004; Xu et al., 2013).

In addition, soil resources in karst areas are extremely scarce, with scarce soil and exposed bedrock embedded and distributed together (Li et al.,2006). The acidity of rainfall in karst areas is high, and the parent rock of carbonate as the main component, has strong permeability and is not resistant to dissolution, resulting in a large number of karst cracks on the rock mass (Dai et al., 2018). After rainfall, it can lead to the loss of surface soil. Traditionally, karst fissures have been thought to exacerbate soil aridity and limit plant growth, especially tall trees (Wang et al. 2004). This is attributed to their adverse effects on the water retention and storage capacity of the soil, leading to water easily seeping downwards, especially during high and prolonged rainfall periods (Fu et al., 2015). Therefore, due to the different thicknesses of karst fissure layers, there are also differences in moisture, nutrients, oxygen, and space in the fissure soil, resulting in heterogeneity in the vertical direction (Li et al., 2018). Against the backdrop of global climate change, the karst areas in southwestern China have shown a trend of unchanged total rainfall and a significant decrease in daily rainfall throughout the year (Feng et al.,2009). As the intensity of a single rainfall increases, the soil is prone to erosion and soil erosion becomes more severe, exacerbating the vertical heterogeneity of soil niche and further affecting plant growth. Studies have shown that karst cracks store 27% of total precipitation, exceeding the contribution of surface soil (21%) to vegetation (Du et al.,2015; Rempe and Dietrich.2018). However, soils filled or formed in surface karst cracks can partially reduce soil erosion rates (Williams 2008), and the presence of cracks enhances surface roughness and soil compaction ability, promoting plant growth (Xiong et al., 2012). Because the accumulation of organic matter and biomass produced by individual plants in photosynthesis is limited, there is a competitive relationship between the biological resources used by plants for survival, growth, or defense. Therefore, plants will balance the input and output of biomass. So in different habitats, plants will change the biomass allocation strategies of different organs, ensuring normal growth of plants based on survival. When plants are subjected to drought stress, different organs will adopt different ways to adapt to drought stress, fully utilize limited resources in the environment, and minimize the harm of stress to plants. Studies have shown that drought can lead to a corresponding decrease in biomass allocation in the aboveground parts, while the biomass input in the roots will increase accordingly, thereby absorbing more water and nutrients (Li et al., 2015). Some graduate students have also found that under mild water stress, plants increase the proportion of biomass input in photosynthetic components, thereby maintaining their ability to assimilate CO2 and produce organic matter. Only after a certain degree of drought, plants increase the input of root biomass and reduce the input of aboveground parts to maintain normal plant growth (Zhang et al., 2017). In addition, rainfall patterns also have an impact on biomass accumulation and distribution. Relevant studies have shown that when rainfall time is prolonged, the accumulation of aboveground biomass in Fraxinus malacophylla H. seedlings is inhibited, while the accumulation of underground biomass is promoted to a certain extent, leading to an increase in root biomass accumulation and thus maintaining plant life activities. At present, global karst desertification control remains an important issue, especially in south-western China. Moreover, the control of rocky desertification in southwestern China can significantly increase the forest area of the region, which is also an important measure for China to increase the total forest carbon sink, strive for development space, and improve its ability to respond to climate change. At the same time, the potential for low-carbon economic development in the region can also be mined. However, Pinus yunnanensis F. and Pinus elliottii E. themselves have physiological characteristics of fast growth and strong adaptability. After the control of rocky desertification, the development of fast-growing and high-yield forests can also meet the demand for wood, thereby developing biomass energy. In addition to generating economic benefits, ecological benefits are also very significant. The control of rocky desertification can fully utilize the functions of forest vegetation in soil and water conservation, soil fixation, fertilizer retention, carbon sequestration, and oxygen release, reducing the occurrence of geological disasters such as soil erosion and debris flows. Therefore, it is crucial to explore the growth strategies of two types of coniferous seedlings in different karst habitats.

Plant roots play an important role in global ecosystems. The root system is an important organ for maintaining vegetation, especially forest growth and development, and connects underground and above-ground ecological processes through the flow of matter and energy (Schenk and Jackson 2002). Biomass and productivity are key factors in global and regional carbon cycling (Cairns et al., 1997), especially in fine roots (Matamala et al., 2003). Roots can protect soil from erosion, and root architecture (root tissue density, root length, root diameter, root surface area, and root volume) effectively reduces soil detachment rate and reduces the erodibility of surface soil in karst mountainous areas (De Baets et al., 2006).

In addition, the uptake of mineral elements (K⁺, Ca²⁺, Na⁺, Mg²⁺) by plants, especially the uptake of root nutrients and water, is affected by environmental stress, leading to delayed plant growth and development (Zhao et al., 2022). K⁺ has important effects on the activation of enzymes, ion balance, cell turgor pressure, water balance, and carbohydrate transport in plants (Wang et al., 2017). Excessive or insufficient Ca²⁺ can affect related physiological and biochemical processes, ultimately leading to plant damage. A low concentration of Ca²⁺ in plants can lead to meristem necrosis, causing cellular physiological imbalances such as weakened plant growth and leaf necrosis (Adams et al.,1994). A study has found that plant leaves develop slowly or curl up at the leaf edges when lacking Ca²⁺ (De Freitas et al., 2016). In karst rocky desertification areas, there are already many carbonates with relatively high Ca²⁺ content, and the Ca²⁺ content varies in different karst habitats. Therefore, exploring the Ca²⁺ content in different karst habitats and precipitation treatments is of great significance. Excessive accumulation of Na⁺ can lead to many secondary effects, such as plant growth restriction, inhibition of leaf photosynthesis and transpiration (Iqbal et al., 2005; Jun et al., 2015). Moreover, excessive Na⁺ content can lead to osmotic stress, ion toxicity, and uneven ion distribution, disrupting plant cell structure, affecting the balanced absorption of mineral nutrients by plant roots, and disrupting the physiological metabolism of plant cells. At the same time, it can also induce the production of a large amount of reactive oxygen species and oxygen free radicals in the plant body, leading to oxidative stress and severely limiting plant growth and development (Zhao et al., 2022). Mg²⁺is considered to be the most abundant free divalent cation in plant cells, playing an important role in stabilizing cell macromolecular structure, maintaining enzyme activity, balancing intracellular reactive oxygen species and ions, and serving as a ligand and activator for over 300 enzymes (Guo et al., 2016). It is also an important component of chlorophyll and plays a crucial role in plant chloroplast photosynthesis (Farhat et al., 2016). At the same time, plants have evolved various mechanisms and strategies to cope with the damage caused by different environmental stresses, including absorbing water through roots and transporting it to leaves to maintain water conditions, accumulating compatible solutes such as proline (Pro) and mineral nutrients, regulating the toxicity of Na⁺ to plants, and maintaining ion distribution balance (Wang et al., 2018). Plants eliminate excess reactive oxygen species and alleviate oxidative stress damage by enhancing the activity of antioxidant enzymes in their bodies, such as superoxide dismutase (SOD) and peroxidase (POD) (Parida et al., 2005). It is also possible to enhance plant stress resistance by activating and inducing intracellular defense genes, such as salt over sensitive (SOS) signaling pathways, by upregulating the expression levels of antioxidant enzymes and SOS-related genes (Talaat et al., 2022). However, the transport system of K⁺ and Na⁺ binding is a key determinant of plant salt tolerance (Liu et al., 2022). Na⁺ competes with K⁺ for binding to root cell sites and transport proteins, leading to translocation, deposition, and partitioning within the plant body. Studies have shown that Chenopodium quinoa W. seedlings adopt self-protection mechanisms such as increasing soluble sugar and proline content, enhancing antioxidant enzyme activity, and reducing malondialdehyde (MDA) content to adapt to salt stress (Yang et al., 2017). Primula forbesii F. seedlings can resist salt damage by increasing soluble protein, proline content, superoxide dismutase, and peroxidase activity (Jia et al., 2022). It is unknown how the two types of coniferous seedlings can resist the stress of different karst fissures through mineral elements and antioxidant systems.

The photosynthetic physiological characteristics of plants can reflect their adaptability to habitat, and studying the light energy utilization efficiency of plants is the key to exploring their productivity (Zhang et al., 2022). Reduced photosynthesis leads to a decrease in CO₂ diffusion into the leaves, as the stomatal conductance (Gs) within the plant body is lower. Due to reduced cell proliferation and restricted leaf growth, it also leads to inhibition of photosynthesis (Lawlor and Tezara 2009; Wu et al., 2018). Moreover, plants in karstic rocky desertification areas are subjected to a variety of natural and anthropogenic factors, which limit their photosynthetic efficiency. As a result, most plant photosynthetic productivity has not reached the ideal state, which is not conducive to the comprehensive management of rocky desertification. Therefore, exploring the photosynthetic physiological characteristics and rainfall response patterns of plants in different karst habitats is the key to improving plant productivity in rocky desertification areas. In addition, the photosynthetic light response curve of plants reveals the corresponding relationship between net photosynthetic rate and photosynthetically active radiation. The physiological parameters obtained from the photosynthetic light response curve (including apparent quantum efficiency, maximum net photosynthetic rate, light compensation point, light saturation point, and dark respiration rate) can directly or indirectly reflect the physiological and ecological processes of plants (Santos Junior et al., 2013). Moreover, the soil thickness in karst areas affects the photosynthetic physiology of plants in terms of soil moisture, nutrients, and underground space availability (Zhang et al., 2020), which in turn affects plant growth. Stomata are physiologically important as they act as water pathways in the physiological processes of photosynthesis, respiration, and transpiration. However, chlorophyll fluorescence parameters are indicators of the response of photosynthesis to different stress environments (Kalaji et al., 2018). The characteristic of chlorophyll fluorescence is a key factor as it is used to measure the quantum yield of photosystem II (PSII) and photo inactivation by determining the quantum yield of light generated under water confinement conditions (Batra et al., 2014). The photosynthesis of plants is significantly affected by water, and although the physiological effects of water deficiency are well documented, it remains a highly important issue in the case of imbalanced precipitation distribution and different karst fissures.

P. yunnanensis F. is a unique species in southwest China, a pioneer tree species for natural regeneration of barren mountains in the Yunnan-Guizhou Plateau, and a major commercial tree species in Yunnan Province of China, accounting for about 52% of the forest area in Yunnan Province. P. yunnanensis F. is crucial for the economic and environmental sustainability of Yunnan forestry. It is widely distributed in the rocky desertification areas of eastern Yunnan, China, and has the characteristics of fast growth and drought resistance. It is a pioneer tree species for afforestation in barren mountains in the southwestern karst region. Since the 1980s, scholars have conducted a large number of studies on (P. yunnanensis F.), mainly focusing on the morphological and growth changes of seedlings under stress (Shen et al., 2020), community structure characteristics (Xu et al., 2016), seed ecological adaptability (Su et al., 2019), pests and diseases (Wang et al., 2015), and response to climate change (Yang and Luo 2011). P. elliottii E., originally from the United States, has the advantages of a fast growth rate, high-fat production, and strong adaptability. It is an excellent timber and landscaping tree species. It was introduced to China in the 1930s and has now become one of the main afforestation tree species for greening barren mountains and ecological protection in 15 provinces in southern China (Dai et al., 2018). The research on P. elliottii E. mainly focuses on aspects such as resin harvesting, seed cultivation, sex line inheritance, and growth equations (Su et al., 2022;Gao et al., 2022). Therefore, based on the fact that karst rocky desertification areas have large karst fissures and are relatively arid. How do P. yunnanensis F. seedlings grow as local tree species in such harsh environments? How to adapt to the growth of the region through its physiological regulatory mechanisms is not yet clear. As an exotic tree species, it is unknown whether and how P. elliottii E. grows normally in rocky desertification areas. The purpose of this study is to (1) elucidate how two types of coniferous seedlings respond to changes in organ biomass, root characteristics, and mineral elements in different karst habitats. (2) Determine how two types of coniferous seedlings respond to different karst fissures and rainfall distribution through their physiological regulatory mechanisms (antioxidant systems and photosynthetic characteristics). (3) Explore whether mild and moderate rocky desertification promotes the growth of two coniferous seedlings. (4) Reveal the trade-off strategies for the growth of each organ of two coniferous seedlings under this stress condition. Therefore, this article studied the physiological growth status of two coniferous seedlings under different degrees of rocky desertification, providing a theoretical reference for the selection of tree species for vegetation restoration in rocky desertification areas. In addition, the impact of rainfall duration combined with different degrees of rocky desertification on two coniferous tree species was also introduced, further improving the efficiency of rocky desertification management.

Research Site and Planting Materials

The research was conducted in a greenhouse at Southwest Forestry University in China (Kunming, Yunnan, 25°03′N、102°46′E). The research area is located in the monsoon climate zone of the subtropical plateau, with an average altitude of 1 954 meters, few frost periods, and a warm climate; The annual average temperature is 16.5 ℃, the annual precipitation is 1 035mm, the relative humidity of the air is 23%~67%, the atmospheric CO₂ concentration is 400–412 ppm, and there is sufficient light. The test plants are 2-year-old P. yunnanensis F. and P. elliottii E. seedlings, with red soil and karst limestone as the test soil and rocks, respectively. Both were taken from Jianshui County, Yunnan Province, China, which has typical karst landforms. The physical and chemical properties of red soil are shown in Table 1.

Table 1

Physical and chemical properties of the tested soil
pH	Volume weight	Organic matter	Organic carbon	Total nitrogen	Total phosphorus	Potassium/	Calcium/	Sodium	Magnesium
5.05	1.08/g·cm^− 3	3.34/g·kg^− 1	31.24/g·kg^− 1	0.68/g·kg^− 1	0.45/g·kg^− 1	0.84/g·kg^− 1	1.02/g·kg^− 1	0.43/g·kg^− 1	0.31/g·kg^− 1

Experimental Design

This study used a two-factor randomized block experiment, setting two factors: karst habitat and rainfall duration. The treatment of karst small habitats is divided into three levels: stone-free soil (S₀), less stone and more soil (S_1/4), and half stone and half soil (S_1/2), to simulate these three levels of karst habitats; The container ensures that the total volume of each habitat is the same (upper diameter 30cm, height 20cm). S₀ habitat is the whole soil. On the top 3/4 of the S_1/4 habitat is the soil layer, and the bottom 1/4 is the karst fissure layer. The upper part half of the S_1/2 habitat is the soil layer, and the lower part half is the karst fissure layer. According to Liu et al., (2022), the annual average number of consecutive days without effective precipitation in Yunnan in the southwestern region is about 6 days. The processing of rainfall time intervals is divided into 3-day rainfall intervals (I_3d), 6-day rainfall intervals (I_6d), and 9-day rainfall intervals (I_9d) to simulate three levels of rainfall allocation. According to the average rainfall of 123.58mm in Kunming City, China from June to December (Li 2016), the irrigation amount was calculated based on natural rainfall and rainfall distribution, and the total rainfall of the three rainfall intervals was kept consistent, thus achieving simulation of total rainfall and rainfall distribution (Table 2). There are 9 treatments for each tree species, totaling 18 treatments. Each treatment is repeated 3 times, with 10 seedlings per replicate, resulting in a total of 540 coniferous seedlings. The experiment was conducted in April 2023 by transplanting seedlings into containers, with one seedling per pot. The seedlings were subjected to seedling refining treatment from April to May, and the experimental research was conducted from June to the end of December 2023.

Table 2

Different rainfall allocation
Handle	Rainfall duration /d	Single irrigation volume/mL	Irrigation frequency/month
I_3d	3	873.09	10
I_6d	6	1746.19	5
I_9d	9	2910.31	3

Sample determination and methods

Plant sampling

On December 22nd, 6 seedlings were randomly selected from each treatment for destructive sampling. Clean the roots with clean water and absorb moisture with filter paper. Place the roots, stems, and leaves of the same plant in envelope bags and mark them accordingly. The roots, stems and leaves were put into an oven with a temperature of 105 ℃ for sterilization for 30min, and then adjusted to 80 ℃ for drying to constant weight. The biomass (g) of the roots, stems, and leaves of two coniferous seedlings was obtained by weighing each organ.

Growth rate

At the beginning of the experiment, ground diameter (mm) was determined with a vernier caliper, and seedling height (cm) was determined with a straightedge for the 2 conifer seedlings in each treatment. At the end of the experiment, the height increment of each treated seedling was measured to calculate the increment of seedling height = measured height - initial height, and the increment of ground diameter = measured ground diameter - initial ground diameter. The sum of the biomass of each organ is the total biomass, and the proportion of root, stem, and leaf biomass is calculated based on the biomass of each organ and the total biomass.

Root morphology characteristics

Immediately use the EPSON Scan root scanner to scan the cleaned roots, and then use WinRHIZO analysis software to obtain the morphological characteristics of the roots, including total root length (RL), root surface area (RS), root diameter (RD), and root volume (RV). Furthermore, the specific root length (cm/g) = root length/root biomass, specific root area (cm²/g) = root surface area/root biomass, and root tissue density (g/cm³) = root biomass/root volume were calculated.

Physiological indicators

After weighing, the plant samples were crushed using a grinder and sieved through a 1.0 mm sieve. An accurate 0.2000 g of the sample was weighed and digested with H₂SO₄-H₂O₂. The K⁺, Ca²⁺, Na⁺, and Mg²⁺contents in each organ were determined using plasma chromatography (Michalski 2006). The antioxidant system, including soluble protein (SP), peroxidase (POD), superoxide dismutase (SOD), malondialdehyde (MDA), and proline (Pro), was determined according to the steps of the Suzhou Greese Biotechnology Co., Ltd. reagent kit.

Photosynthetic pigments and fluorescence parameters

Measurement of photosynthetic pigment content (mg/g): Take 0.2g of ground leaf powder, put it into a mortar, add a small amount of SiO₂, and then add 2–3 mL of 95% ethanol. Grind the homogenate and make up to 25 mL with 95% ethanol. Pour the pigment extraction solution into a colorimetric dish and measure the absorbance values at wavelengths 665, 649, and 470 nm, using 95% ethanol as the control. Calculate the chlorophyll a (Chla), chlorophyll b (Chlb), carotenoids (Car), chlorophyll a + b, and chlorophyll a/b of the leaves (Jaleel et al., 2008).

Chlorophyll fluorescence parameter determination: Select the coniferous leaves under each growth point of P. yunnanensis F. and P. elliottii E., and after dark adaptation for 30 minutes, use the LI-6800 portable photosynthetic analyzer to obtain the initial fluorescence value (Fo), maximum fluorescence value (Fm), and variable fluorescence (Fv) under dark conditions; Chlorophyll fluorescence parameters include maximum fluorescence value (Fm') under light conditions, maximum photochemical quantum efficiency (Fv/Fm) of PSII under dark adaptation, the potential activity of PSII (Fv/Fo), excitation energy capture efficiency of PSII reaction center (Fv '/Fm'), non-photochemical quenching coefficient (NPQ), photochemical quenching coefficient (qP), and electron transfer rate (ETR).

Photosynthetic gas exchange and response curve

The photosynthetic indexes of P. yunnanensis F. and P. elliottii E. needles were measured by Li-6800 portable photosynthesis tester (LI-COR, USA) in each treatment.

Measurement of photosynthetic gas exchange parameters: from 9:00 am to 11:30 am on a sunny day at a light intensity of 1800µmol·m^− 2·s^− 1, CO₂ concentration of 400µmol·mol^− 1. Measure the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO₂ concentration (Ci) of P. yunnanensis F. and P. elliottii E. needles under different treatments at a temperature of 25 ℃.

Measurement of light response curve: At 9:00–11:30, set the light intensity gradient to be 1800, 1600, 1400, 1200, 1000, 800, 600, 400, 300, 200, 150, 100, 50, 30, and 0µmol·m^− 2·s^− 1 in sequence, CO₂ concentration set at 400µmol·mol^− 1.

Determination of CO₂ response curve: CO₂ concentration gradients are set at 400, 300, 200, 100, 50, 20, 10, 400, 400, 600, 800, 1000, 1200, 1400, 1600, and 1800µmol·mol^− 1, PAR value set to 1800µmol·m^− 2·s^− 1.

Referring to Ye (2010) analysis, two types of needle parameters were obtained: apparent quantum efficiency (AQY), maximum net photosynthetic rate under light (L_Pnmax), light saturation point (LSP), and light compensation point (LCP); Maximum net photosynthetic rate (Pnmax) and carboxylation efficiency(α)、 Dark respiration rate (Rd), photorespiration rate (Rp), CO₂ saturation point (CSP), CO₂ compensation point (CCP).

Statistical analysis

Utilizing Amos 23.0 Construct a structural equation model to explore the degree of influence between physiological growth and photosynthetic characteristics of two types of coniferous seedlings. Organize and calculate data using Excel 2010, and conduct statistical analysis using SPSS 25.0 (SPSS Inc., Chicago, IL, USA) and Origin 2021.0 (OriginLab Co., Northampton, MA, USA).

Growth and biomass

Growth rate

According to Table 3, there were no significant differences (P > 0.05) in seedling height, diameter, and needle length between P. yunnanensis F. and P. elliottii E. under different karst habitats and rainfall distribution before the experiment began, indicating that there was not much difference in growth among all seedlings before the experiment began. However, in terms of seedling height and needle length, P. elliottii E. is significantly higher than (P. yunnanensis F.), while the ground diameter is lower than (P. yunnanensis F.). From Table 4, there was a significant difference in needle length between the two types of coniferous seedlings under the interaction between rainfall duration and karst habitat, except for the needle length of P. elliottii E., while the other growth rates show significant differences (P < 0.05) under different rainfall duration, karst habitat, and their interaction. Specifically, under the same rainfall duration, the growth of P. yunnanensis F. shows a significant pattern of first increasing and then decreasing with the increase of karst cracks, reaching its maximum in the S_1/4 karst habitat. However, extending the rainfall duration (I_9d) has a certain inhibitory effect on the growth of P. yunnanensis F. Under I_3d treatment, the height growth of seedlings under the S_1/4 karst fissure reached 9.105cm, the ground diameter increased by 15.530mm, and the needle length increased the fastest (21.958cm). Under the I_6d treatment, the growth rate of seedling height under the S_1/4 karst fissure was 1.631 times that of the S₀ habitat, with a ground diameter of 1.307 times and a needle length of 1.100 times. Under the I_9d treatment, the height of seedlings under the S1/4 karst fissure increased by 0.561cm compared to S₀, with a ground diameter of 0.902mm and a needle length of 1.633cm. However, the performance of P. elliottii E. is not entirely the same. The growth of P. elliottii E. significantly decreases with the increase of karst cracks under the same rainfall duration (P < 0.05), and also decreases with the increase of rainfall duration. The growth rates of seedling height, ground diameter, and needle length were highest in the 3d rainfall duration and the whole soil habitat, with values of 36.117cm, 10.853mm, and 12.267cm, respectively. The growth of P. elliottii E. is significantly inhibited by extending the duration of rainfall.

Table 3

Initial growth of *P. yunnanensis* F. and *P. elliottii* E.
Rainfall duration	Karst habitat	Pinus yunnanensis F.			Pinus elliottii E.
Rainfall duration	Karst habitat	Seedling height/cm	Ground diameter/mm	Needle length/cm	Seedling height/cm	Ground diameter/mm	Needle length/cm
I_3d	S₀	10.975 ± 0.237Aa	6.597 ± 0.783Aa	7.100 ± 0.276Aa	31.433 ± 1.061Aa	4.190 ± 0.222Aa	9.567 ± 0.561Aa
	S_1/4	10.928 ± 0.319Aa	6.835 ± 0.794Aa	7.025 ± 0.172Aa	30.883 ± 0.880Aa	4.110 ± 0.251Aa	9.600 ± 0.620Aa
	S_1/2	10.957 ± 0.241Aa	6.758 ± 0.467Aa	6.848 ± 0.413Aa	30.633 ± 0.671Aa	4.118 ± 0.243Aa	9.733 ± 0.589Aa
I_6d	S₀	10.543 ± 0.551Aa	7.017 ± 0.508Aa	7.233 ± 0.476Aa	31.117 ± 1.055Aa	4.065 ± 0.255Aa	9.267 ± 0.656Aa
	S_1/4	10.903 ± 0.619Aa	7.208 ± 0.881Aa	7.217 ± 0.412Aa	30.917 ± 0.744Aa	4.195 ± 0.292Aa	9.933 ± 0.455Aa
	S_1/2	11.062 ± 0.364Aa	7.447 ± 0.392Aa	7.383 ± 0.508Aa	31.050 ± 0.704Aa	4.168 ± 0.193Aa	9.517 ± 0.703Aa
I_9d	S₀	10.517 ± 0.713Aa	6.443 ± 0.905Aa	7.050 ± 0.187Aa	30.717 ± 0.821Aa	4.117 ± 0.228Aa	9.867 ± 0.32Aa
	S_1/4	11.148 ± 0.679Aa	6.790 ± 0.391Aa	7.250 ± 0.274Aa	30.717 ± 0.801Aa	4.200 ± 0.186Aa	9.900 ± 0.522Aa
	S_1/2	11.018 ± 0.516Aa	7.157 ± 0.430Aa	7.117 ± 0.256Aa	30.717 ± 0.440Aa	4.198 ± 0.262Aa	9.800 ± 0.718Aa
F value	I	0.245^ns	3.056^ns	3.010^ns	0.762^ns	0.099^ns	1.174^ns
	S	2.510^ns	2.037^ns	0.091^ns	0.661^ns	0.181^ns	0.790^ns
	I\(\:\times\:\)S	0.914^ns	0.318^ns	0.821^ns	0.467^ns	0.378^ns	0.695^ns
Note: The values in the table are mean ± standard deviation. Different uppercase letters in the same column indicate significant differences between different karst habitats under the same rainfall interval duration (P < 0.05), while different lowercase letters indicate significant differences between different rainfall intervals under the same karst habitat (P < 0.05). I: Represents rainfall duration, S: represents karst habitat, I x S: represents the interaction between rainfall duration and karst habitat. ns: represents P > 0.05, * P < 0.05, * * P < 0.01. Same below.

Table 4

Effects of different karst habitats and rainfall distribution on the growth of two coniferous Seedlings
Rainfall duration	Karst habitat	Pinus yunnanensis F.			Pinus elliottii E.
Rainfall duration	Karst habitat	Seedling height/cm	Ground diameter/mm	Needle length/cm	Seedling height/cm	Ground diameter/mm	Needle length/cm
I_3d	S₀	7.775 ± 0.519Ba	12.412 ± 0.63Ba	19.1 ± 0.369Bb	36.117 ± 1.852Aa	10.853 ± 0.497Aa	12.267 ± 3.157Aa
	S_1/4	9.105 ± 0.673Ab	15.53 ± 0.801Ab	21.958 ± 0.683Ab	28.117 ± 2.006Ba	9.55 ± 0.642Ba	7.033 ± 3.834Ba
	S_1/2	4.077 ± 0.688Ca	10.68 ± 0.863Ca	17.085 ± 0.853Cb	21.917 ± 1.484Ca	6.113 ± 0.312Ca	6.233 ± 0.674Ba
I_6d	S₀	7.49 ± 0.943Ba	13.018 ± 0.597Ba	21.633 ± 1.029Ba	35.333 ± 2.085Aa	11.355 ± 0.602Aa	11.783 ± 0.313Aa
	S_1/4	12.213 ± 0.37Aa	17.098 ± 1.103Aa	23.767 ± 0.476Aa	21.633 ± 0.909Bb	6.977 ± 0.514Bb	6.183 ± 0.833Ba
	S_1/2	5.572 ± 0.873Ca	10.5 ± 0.647Ca	19.25 ± 1.155Ca	16.767 ± 1.642Cb	5.687 ± 0.259Ca	4.183 ± 0.864Cb
I_9d	S₀	7.917 ± 0.935Aa	13.218 ± 1.055Aa	18.400 ± 0.876Ab	29.367 ± 0.72Ab	8.825 ± 0.664Ab	8.167 ± 0.731Ab
	S_1/4	8.568 ± 0.978Ab	14.120 ± 0.637Ac	20.033 ± 0.997Ac	18.417 ± 1.143Bc	5.938 ± 0.603Bc	4.317 ± 0.85Ba
	S_1/2	4.365 ± 1.837Ba	7.138 ± 2.427Bb	13.067 ± 2.582Bc	11.033 ± 0.516Cc	2.817 ± 0.812Cb	1.300 ± 0.805Cc
F value	I	14.005**	15.792**	62.857**	171.869**	131.284**	23.054**
	S	139.528**	137.363**	98.416**	614.443**	416.337**	70.332**
	I\(\:\times\:\)S	7.991**	7.499**	3.954**	7.224**	12.781**	0.850^ns

Biomass

As shown in Fig. 1, there are significant differences (P < 0.05) in the biomass of each organ of the two coniferous seedlings under different karst habitats and rainfall allocation treatments. There were significant differences in root, stem, leaf, above-ground, and total biomass of P. yunnanensis F. under different karst habitats, different rainfall duration, and their interaction (P < 0.01). The biomass of various organs and the total biomass of P. yunnanensis F. show a significant change pattern of first increasing and then decreasing with the deepening of karst cracks under the same rainfall duration; Under the same karst habitat, there is a significant change pattern of first increasing and then decreasing with the extension of rainfall duration, and both extending or shortening rainfall duration have inhibitory effects. Under the 3d rainfall duration, the biomass accumulation of roots, stems, and leaves increased by 4.718g, 3.517g, and 5.823g in the S_1/4 karst habitat treatment compared with the S₀ karst habitat, respectively. Under 6 days of rainfall, the biomass of roots, stems, and leaves were highest under the S_1/4 karst habitat treatment, with values of 14.167g, 15.249g, and 21.858g, respectively. Under 9 days of rainfall, the biomass accumulation of roots, stems, and leaves increased by 6.634%, 9.065%, and 1.520% in the S_1/4 karst habitat treatment compared with the S₀ karst habitat, respectively. However, under the same rainfall duration, the biomass of various organs and total biomass of P. elliottii E. show a significant decrease with the deepening of karst cracks; Under the same karst habitat, there is a significant decrease in biomass with the extension of rainfall duration. The increase in rainfall duration significantly inhibits the accumulation of biomass in various organs, while the decrease in rainfall duration significantly promotes the accumulation of biomass in (P. elliottii E.). In the S₀ karst habitat, the biomass of roots, stems, and leaves during 9d rainfall are 3.112g, 1.603g, and 9.369g lower than the 6-day rainfall duration, respectively. In the S1/4 karst habitat, the biomass of the root, stem, and leaf decreased by 36.176%, 22.639%, and 141.954% in the 9-day rainfall duration compared to 6 days of rainfall, respectively. In the S_1/2 karst habitat, the biomass of roots, stems, and leaves was the lowest after 9 days of rainfall, with values of 7.297g, 10.314g, and 5.268g, respectively. In conclusion, S_1/4 karst fissures (mild rocky desertification) promote the accumulation of biomass in all organs of P. yunnanensis F., while the accumulation of biomass in all organs of P. elliottii E. is inhibited by karst fissures.

The differences in biomass allocation between the roots, stems, and leaves of two coniferous seedlings under different karst habitats and rainfall distribution (Table 5). Specifically, except for no significant difference in the proportion of stem biomass between P. yunnanensis F. seedlings under rainfall duration and karst habitat, there were extremely significant differences in the proportion of root and leaf biomass (P < 0.01), and only significant differences in the proportion of stem biomass existed under the interaction between the two (P < 0.05). For P. elliottii E., the proportion of biomass in each organ showed significant differences (P < 0.05) except for the proportion of root biomass under the interaction of rainfall duration and karst habitat.

The effects of the proportion of each organ in two types of coniferous seedlings on rainfall duration and karst habitat are different (Fig. 2). For P. yunnanensis F. seedlings, the allocation of leaf biomass is significantly higher than that of roots and stems, and with the increase of karst cracks, the proportion of root, stem, and leaf biomass shows a trend of first increasing and then decreasing. The proportion of biomass in each organ shows a trend of leaves > stems > roots. For P. elliottii E. seedlings, the proportion of biomass in each organ shows a decreasing trend with the extension of rainfall duration, and the proportion of biomass in each organ shows a trend of leaves > stems > roots. In summary, the biomass accumulation in each organ of the two coniferous seedlings showed a trend of leaf > stem > root, and plant growth tended to accumulation of leaf biomass, reflecting the growth strategy of the plant.

Table 5

Analysis of variance in biomass allocation of different organs of two coniferous seedlings under different karst habitats and rainfall distribution
Varieties of trees	Handle	Root	Stem	Leaf
P. yunnanensis F.	I	6.835^**	0.564^ns	5.547^**
	S	10.366^**	2.166^ns	6.791^**
	I\(\:\times\:\)S	2.457^ns	3.496^*	0.850^ns
P. elliottii E.	I	48.254^**	311.360^**	658.570^**
	S	3.450^*	14.673^**	6.158^**
	I\(\:\times\:\)S	2.201^ns	5.818^**	5.300^**

Root system characteristics

As shown in Fig. 3, the root morphology of the two coniferous seedlings showed significant changes (P < 0.05) under different karst fissures and rainfall durations. For P. yunnanensis F. seedlings, there were significant differences in root morphology between karst cracks and rainfall duration treatments (P < 0.01), while only root length had no significant difference under their interaction (P > 0.05). The root characteristics all thicken with the deepening of karst cracks under the same rainfall duration, and the rainfall effect of 6d under each karst crack is the best. Specifically, the root length, root surface area, root volume, and root diameter of P. yunnanensis F. are 1.856 times, 2.880 times, 4.369 times, and 2.461 times higher in S_1/2 karst fractures than in S₀ karst habitats, respectively, under a rainfall duration of 3 days. The root length, root surface area, root volume, and root diameter of the S_1/2 karst habitat were the largest under a rainfall duration of 6 days., and their values were 3557.752cm, 3964.692cm², 552.708cm³, and 4.590mm, respectively. However, root length, root surface area, root volume, and root diameter were inhibited under a rainfall duration of 9 days, but they also increased with the increase of karst fractures. For P. elliottii E. seedlings, there were significant differences in root morphology in karst fissures and rainfall duration treatment (P < 0.01), and there were significant differences in other indicators except for root surface area and specific root area under the interaction of both treatments (P < 0.05). The root characteristics decrease with the deepening of karst cracks under the same rainfall duration, and the best rainfall effect of 3d is observed under each karst crack. Specifically, root length, root surface area, root volume, and root diameter of P. elliottii E. increased by 19.622%, 48.207%, 120.599%, and 92.373%, respectively, under all-soil habitats (S₀) for 3d rainfall duration compared to 6d rainfall duration; However, the root length, root surface area, root volume, and root diameter were 434.044cm, 773.848cm², 159.699cm³, and 1.066mm lower under a 9-day rainfall duration than under a 6-day rainfall duration, respectively. In summary, increasing the thickness of karst cracks promotes the growth of root characteristics in P. yunnanensis F. seedlings, which also indicates that P. yunnanensis F. can adapt to certain karst habitats. However, prolonged rainfall duration (9 days) or high-frequency rainfall (3 days) are not conducive to the growth of P. yunnanensis F. root systems. However, the root system characteristics of P. elliottii E. are radically different. Deepening karst cracks have a significant inhibitory effect on root characteristics, and high-frequency rainfall has the best effect on root growth.

K⁺、Ca²⁺、Na⁺、Mg²⁺

As shown in Fig. 4, the accumulation of mineral elements in different organs of the two coniferous seedlings showed significant changes (P < 0.05) under different karst fissures and rainfall durations. For P. yunnanensis F. seedlings, the content of K⁺, Ca²⁺, Na⁺, and Mg²⁺ in various organs showed extremely significant differences (P < 0.01) under different karst fissures and rainfall durations. Under the interaction of the two, except for significant differences in leaf K⁺, Ca²⁺, and Mg²⁺ content, there was no significant difference in the accumulation of other contents (P > 0.05). Under the same rainfall duration, the accumulation of mineral element content in various organs shows a significant change pattern of first increasing and then decreasing with the deepening of karst fractures (P < 0.05). Under the same karst fissure, the accumulation of mineral element content in various organs also shows a significant change pattern of first increasing and then decreasing with the increase of rainfall duration (P < 0.05) (except for the K⁺, Ca²⁺, and Mg²⁺ content in leaves in the S_1/4 karst fissure). The values of K⁺/Na⁺ in roots, stems, and leaves varied between 1.522 ~ 2.252, 4.100 ~ 11.579, and 7.045 ~ 20.716 under different treatments, respectively. The K⁺/Na⁺ values in stems and leaves were relatively high, indicating that P. yunnanensis F. seedlings have certain salt tolerance. The accumulation of K⁺, Ca²⁺, and Mg²⁺ content in various organs is as follows: leaves > roots > stems, while Na⁺ is as follows: roots > leaves > stems. For P. elliottii E. seedlings, under the same rainfall duration, the content of K⁺, Na⁺, and Mg²⁺ in each organ showed a significant decrease with the deepening of karst cracks (P < 0.05), while the content of Ca²⁺ showed a changing pattern of first increasing and then decreasing, and the effect was best under a 3-day rainfall duration. The K⁺/Na⁺ values in the roots were highest in the karst habitat of S_1/2 and rainfall duration of 6 days (6.330), while the K⁺/Na⁺ values in the stems and leaves were highest in the habitat of S₀ with a rainfall duration of 9 days, with values of 7.727 and 9.979, respectively. Moreover, the accumulation of K⁺, Ca²⁺, Na⁺, and Mg²⁺ content in various organs is manifested as root > stem > leaf, with mineral element content more inclined towards root accumulation. In summary, the accumulation of mineral elements in various organs of the two types of coniferous seedlings showed a trend of Ca²⁺>Mg²⁺>K⁺>Na⁺. The overall accumulation of mineral elements in various organs of P. yunnanensis F. seedlings was higher than that of P. elliottii E.

Antioxidant system

From Fig. 5, different karst habitats and rainfall distribution affected the enzyme activities of the two conifer seedlings, which could ameliorate the damage caused by karst fissure stress on the two conifer seedlings. For P. yunnanensis F. seedlings, under the same rainfall duration, the activities of SOD and POD antioxidant enzymes and soluble protein content significantly increased with the deepening of karst cracks (P < 0.05). Under a rainfall duration of 3 days, SOD, POD, and SP all reached their maximum values in the S_1/2 karst habitat, with values of 821.938 U·g^− 1, 146.833 U·g^− 1, and 54.057 mg·g^− 1, respectively. Under 6 days of rainfall, the activities of SOD, POD, and SP in the S_1/2 karst habitat were significantly higher than those in the S0 habitat by 8.325%, 31.684%, and 82.289%. Under a 9-day rainfall duration, the activities of SOD, POD, and SP were the lowest in the S_1/2 karst habitat. Both MDA and Pro activities were highest under the 9-day rainfall duration S_1/2 karst habitat treatment, with values of 19.309 U·g^− 1 and 61.395 U·g^− 1, respectively, indicating that the structure and function of the biofilm of P. yunnanensis F. seedlings were damaged under this treatment. For P. elliottii E. seedlings, under different treatments, the activities of SOD, POD, and SP significantly decreased with the increase of karst cracks and rainfall duration (P < 0.01). All indicators were highest under the S₀ treatment with a rainfall duration of 3 days, with values of 434.394 U·g^− 1, 146.833 U·g^− 1, and 62.450 mg·g^− 1, respectively. Both MDA and Pro activities were highest under the S_1/2 karst habitat treatment with 9 days of rainfall, with values of 26.239 U·g^− 1 and 71.799 U·g^− 1, respectively. This value is higher than that of P. yunnanensis F. seedlings, indicating that the structure and function of the biofilm in P. elliottii E. seedlings have been severely damaged. However, the levels of SOD, POD, and SP in P. elliottii E. seedlings are lower than those in P. yunnanensis F. seedlings, indicating that P. elliottii E. seedlings have poorer ability to resist karst cracks and rainfall duration compared to P. yunnanensis F. In summary, P. elliottii E. has greater biofilm destruction than P. yunnanensis F., and its antioxidant enzyme activity is lower than P. yunnanensis F.

Photosynthetic pigments and fluorescence parameters

Photosynthetic pigment content

As shown in Fig. 6, the significant differences in photosynthetic pigment content between the two coniferous seedlings were different for different karst cracks and rainfall duration (P < 0.05). For P. yunnanensis F. seedlings, the content of Chla, Chlb, Car, and Chla + b showed extremely significant differences (P < 0.01) under different karst fissures and rainfall durations, while there was no significant difference (P < 0.05) under the interaction of the two. Under the same rainfall duration, the content of photosynthetic pigments increases with the deepening of karst cracks. Increasing karst cracks can to some extent promote the synthesis of photosynthetic pigments. The content of Chla, Chlb, Car, and Chla + b reaches their maximum under the S_1/2 karst habitat treatment with a rainfall duration of 6 days, with values of 1.043mg·g^− 1, 0.302mg·g^− 1, 0.208mg·g^− 1, and 1.345mg·g^− 1, respectively. For P. elliottii E. seedlings, there were significant differences in photosynthetic pigment content (P < 0.05) under different karst cracks, rainfall duration, and their interaction. Under the same rainfall duration, the content of photosynthetic pigments shows a significant trend of first increasing and then decreasing with the deepening of karst cracks. S_1/4 karst crack treatment has a certain promoting effect on the synthesis of photosynthetic pigment content in P. elliottii E. seedlings while prolonging rainfall duration inhibits the synthesis of photosynthetic pigments. Under different treatments, the Chlb synthesis of the two coniferous seedlings is the same, but the synthesis of Car in P. elliottii E. is significantly higher than that in P. yunnanensis F. (Fig. 6-C). In summary, mild (S_1/4) and moderate rocky desertification (S_1/2) are beneficial for the synthesis of photosynthetic pigments in P. yunnanensis F., while the photosynthetic pigment content in P. elliottii E. increases under mild rocky desertification (S_1/4) treatment, while it decreases under moderate rocky desertification (S_1/2) treatment.

Chlorophyll fluorescence parameters

The effects of karst cracks and rainfall duration on the fluorescence parameters of two types of coniferous seedlings are shown in Fig. 6. For P. yunnanensis F. seedlings, different karst cracks, rainfall duration, and their interaction have a significant limit effect on all indicators (P < 0.01). Under the rainfall duration of 3d and 6d, Fm, Fv, Fo/Fv, Fv/Fm, Fv '/Fm', qP, ETR, and NPQ all significantly increased with the deepening of karst fissures (P < 0.05), indicating that the photosynthetic system of P. yunnanensis F. seedlings was less or not damaged with the deepening of karst fissures during 3d and 6d rainfall. The increase in their values also indicates that the photosynthetic capacity of plants is enhanced, utilizing their photosynthesis. On the contrary, under the 9-day rainfall duration, these indicators showed a downward trend, indicating that the treatment of karst cracks during the 9-day rainfall duration is not conducive to the improvement of plant photosynthetic capacity. The maximum Fo was observed under the S_1/2 rainfall duration of 9 days (720.667), indicating that the photosynthetic system response center of P. yunnanensis F. plants was damaged to some extent under this treatment (Fig. 6-A). For P. elliottii E. seedlings, under the same rainfall duration, Fo increases with the deepening of karst cracks, reaching a maximum value of (1238.000), while Fm, Fv, Fo/Fv, Fv/Fm, Fv '/Fm', qP, ETR, and NPQ show a significant downward trend. Reducing rainfall duration (3d) significantly promotes their increase. Under the full soil (S₀) treatment, the 3d rainfall duration P. elliottii E. Fm, Fv, Fo/Fv, Fv/Fm, Fv '/Fm', qP, ETR, and NPQ increased by 13.274%, 62.459%, 208.498%, 43.169%, 55.692%, 53.315%, 9.081%, and 90.092%, respectively, compared to the 6-day rainfall duration; The duration of rainfall for 9 days is significantly lower than that for 6 days, with values of 197.166, 218.667, 0.552, 0.221, 0.274, 0.240, 30.092, and 0.261 respectively. In summary, under normal rainfall duration (6d) and reduced rainfall duration (3d), the increase of karst cracks to some extent promotes the photosynthesis of P. yunnanensis F. seedlings, thereby enabling the plant to produce more organic matter to provide energy for its growth and development. However, the photosynthetic system of P. elliottii E. seedlings is relatively complete in the whole soil habitat with a duration of 3d rainfall, and plant photosynthesis is the strongest, synthesizing the most organic matter. Therefore, reducing the duration of rainfall is beneficial for the growth of P. elliottii E. seedlings. Extending the duration of rainfall has an inhibitory effect on the growth and development of P. yunnanensis F. and P. elliottii E.

Photosynthetic characteristics

Gas exchange parameters

According to Table 6, the changes in photosynthetic gas parameters of the two types of coniferous seedlings are different under different treatments. As for P. yunnanensis F. seedlings, there were significant differences in Tr, Pn, Ci, and Gs under different karst cracks, rainfall duration, and their interaction (P < 0.01). Under the rainfall duration of 3 days and 6 days, Tr, Pn, and Gs all significantly increased with the deepening of karst fractures. Under the rainfall duration of 9 days, they showed a significant change pattern of first increasing and then decreasing (P < 0.05). At the rainfall duration of 6 days and S_1/2 karst fractures, Tr, Pn, and Gs reached their maximum values, which were 0.335 mol·m⁻²·s⁻¹, 14.358µmol·m⁻²·s⁻¹, and 0.273 mol·m⁻²·s⁻¹, respectively. However, the concentration of Ci does not show different patterns. Under the rainfall duration of 3 and 6 days, the concentration of Ci shows a significant downward trend with the deepening of karst cracks, and under the rainfall duration of 9 days, it shows a trend of first decreasing and then increasing. This phenomenon indicates that P. yunnanensis F. seedlings consume the most CO₂ concentration during photosynthesis, resulting in the highest Pn. As for P. elliottii E. seedlings, Tr, Pn, Ci, and Gs showed extremely significant differences (P < 0.01) under different karst fissures and rainfall duration treatments, with only Tr and Pn showing extremely significant differences in their interaction (P < 0.01). Under the same rainfall duration, the values of Tr, Pn, and Gs decrease with the increase of karst fractures, while the performance of Ci is exactly the opposite. In the whole soil (S₀) habitat, the concentrations of Tr, Pn, and Gs in P. elliottii E. seedlings were higher in 3d rainfall than in 6d by 0.136 mol·m⁻²·s⁻¹, 0.439µmol·m⁻²·s⁻¹, and 0.110 mol·m⁻²·s⁻¹. Under mild rocky desertification (S_1/4), the concentrations of Tr, Pn, and Gs were 167.925%, 35.636%, and 53.731% higher under 3d rainfall than 6d. Under moderate rocky desertification (S_1/2), the concentrations of Tr, Pn, and Gs were the lowest 6 days of rainfall, with values of 0.032 mol·m⁻²·s⁻¹, 2.942µmol·m⁻²·s⁻¹, and 0.024 mol·m⁻²·s⁻¹. Research has shown that increasing the consumption of Ci concentration in P. yunnanensis F. seedlings through karst cracks can enhance the photosynthetic capacity of plants and promote the synthesis of Pn, Tr, and Gs. Reducing rainfall duration promotes the synthesis of Pn, Tr, and Gs in P. elliottii E. seedlings, thereby improving the photosynthetic capacity of plants, increasing karst cracks, inhibiting photosynthesis in P. elliottii E. seedlings, and promoting the accumulation of Ci concentration.

Table 6

Effects of different karst fissures and rainfall distribution on photosynthetic gas exchange parameters of two coniferous seedlings
Varieties of trees	Rainfall duration	Karst habitat	Tr/mol·m⁻²·s⁻¹	Pn/µmol·m⁻²·s⁻¹	Ci/µmol·mol⁻¹	Gs/mol·m⁻²·s⁻¹
P. yunnanensis F.	I_3d	S₀	0.089 ± 0.017Bb	3.301 ± 0.934Bb	346.337 ± 5.623Ab	0.039 ± 0.001Cb
		S_1/4	0.114 ± 0.013Ab	4.957 ± 0.845Ab	341.776 ± 10.27Ab	0.06 ± 0.007Bb
		S_1/2	0.121 ± 0.016Ab	5.639 ± 1.014Ab	263.651 ± 16.472Bb	0.115 ± 0.006Ab
	I_6d	S₀	0.24 ± 0.026Ba	8.199 ± 0.78Ca	450.736 ± 18.598Aa	0.23 ± 0.019Ca
		S_1/4	0.317 ± 0.022Aa	11.245 ± 0.976Ba	439.214 ± 31.338Aa	0.252 ± 0.011Ba
		S_1/2	0.335 ± 0.026Aa	14.358 ± 2.92Aa	352.942 ± 12.122Ba	0.273 ± 0.016Aa
	I_9d	S₀	0.058 ± 0.012Bc	2.884 ± 0.359Ab	282.784 ± 78.672Ab	0.024 ± 0.013Ab
		S_1/4	0.074 ± 0.018Ac	3.058 ± 0.553Ac	277.245 ± 15.885Ac	0.044 ± 0.037Ab
		S_1/2	0.041 ± 0.007Cc	1.619 ± 0.402Bc	337.403 ± 33.605Ab	0.030 ± 0.009Ac
F value		I	852.519^**	253.760^**	65.701^**	921.567^**
		S	25.948^**	18.410^**	25.549^**	29.832^**
		I\(\:\times\:\)S	14.662^**	14.229^**	4.710^**	8.943^**
P. elliottii E.	I_3d	S₀	0.290 ± 0.024Aa	16.565 ± 2.178Aa	254.718 ± 24.004Ba	0.253 ± 0.044Aa
		S_1/4	0.284 ± 0.013Aa	15.940 ± 2.189ABa	309.622 ± 150.809Aa	0.206 ± 0.032Ba
		S_1/2	0.196 ± 0.014Ba	13.502 ± 1.682Ba	344.865 ± 12.514Aa	0.176 ± 0.016Ba
	I_6d	S₀	0.154 ± 0.024Ab	16.126 ± 1.006Aa	204.584 ± 16.225Ab	0.143 ± 0.008Ab
		S_1/4	0.106 ± 0.006Bb	11.752 ± 0.481Bb	230.868 ± 16.168Bab	0.134 ± 0.018Ab
		S_1/2	0.085 ± 0.007Cb	9.779 ± 0.548Cb	293.237 ± 8.506Ab	0.105 ± 0.001Bb
	I_9d	S₀	0.050 ± 0.007Ac	7.601 ± 0.809Ab	147.743 ± 11.683Bc	0.086 ± 0.008Ac
		S_1/4	0.040 ± 0.006Bc	5.993 ± 0.749Bc	151.196 ± 26.897ABb	0.075 ± 0.013Ac
		S_1/2	0.032 ± 0.006Cc	2.942 ± 0.722Cc	171.777 ± 10.504Ac	0.024 ± 0.012Bc
F value		I	1133.991^**	261.482^**	34.908^**	228.063^**
		S	87.620^**	57.007^**	7.460^**	35.827^**
		I\(\:\times\:\)S	19.851^**	5.893^**	0.912^ns	2.208^ns

Pn-PAR and Pn-CO₂ curves

From Fig. 8 and Table 7, it can be seen that there are differences in the Pn-PAR response curves of two types of coniferous seedlings under different treatments. For P. yunnanensis F. seedlings, the fitting degree of Pn-PAR response curves varies between 0.9136 and 0.9949 under each treatment. The fitting degrees of I_6dS_1/4 and I_6dS_1/2 treatments are the highest, both reaching above 0.99. Under 9 treatments, with the increase of PAR, the Pn of P. yunnanensis F. seedlings showed a trend of first increasing and then stabilizing, with PAR ranging from 0 to 200µmol·m^− 2·s^− 1, the Pn values of the 9 treatments showed a rapid increase trend with the increase of PAR. When PAR > 200µmol·m^− 2·s^− 1, the Pn of P. yunnanensis F. seedlings gradually increased and tended to plateau under each treatment (Fig. 8-A). In addition, under the I_6dS_1/2 treatment, the maximum net photosynthetic rate of P. yunnanensis F. seedlings reached its maximum value (14.951µmol·m^− 2·s^− 1), and under this treatment, the LSP value is the highest (14.951µmol·m^− 2·s^− 1), Rd and LCP have the lowest values of 0.590 and 21.395µmol·m^− 2·s^− 1, respectively, indicates that P. yunnanensis F. seedlings have the strongest photosynthetic ability under this treatment, with a shorter dark response time, and can undergo light response under lower light conditions (Table 7). For P. elliottii E. seedlings, the fitting degree of Pn-PAR response curves under each treatment varied between 0.9027 and 0.9952, and the fitting degree was good. When PAR is between 0 and 400µmol·m^− 2·s^− 1, the Pn value of P. elliottii E. seedlings under 9 treatments showed a sharp increasing trend with the increase of PAR. When PAR > 400µmol·m^− 2·s^− 1, the Pn of P. elliottii E. seedlings gradually increased and tended to stabilize under each treatment (Fig. 8-B). However, under the treatment of I_3dS₀ and I_6dS₀, the LSP of P. elliottii E. seedlings was the highest, with values of 1081.100 and 1019.310µmol·m^− 2·s^− 1, respectively, indicating that the maximum saturated light intensity of P. elliottii E. seedlings in this environment is 1081.100µmol·m^− 2·s^− 1, exceeding this light intensity will inhibit plant photosynthesis, thereby affecting plant growth and development (Table 7). Moreover, under I_3dS₀ treatment, the net photosynthetic rate of P. elliottii E. seedlings reached its maximum (17.771µmol·m^− 2·s^− 1).

Table 7

Effects of different karst habitats and rainfall distribution on the characteristic parameters of Pn-PAR curves in two coniferous seedlings
Varieties of trees	Handle	AQY	R_d	Lp_max	LCP	LSP	R²
P. yunnanensis F.	I_3dS₀	0.021	2.787	3.320	89.608	845.742	0.9701
	I_3dS_1/4	0.029	3.818	4.654	74.306	965.870	0.9860
	I_3dS_1/2	0.030	2.431	5.154	44.125	1000.550	0.9523
	I_6dS₀	0.028	2.366	8.545	59.295	1024.250	0.9877
	I_6dS_1/4	0.033	1.583	11.449	27.266	1253.210	0.9949
	I_6dS_1/2	0.022	0.590	14.951	21.395	1624.530	0.9926
	I_9dS₀	0.016	2.697	2.768	97.060	714.156	0.9613
	I_9dS_1/4	0.017	2.390	2.815	119.431	940.440	0.9136
	I_9dS_1/2	0.032	4.639	1.925	164.313	674.378	0.9517
P. elliottii E.	I_3dS₀	0.045	1.532	17.771	27.148	1081.100	0.9952
	I_3dS_1/4	0.038	4.035	16.059	46.563	911.510	0.9670
	I_3dS_1/2	0.019	2.694	13.007	61.736	882.096	0.9324
	I_6dS₀	0.070	3.577	17.241	36.285	1019.310	0.9632
	I_6dS_1/4	0.039	3.874	12.601	51.209	880.186	0.9027
	I_6dS_1/2	0.039	4.603	10.764	68.021	729.511	0.9391
	I_9dS₀	0.038	6.721	8.290	94.572	696.062	0.9208
	I_9dS_1/4	0.058	7.709	6.172	105.775	586.439	0.9028
	I_9dS_1/2	0.064	10.541	3.770	127.868	578.686	0.9486

According to Fig. 9 and Table 8, there are differences in the effects of different treatments on the Pn-CO₂ response curves of two coniferous seedlings. For P. yunnanensis F., the fitting degree of all 9 treatments is higher than 0.94, with a maximum fitting degree of 0.9876, indicating good fitting effects. Ci between 0 and 400µmol·m^− 1, the Pn values of the 9 treatments showed an accelerated increase trend with the increase of Ci. When Ci > 400µmol·m^− 1, the Pn of P. yunnanensis F. seedlings gradually increased and tended to plateau under various treatments (Fig. 9-A). In addition, the I_6dS_1/2 treatment CSP was significantly higher than I_6dS₀ by 1086.610µmol·m^− 1, while under I₉S_1/2 treatment, CSP and Rp were the lowest, with values of 690.507µmol·m^− 1 and 12.007µmol·m^− 2·s^− 1(Table 8), respectively, indicates that under this treatment, P. yunnanensis F. seedlings have a lower ability to utilize CO₂ and weak photosynthesis. For P. elliottii E. seedlings, the fitting degree of all 9 treatments was higher than 0.9338, with a maximum fitting degree of 0.9874, indicating good fitting effects. Under each treatment, the Pn value of P. elliottii E. seedlings showed a significant trend of first increasing sharply and then decreasing with the increase of Ci (Fig. 9-B). However, under the treatment of I₃S₀ and I₃S_1/4, the P. elliottii E. seedlings showed the best utilization efficiency of CO₂, indicating that the photosynthetic capacity of the treated plants was the best.

Table 8

Effects of different karst habitats and rainfall distribution on the characteristic parameters of Pn-CO₂ curves of two conifer seedlings
Varieties of trees	Handle	P_max	α	R_p	CCP	CSP	R²
P. yunnanensis F.	I_3dS₀	16.565	0.050	3.499	74.120	981.047	0.9784
	I_3dS_1/4	19.470	0.031	1.832	61.101	1439.610	0.9801
	I_3dS_1/2	22.828	0.121	4.637	44.078	1710.270	0.9414
	I_6dS₀	20.980	0.060	2.954	51.877	1084.460	0.9565
	I_6dS_1/4	22.672	0.065	2.589	42.581	1624.970	0.9685
	I_6dS_1/2	25.331	0.089	2.152	25.791	2171.070	0.9876
	I_9dS₀	12.393	0.186	10.730	85.394	723.405	0.9477
	I_9dS_1/4	12.939	0.541	18.505	71.890	1002.800	0.9685
	I_9dS_1/2	8.689	0.212	12.007	100.719	690.557	0.9565
P. elliottii E.	I_3dS₀	27.579	0.073	2.619	37.756	1444.840	0.9856
	I_3dS_1/4	25.590	0.058	3.720	67.581	1203.140	0.9643
	I_3dS_1/2	23.315	0.056	4.376	82.595	1119.800	0.9765
	I_6dS₀	25.619	0.073	4.605	67.312	1123.250	0.9338
	I_6dS_1/4	19.821	0.092	7.787	98.477	989.411	0.9874
	I_6dS_1/2	15.453	0.128	10.010	103.280	885.279	0.9764
	I_9dS₀	12.356	0.138	10.140	104.657	836.126	0.9865
	I_9dS_1/4	10.231	0.120	9.171	109.460	748.843	0.9523
	I_9dS_1/2	7.941	0.201	13.362	122.892	674.649	0.9394

Structural Equation Modeling

The structural equation model (Fig. 10) shows that under different treatments, P. yunnanensis F. G, Pro, and RV all have a direct effect on SOD, with path coefficients of 0.46, 0.78 (P < 0.05), and 0.74 (P < 0.001), respectively. Pro and POD have indirect effects on RB, while SOD, H, and SP all have direct effects on RB, with path coefficients of 0.46 (P < 0.05), 1.51 (P > 0.05), and − 0.92 (P > 0.05), respectively. G and Pro have a direct effect on H, with path coefficients of 0.97 and 0.40, respectively (P < 0.001). LB, RB, and RD all have a direct effect on RV, with path coefficients of 0.68 (P < 0.05), -0.90 (P < 0.01), and 1.18 (P < 0.001), respectively. This fully demonstrates the high correlation between the physiological growth of P. yunnanensis F. seedlings, and the plant's seedling height, ground diameter, biomass, and root characteristics have a significant impact on antioxidant enzymes. For example, as the diameter of the ground increases, the SOD activity of plants will be enhanced, and the increase in root biomass promotes the increase in root volume, which in turn promotes the accumulation of stem and leaf biomass; However, the enhancement of antioxidant activity can in turn promote the accumulation of plant biomass and growth (Fig. 10-A). From Fig. 10-B, it can be seen that there are certain direct or indirect effects between the photosynthetic pigments, fluorescence parameters, and photosynthetic gas exchange parameters of P. yunnanensis F. seedlings. Car, Chlb, Fv, and Fo all have a direct effect on Fm, with path coefficients of 0.40 (P < 0.01), 0.11 (P < 0.01), 0.82 (P < 0.001), and − 0.29 (P < 0.05), respectively. Tr and qP have a direct effect on Chla, with path coefficients of 0.34 (P < 0.01) and 0.40 (P < 0.01), respectively. Pn and Ci have a positive direct effect on GS and Ci has a positive effect on Pn. Although Fm has no direct effect on Pn, it indirectly affects Pn through Ci. These fully demonstrate that the increase in photosynthetic pigment content and fluorescence parameters in P. yunnanensis F. seedlings can indirectly or directly reflect the net photosynthetic rate of the plant, thereby reflecting the photosynthetic capacity of the plant.

However, for P. elliottii E. seedlings, H and SOD have a direct effect on G, with path coefficients of 0.94 (P < 0.01) and 0.09 (P > 0.05), respectively. MDA, Pro, and H all have a negative direct effect on RS, indicating that an increase in MDA, Pro, and H reduces RS. RV and LB have a positive direct effect on RD, while RV has a positive direct effect on SOD and SP has a positive direct effect on LB, with path coefficients of 0.36 (P > 0.05) and 0.39 (P < 0.05), respectively. RB has no direct effect on RD, but it can indirectly affect RD through RV. Therefore, under different treatments, the physiological growth of P. elliottii E. seedlings will adapt to changes in different environments through the adjustment of their physiological indicators, to maintain their growth and development (Fig. 10-C). In addition, from Fig. 10-D, it can be seen that Chla has a direct effect on Fo, Pn, and Chlb, with path coefficients of -1.22 (P < 0.01), 0.31 (P < 0.05), and 0.61 (P < 0.001), respectively. Pn has a direct effect on Chlb, Tr, and Ci, indicating that as the net photosynthetic rate of plants increases, Chlb, Tr, and Ci also increase. Ci can indirectly affect Tr through Gs, and Chla can also indirectly affect Car. These studies can all demonstrate that P. elliottii E. seedlings can directly or indirectly affect the photosynthetic capacity of plants through the conditions of photosynthetic pigment content or fluorescence parameters. The relationship between photosynthetic pigment, fluorescence parameters, and gas exchange parameters of P. elliottii E. seedlings can also be intuitively seen through this graph.

Fuzzy comprehensive analysis

According to fuzzy membership analysis (Table 9). The average membership function values of P. yunnanensis F. seedlings under different karst fissures and rainfall distribution treatments are I_6dS_1/4>I_6dS_1/2>I_3dS_1/4>I_3dS_1/2>I_6dS₀>I_9dS_1/4>I_3dS₀>I_9dS₀>I_9dS_1/2. From this, it can be seen that the optimal treatment group is the I_6dS_1/4 treatment, indicating that under natural rainfall duration, increasing karst cracks can promote the growth of P. yunnanensis F. seedlings. Combined with previous analysis, it can also be seen that the antioxidant system, photosynthetic pigments, root characteristics, and other indicators of P. yunnanensis F. seedlings in this treatment are the best; Secondly, the I_6dS_1/2 treatment indicates that under moderate rocky desertification, P. yunnanensis F. seedlings can also grow and develop normally. However, the average membership function values of P. elliottii E. seedlings under different treatments were I_3dS₀ > I_3dS_1/4>I_6dS₀>I_3dS_1/2>I_6dS_1/4>I_9dS₀>I_6dS_1/2>I_9dS_1/4>I_9dS_1/2. From this, it can be seen that reducing rainfall duration significantly promotes the growth of P. elliottii E. seedlings, and I_3dS₀ treatment has the best effect, followed by I_3dS_1/4. This also indicates that the increase of karst cracks inhibits the growth and development of P. elliottii E. seedlings, which is consistent with previous research results.

From Fig. 11-A, it can be seen that under different karst fissures and rainfall distribution treatments, the physiological growth indicators of P. yunnanensis F. seedlings exhibit different trends, but overall, the I_6dS_1/2 treatment has the best effect, with multiple indicators such as Fv, Fm, Chlb, qP, ETR reaching their maximum values under this treatment. Under each treatment, Fv is the highest, with a value of 3.435. The second best treatment is I_6dS_1/4, which is consistent with the analysis results in Table 9. However, P. elliottii E. seedlings showed different trends of change (Fig. 11-B). Under different treatments, the overall physiological growth indicators of P. elliottii E. seedlings showed the best effect under the I_3dS₀ treatment, followed by the I_3dS₀ treatment. These analyses further validate the fuzzy membership analysis.

Table 9

Membership analysis of physiological growth indicators of two coniferous seedlings under different karst fissures and rainfall distributions
Varieties of trees	Index	I_3dS₀	I_3dS_1/4	I_3dS_1/2	I_6dS₀	I_6dS_1/4	I_6dS_1/2	I_9dS₀	I_9dS_1/4	I_9dS_1/2
P. yunnanensis F.	G	0.4713	0.8061	0.3140	0.5733	1.0000	0.3787	0.5360	0.6607	0.0000
	H	0.4639	0.6186	0.0000	0.3711	1.0000	0.1814	0.4206	0.5794	0.0433
	RB	0.3546	0.7956	0.1864	0.6184	1.0000	0.4385	0.2169	0.2527	0.0000
	SB	0.4428	0.7360	0.2007	0.6322	1.0000	0.4001	0.1424	0.2038	0.0000
	LB	0.4201	0.7783	0.1954	0.5639	1.0000	0.4253	0.1703	0.1815	0.0000
	RL	0.1612	0.4609	0.7192	0.4249	0.7172	1.0000	0.0000	0.1663	0.4357
	RS	0.0968	0.6646	0.7443	0.4183	0.8138	1.0000	0.0000	0.1805	0.5150
	RV	0.0996	0.4586	0.9632	0.3119	0.6857	1.0000	0.0000	0.1235	0.3231
	RD	0.0958	0.3230	0.6650	0.3503	0.8043	1.0000	0.0000	0.1277	0.3003
	RK⁺	0.1438	0.7467	0.4124	0.4460	1.0000	0.7520	0.0000	0.4114	0.2387
	RCa²⁺	0.4671	0.7350	0.5622	0.5476	1.0000	0.6254	0.0000	0.1197	0.0534
	RNa⁺	0.3358	0.5262	0.1602	0.3072	1.0000	0.4239	0.0000	0.3757	0.2637
	RMg²⁺	0.3516	0.9433	0.7295	0.6634	1.0000	0.8375	0.0000	0.5584	0.2358
	SK⁺	0.3296	0.7620	0.3911	0.6399	1.0000	0.7391	0.0000	0.4468	0.2957
	SCa²⁺	0.5705	0.9549	0.7195	0.5334	1.0000	0.8520	0.0000	0.2387	0.0292
	SNa⁺	0.0959	0.5618	0.3752	0.3632	1.0000	0.6466	0.0000	0.4938	0.1821
	SMg²⁺	0.0656	1.0000	0.3614	0.5998	0.9017	0.8708	0.0000	0.4687	0.0625
	YK⁺	0.1399	1.0000	0.3797	0.2213	0.6155	0.4383	0.0000	0.2856	0.0881
	YCa²⁺	0.4663	1.0000	0.6602	0.5996	0.9337	0.7580	0.0000	0.4836	0.1667
	YNa⁺	0.0804	0.8026	0.4614	0.4005	1.0000	0.5296	0.0000	0.6229	0.2065
	YMg²⁺	0.1558	1.0000	0.5938	0.3258	0.6563	0.4618	0.0000	0.4678	0.2112
	SOD	0.5079	0.7826	0.9188	0.8849	0.9732	1.0000	0.2913	0.3264	0.0000
	Pro	0.4657	0.8670	0.9407	0.7494	0.9920	1.0000	0.4286	0.5488	0.0000
	SP	0.2867	0.3156	0.5584	0.3259	0.9417	1.0000	0.1890	0.2265	0.0000
	MDA	0.4994	0.5526	0.6589	0.8469	0.9817	1.0000	0.3980	0.4447	0.0000
	POD	0.2821	0.3192	0.4244	0.5897	0.9590	1.0000	0.2038	0.2231	0.0000
	Chla	0.4738	0.6043	0.7738	0.6025	0.8762	1.0000	0.0000	0.1274	0.2124
	Chlb	0.1007	0.5118	0.7765	0.3692	0.9124	1.0000	0.0000	0.1447	0.3090
	Car	0.1661	0.3279	0.5863	0.3666	0.7871	1.0000	0.0000	0.1465	0.3608
	Fo	0.0014	0.0000	0.0620	0.0118	0.0207	0.0794	0.1528	0.6813	1.0000
	Fm	0.2088	0.3736	0.4586	0.3530	0.6496	1.0000	0.1416	0.0729	0.0000
	Fv/Fm	0.5198	0.6117	0.7289	0.6705	0.8772	1.0000	0.4027	0.2926	0.0000
	FV	0.1506	0.2754	0.3782	0.2821	0.6006	1.0000	0.0925	0.0466	0.0000
	Fv/Fo	0.1805	0.3245	0.3944	0.3240	0.6676	1.0000	0.0886	0.0250	0.0000
	Fv'/Fm'	0.4145	0.5495	0.7542	0.5137	0.7643	1.0000	0.4299	0.3689	0.0000
	qP	0.3033	0.5606	0.7169	0.4946	0.9196	1.0000	0.2834	0.1162	0.0000
	ETR	0.1997	0.4868	0.8385	0.3316	0.5245	1.0000	0.6591	0.0352	0.0000
	NPQ	0.1190	0.4047	0.6754	0.2202	0.7702	1.0000	0.1902	0.1114	0.0000
	Tr	0.1586	0.2578	0.2748	0.6969	0.9564	1.0000	0.0493	0.1073	0.0000
	Pn	0.1321	0.2620	0.3156	0.5165	0.7557	1.0000	0.0993	0.1129	0.0000
	Ci	0.0000	0.4176	0.4420	0.4773	0.9384	1.0000	0.1023	0.3942	0.0727
	Gs	0.0601	0.1478	0.3673	0.8274	0.9173	1.0000	0.0000	0.0808	0.0266
Mean value		0.2628	0.5864	0.5200	0.4849	0.8551	0.8057	0.1354	0.2877	0.1341
Stor		7	3	4	5	1	2	8	6	9
P. elliottii E.	G	0.9552	0.7906	0.3827	1.0000	0.4945	0.3379	0.7051	0.3716	0.0000
	H	1.0000	0.6686	0.4186	0.9574	0.4186	0.2351	0.7106	0.2862	0.0000
	RB	1.0000	0.7357	0.5128	0.7164	0.5450	0.3582	0.4772	0.2512	0.0000
	SB	1.0000	0.6346	0.4092	0.6082	0.4427	0.3260	0.4841	0.2138	0.0000
	LB	1.0000	0.7233	0.5836	0.7242	0.6528	0.4767	0.2655	0.1185	0.0000
	RL	1.0000	0.8594	0.6801	0.7864	0.7047	0.5047	0.6315	0.1335	0.0000
	RS	1.0000	0.8695	0.5527	0.5753	0.2549	0.1384	0.2716	0.1069	0.0000
	RV	1.0000	0.7634	0.3871	0.3880	0.1700	0.0738	0.0978	0.0454	0.0000
	RD	1.0000	0.7206	0.3313	0.4198	0.1175	0.0897	0.1916	0.0698	0.0000
	RK⁺	1.0000	0.6820	0.5394	0.7217	0.4157	0.2334	0.5256	0.1543	0.0000
	RCa²⁺	0.6538	1.0000	0.5101	0.4174	0.8003	0.2563	0.1601	0.6226	0.0000
	RNa⁺	1.0000	0.8495	0.6789	0.5872	0.3796	0.0483	0.5184	0.1127	0.0000
	RMg²⁺	1.0000	0.5805	0.4895	0.6401	0.3524	0.2654	0.5209	0.1680	0.0000
	SK⁺	1.0000	0.6975	0.2781	0.6988	0.3590	0.1555	0.9346	0.2348	0.0000
	SCa²⁺	0.6871	1.0000	0.2410	0.4322	0.7701	0.1755	0.1656	0.6299	0.0000
	SNa⁺	1.0000	0.8438	0.6207	0.3685	0.1738	0.0201	0.2114	0.1059	0.0000
	SMg²⁺	1.0000	0.6230	0.4250	0.7256	0.2674	0.0854	0.6285	0.1328	0.0000
	YK⁺	1.0000	0.7676	0.3661	0.7492	0.4138	0.0393	0.8925	0.1116	0.0000
	YCa²⁺	0.6935	1.0000	0.5055	0.5071	0.7764	0.0000	0.3880	0.7762	0.3430
	YNa⁺	1.0000	0.6870	0.4446	0.9160	0.2665	0.1495	0.4784	0.2897	0.0000
	YMg²⁺	1.0000	0.6710	0.4543	0.7831	0.3023	0.0000	0.6730	0.0832	0.0082
	SOD	1.0000	0.9733	0.5384	0.6735	0.6192	0.3276	0.2256	0.1016	0.0000
	Pro	1.0000	0.9489	0.7101	0.8102	0.6164	0.3719	0.5475	0.2114	0.0000
	SP	1.0000	0.5702	0.3568	0.6926	0.3784	0.1552	0.0850	0.0769	0.0000
	MDA	1.0000	0.8641	0.7056	0.8679	0.8616	0.6458	0.3457	0.1847	0.0000
	POD	1.0000	0.9880	0.8149	0.6192	0.3635	0.1957	0.2517	0.0879	0.0000
	Chla	0.8056	1.0000	0.5371	0.4043	0.5603	0.3355	0.0182	0.0584	0.0000
	Chlb	0.7094	1.0000	0.6043	0.4083	0.7140	0.1301	0.1471	0.0750	0.0000
	Car	1.0000	0.8263	0.6404	0.4690	0.3406	0.1890	0.0771	0.0163	0.0000
	Fo	0.0000	0.0985	0.3336	0.2087	0.3005	0.5182	0.4227	0.5149	1.0000
	Fm	1.0000	0.7350	0.6248	0.7789	0.5575	0.3500	0.3966	0.1972	0.0000
	Fv/Fm	1.0000	0.8277	0.6209	0.6696	0.4471	0.3120	0.2115	0.1435	0.0000
	FV	1.0000	0.7122	0.5031	0.5998	0.3519	0.2096	0.1538	0.0865	0.0000
	Fv/Fo	1.0000	0.5164	0.2170	0.3304	0.1644	0.0706	0.0618	0.0324	0.0000
	Fv'/Fm'	1.0000	0.8117	0.5933	0.6300	0.4257	0.3113	0.0701	0.0695	0.0000
	qP	1.0000	0.6559	0.2023	0.5999	0.3971	0.0963	0.3509	0.0869	0.0000
	ETR	1.0000	0.9659	0.8618	0.9055	0.7627	0.5791	0.5615	0.0582	0.0000
	NPQ	1.0000	0.7795	0.5001	0.3798	0.2877	0.2513	0.2703	0.2520	0.0000
	Tr	1.0000	0.9670	0.6411	0.4783	0.2923	0.2139	0.0812	0.0417	0.0000
	Pn	0.9678	0.9542	0.7752	1.0000	0.6467	0.5018	0.3420	0.2240	0.0000
	Ci	1.0000	0.8212	0.5427	0.7381	0.4217	0.2884	0.1219	0.0175	0.0000
	Gs	1.0000	0.7961	0.6635	0.5184	0.4819	0.3536	0.2724	0.2208	0.0000
Mean value		0.9398	0.7852	0.5190	0.6311	0.4540	0.2471	0.3559	0.1851	0.0322
Stor		1	2	4	3	5	7	6	8	9

Growth and root characteristics

The ecosystem in KRD areas is unstable, prone to degradation, nutrient loss, and decreased water-holding capacity, ultimately leading to soil erosion. Due to human activities, it is difficult for karst ecosystems to recover from desertification. Therefore, karst is becoming increasingly common in southwestern China (Peng et al., 2013;Tang et al., 2013). The changes in resource availability have significant differences in the allocation and coordination processes among plant organs (Poorter et al., 2015). The accumulation of aboveground biomass is mainly driven by the capture of light energy by plants, thereby driving the adjustment of aboveground biomass (Poorter et al., 2015༛ Freschet et al., 2015). In contrast, the transformation of the underground section is mainly driven by nutrients and water (Freschet et al., 2018). Moreover, biomass is an important indicator of plant energy accumulation, and the distribution differences of biomass in various organs can reflect the growth strategy of plants (Westoby et al., 2002). In this study, with the deepening of karst cracks, the seedling height, ground diameter, and biomass of various organs of P. yunnanensis F. seedlings showed a significant trend of first increasing and then decreasing(P < 0.05). Properly increasing karst cracks promoted the growth of P. yunnanensis F. seedlings, and P. yunnanensis F. seedlings did not have a good effect on mild rocky desertification in the whole soil habitat. This is similar to the research findings of Zong and Shi (2020). This may be because under full soil treatment, although plants can obtain the most nutrients and living space, the permeability of karst soil is poor, and the soil in this habitat is prone to compaction, resulting in certain deep water stress (Zhang et al., 2019); The soil permeability in habitats with fewer rocks and more soil allows plants to have better contact with oxygen, which is beneficial for plant growth. However, P. elliottii E. seedlings showed a significant downward trend with the deepening of karst cracks, and increasing karst cracks significantly inhibited the growth of P. elliottii E. seedlings, consistent with Wu et al., (2008) study on the effect of drought stress on Sophora davidii K.seedlings. This may be because increasing karst fissures improves soil permeability, but it also leads to rapid soil water loss, causing drought problems. In addition, as P. elliottii E. is an exotic tree species, its physiological mechanisms have not yet adapted to changes in environmental conditions. Therefore, under the whole soil treatment, P. elliottii E. has the best biomass accumulation.

However, plants adopt different survival strategies to adapt to environmental changes during their growth and evolution, and the allocation of biomass to each organ is one of the most basic survival strategies. Plants prioritize the allocation of biomass to organs with limited resources by changing the proportion of biomass allocated between roots, stems, and leaves, to enhance their competition for nutrients, water, and light resources and maximize their growth rate (Poorter et al., 2012). Environmental change is an important factor affecting plant biomass allocation and plays a dominant role in root and leaf biomass allocation (Xie et al., 2012). This study found that under different karst fissures and rainfall treatments, the biomass of various organs in P. yunnanensis F. and P. elliottii E. seedlings showed that leaves > stems > roots, and plants preferentially allocated nutrients to leaf organs, which is inconsistent with the research results of Peng et al., (2023) and Zheng et al., (2024), they found that plants preferentially allocated nutrients to the underground part, but consistent with Li and Kang (2002) study. This is likely related to our irrigation and karst fissures. The leaching phenomenon occurs during watering, which can squeeze out the air and cause poor root respiration, thereby affecting the growth and development of the roots. Secondly, the root system of plants usually supplies nutrients to the stems and leaves, while the roots do not have much energy supply and insufficient energy reserves from the stems and leaves, so the accumulation of root biomass is not as high as that of the stems and leaves. Finally, leaves are the organs for plants to absorb light resources for photosynthesis. The allocation of higher leaf biomass can increase the capture of light resources by two types of coniferous seedlings, improve their photosynthesis, produce more photosynthetic products, and thus transport material capacity through the stem to the roots, enabling normal root growth.

Root characteristics (root length, root diameter, root volume, root surface area, specific root length, etc.) are comprehensive indicators reflecting the absorption, synthesis, oxidation, and reduction abilities of roots, as well as the growth status of plants (Hongying 2001). The root system structure can effectively reduce the detachment rate of soil and the erodibility of surface soil saturated with root infiltration in karst mountainous areas (De Baets et al., 2006). In this study, the root characteristics of P. yunnanensis F. seedlings increased in thickness with the deepening of karst cracks, and prolonged rainfall duration (I_9d) inhibited root growth, which is similar to previous research results (Urbanavičiūtė et al., 2022; Jangpromma et al., 2012). Our research indicates that by increasing the thickness of karst cracks, the root activity of P. yunnanensis F. seedlings is higher, which also indicates the adaptability of P. yunnanensis F. seedlings to karst habitat stress. However, when prolonging the duration of rainfall, the root activity of most seedlings will gradually lose vitality, thereby inhibiting root growth and ultimately leading to death. In this study, we can conclude that by increasing karst cracks, P. yunnanensis F. seedlings maintained high root activity, which is also the reason why P. yunnanensis F. seedlings can survive in KRD areas. However, the root growth of P. elliottii E. seedlings decreases with the increase of karst cracks. This may be determined by the physiological characteristics of P. elliottii E. seedlings themselves, which increase karst cracks, cause rapid soil water loss, and result in less water absorption by the roots, which is not conducive to the development of the roots. The results of this study also indicate that reducing rainfall duration (3d) has a promoting effect on the growth of the root structure of P. elliottii E. seedlings, which fully indicates that P. elliottii E. seedlings have a high water demand.

Physiological growth and antioxidant system

K⁺, Ca²⁺, Na⁺, and Mg²⁺ are essential elements in plants and play important roles in plant growth and development. Ca²⁺ can act as a second messenger to participate in various physiological processes in plants (Gilroy et al., 2016). K⁺ and Mg²⁺ have important effects on the transportation of photosynthetic products between source-sink, and changes in the distribution of photosynthetic products are the main reason for differences in plant dry matter distribution under water stress conditions (Li et al., 2018). Studies have shown that K⁺ and Mg²⁺ deficiencies significantly inhibit root biomass accumulation (Xue et al., 2020). This study also found that P. yunnanensis F. seedlings and P. elliottii E. seedlings had the lowest K⁺ and Mg²⁺ content and less root biomass accumulation in karst habitats of S_1/2 with a rainfall duration of 9 days. The main reason for this phenomenon may be related to karst stress and longer rainfall intervals. The environment where plants with large karst cracks are located has less water, and the plants are affected by water deficiency, which leads to the loss of K⁺ and thus affects the accumulation of plant root biomass. In addition, the mineral element content of the two conifer seedlings showed that Ca²⁺>Mg²⁺>K⁺>Na⁺, with the highest Ca²⁺ content. This is likely related to KRD habitats, where there is more highly weathered carbonate bedrock exposed to the surface, and carbonates are already rich in high Ca²⁺ (Jiang et al., 2014). Moreover, the accumulation of element content in various organs of P. yunnanensis F. showed that leaves > roots > stems, while P. elliottii E. showed that roots > stems > leaves.The nutrient accumulation of P. elliottii E. mainly tends to be in the roots, indicating that to grow in rocky desertification areas, P. elliottii E. distributes more nutrients to the roots, thereby ensuring their growth and development. From the root characteristics, it can also be seen that the root length, root volume, and root surface area of P. elliottii E. seedlings grow better than those of P. yunnanensis F. Na⁺ is harmful to most plants, as excessive absorption can cause osmotic stress and inhibit K⁺ uptake (Wang et al., 2012), reduce enzyme activity, and disrupt cellular metabolic processes (Flowers et al., 2015). K⁺ is involved in the control of cell turgor pressure, photosynthetic metabolism, and enzyme activation. The decrease in its content can lead to a slowdown in plant growth (Kanawapee et al., 2012). The K⁺/Na⁺ ratio is an important indicator of plant salt tolerance, and maintaining an appropriate K⁺/Na⁺ ratio is a necessary prerequisite for plants to ensure normal stomatal function and some physiological metabolism (Shabala and Cuin 2007). A higher K⁺/Na⁺ ratio usually indicates stronger salt tolerance and salt-tolerant plants often absorb sufficient K⁺ content to cope with salt damage (Maathuis et al., 1999). In this study, both coniferous seedlings had the highest K⁺/Na⁺ concentration in the leaves, indicating that the leaves have a strong selective absorption ability for K⁺. Under karst stress, more K⁺ is transported to the leaves, which is beneficial for reducing osmotic stress in the leaves (Guo et al., 2016).

Meanwhile, plants accumulate beneficial osmoregulatory substances such as proline, soluble proteins, and soluble sugars in their cells when facing adversity stress, to maintain the osmotic equilibrium within the cells and reduce the damage of stress to plants (Kumari et al., 2015). In addition, the accumulation of a large amount of reactive oxygen species in the plant body can lead to membrane lipid peroxidation, production of MDA, disruption of membrane system integrity and cellular structure, and damage to chloroplast and thylakoid structures, resulting in the degradation of photosynthetic pigments and affecting plant photosynthesis (Sun et al., 2010). Antioxidant enzyme activity is a major component of plant antioxidant defense systems (Azooz et al., 2010). SOD is toxic to H2O2, which must be converted into O2 and H2O through antioxidant enzymes (e.g., POD), and SOD and POD belong to enzymatic clearance mechanisms (Guo et al., 2012; You et al., 2015). However, when plants are subjected to mitigating stress, a large amount of soluble proteins are synthesized to reduce the osmotic potential of plant cells and protect the plant from environmental toxicity (Bonifacio et al., 2016). Moreover, the activity of antioxidant enzymes is related to the plant's ability to resist adverse environments (Yan et al., 2016). In this study, the content of SOD, POD, and SP in P. yunnanensis F. seedlings gradually increased with the increase of karst cracks under the rainfall duration of 3 and 6 days. The extension of rainfall interval (9 days) first increased and then decreased, which is consistent with previous research results (Yin et al., 2016; Nunkaew et al., 2014). This may be because P. yunnanensis F. seedlings actively regulate their protective enzyme activity to eliminate the damage caused by rocky desertification and karst cracks. Secondly, P. yunnanensis F. seedlings reduce their cell osmotic potential by increasing SP content, thereby promoting normal plant growth. Mild rocky desertification (S_1/4) alleviated the damage of P. yunnanensis F. seedlings to karst cracks under 9 days of rainfall, which is consistent with previous results on nitrogen fertilizer alleviating stress-induced membrane damage (Wang et al., 2022). On the contrary, the POD, SOD, and SP of P. elliottii E. seedlings gradually decrease with the increase of karst cracks. P. elliottii E. seedlings did not increase the activity of antioxidant enzymes to alleviate the damage caused by stress under this environmental stress, which was different from the previous studies (Yin et al., 2016; Nunkaew et al., 2014). This situation is likely due to the inability of P. elliottii E. seedlings to adapt to environmental changes through antioxidant enzymes due to their genetic factors under karst and long-term rainfall stress. Secondly, insufficient water leads to redox imbalance within plant leaf cells, thereby increasing the likelihood of oxidative damage. Moreover, the increase of karst fissures accelerates soil moisture loss, resulting in low soil moisture content. Therefore, the antioxidant enzymes of P. elliottii E. in karst fissure habitats are lower than those in whole soil habitats. Finally, it is also possible that karst stress inhibits the transcription or genetic mutations of genes related to P. elliottii E. seedlings, thereby affecting the incomplete expression of antioxidant enzyme activity (Limaye et al., 2003). Of course, this reason still needs further verification from us. In addition, this study also found that the MDA and Pro levels of two coniferous seedlings were significantly higher under the I9dS1/2 treatment than other treatments (Fig. 5), indicating that prolonged rainfall duration and S1/2 karst habitat led to an increase in MDA content in the bodies of the two coniferous seedlings. Karst fissure stress has already caused damage to the membrane system, exacerbating membrane lipid peroxidation, which is consistent with the results of Li et al., (2020). In summary, an appropriate increase of karst fissures could increase the antioxidant enzyme activities of P. yunnanensis F. seedlings, thereby reducing the content of MDA and Pro; The P. elliottii E. seedlings have the highest antioxidant enzyme activity in the whole soil habitat.

Photosynthetic pigments

Karst stress affects the absorption and utilization of water and nutrients by plants (Leung et al., 1995), which in turn affects the generation of photosynthetic chemical energy ATP and NADPH, leading to a decrease in the photosynthetic capacity of plants in the region and ultimately affecting their growth and development. The key factors affecting the normal growth of plants in karst areas are not limited to factors such as light and CO₂ concentration but are often influenced by water conditions, nutrient composition, soil acidity, and alkalinity (Nie et al., 2014). Chlorophyll a and chlorophyll b are both sensitive to the decrease in soil water potential. Chlorophyll is one of the most important components of plant photosynthesis, and its content significantly decreases under water scarcity conditions (Xiao et al., 2008). Under adverse conditions, increasing chlorophyll enzyme activity can alter the chlorophyll structure and lipoprotein complexes in the membrane, and reduce the inactivation of PSII-related photopigment proteins and Calvin cycle enzymes (Ahmadizadeh 2013). Carotenoids play an important role in photosynthesis and drought resistance (Jaleel et al., 2009). They are important molecules involved in photoprotection during photosynthesis, as well as antioxidants and single oxygen scavengers, preventing lipid peroxidation (Xiao et al., 2008). In this study, the photosynthetic pigment content (Chla, Chlb, Car) of P. elliottii E. seedlings showed a trend of first increasing and then decreasing with the increase of karst habitat, and gradually decreasing with the extension of rainfall time, which is consistent with the research results of Ahmadizadeh (2013). It is possible that low-frequency heavy rainfall (9d) in a semi-soil and semi-stone habitat can lead to leaching, causing nutrient loss in the soil with the loss of rainfall. It is also possible that the increase of karst cracks causes plants to experience certain droughts, leading to excessively high temperatures and inhibiting the formation of chlorophyll esters in the plant body, resulting in a decrease in chlorophyll formation. The content of photosynthetic pigments in P. yunnanensis F. seedlings gradually increases with the increase of karst cracks (except for 9 days of rainfall).

Fluorescence and gas exchange parameters

Chlorophyll fluorescence parameters are a set of variables or constants used to describe the photosynthetic physiological status and mechanism of plants, reflecting that the greater the electron transfer activity of plant PSII, the greater the light energy utilization efficiency. To a certain extent, ETR is positively correlated with the photochemical efficiency of Photosystem II, and Fv/Fm reflects the potential maximum photosynthetic capacity of plants, which is the most obvious feature of plant photoinhibition. It can provide a relative measure of the maximum quantum yield of primary photochemistry in Photosystem II, and the Fv/Fm value of non-stressed leaves is about 0.83; qP is a decrease in fluorescence caused by photochemical reactions (Cai et al., 2018; Masacci et al., 2008). The level of Fo is related to the state of the reaction center. An increase in Fo indicates severe damage or inactivation of the PSII reaction center, while a decrease indicates increased heat dissipation in the plant body, which is a photoprotective mechanism (Xu et al., 1999). Fm reflects the electron-transport ability of PSII, while a decrease in Fm indicates a decrease in the conversion of light energy to chemical energy (Goltsev et al., 2016). Studies have shown that different environmental stresses have varying effects on the Fo, Fm, and Fv/Fm of bryophyte. As the density increases, the Fo and Fm of Sphagnum palustre L. significantly increase, while Fv/Fm first decreases and then tends to stabilize (Shi et al., 2021). Low-temperature stress can cause a decrease in Fo, Fm, and Fv/Fm of Symtrichia caninervis M. (Zhang et al., 2011). Drought stress can cause an increase in Fo and a decrease in Fm and Fv/Fm in red tooth moss, Barbula unguiculata H., and Bryum argenteum H. (Cong et al., 2021). In this study, with the thickening of karst cracks, the Fm, Fv, Fo/Fv, Fv/Fm, Fv'/Fm', qP, ETR, and NPQ of P. yunnanensis F. seedlings gradually increased, while Fo gradually decreased. Under half-soil and half-stone habitats with extended rainfall duration, these indicators all decreased. The fluorescence indicators of P. elliottii E. seedlings gradually decrease and Fo gradually increases with the thickness of karst, which is not completely consistent with previous research. The decrease in fluorescence parameters indicates that the deterioration of rocky desertification habitats and the extension of rainfall intervals can affect the light-harvesting protein complexes and activity of enzymes, leading to an impact on the activity of PSII reaction centers, inhibiting the photosynthetic pigments from converting the captured light energy into chemical energy (Zhang et al., 2014). The decrease in Fo indicates that the PSII reaction center has not been completely deactivated, and more energy is dissipated in the form of heat (Masacci et al., 2008). NPQ is the excess light energy dissipated in thermal form by the PSII reaction center (Brestic et al., 2016; Baker et al., 2008). The qP and NPQ of P. yunnanensis F. increase with the deepening of rocky desertification, while those of P. elliottii E. gradually decrease. This indicates that in habitats with lower levels of rocky desertification, the electron transfer activity of P. elliottii E. is higher, but with the reverse succession of rocky desertification ecosystems, the habitat further deteriorates. The opening ratio of PSII reaction centers and the energy involved in photochemical reactions will be suppressed, causing plants to dissipate excess light energy in the form of heat, reducing damage to photosynthetic structures. In addition, beyond increasing heat dissipation and other regulatory mechanisms, plants can also consume excess light energy through processes such as photorespiration and nitrogen metabolism, avoiding burns to photosynthetic organs.

However, photosynthesis is the foundation of plant growth and development, and the light response curve reveals the corresponding relationship between net photosynthetic rate and photosynthetic effective radiation. The light response parameters reflected by it are important indicators for evaluating photosynthesis. The light saturation point reflects the plant's ability to utilize strong light, while a high value reflects the plant's growth and development are less likely to be inhibited in a strong light environment. The LCP and AQY reflect the plant's ability to utilize weak light (Wang et al., 2006). Stomatal conductance (Gs) also plays an important role in photosynthesis by absorbing CO₂ and controlling water loss (Lammertsma et al., 2011). Plant species with higher Gs are expected to have higher maximum CO₂ assimilation (Messinger et al., 2006). This study found that P. yunnanensis F. seedlings generally exhibited higher Gs and transpiration rate (Tr) compared to P. elliottii E. (Table 6). According to other studies, the higher the maximum photosynthetic rate of plants, the higher the Gs value (Marenco et al., 2001), and the results of this study also indicate this pattern (Table 6).

In this study, P. yunnanensis F. seedlings showed lower LCP and higher AQY under I_6dS_1/4 treatment (Table 7), which is consistent with previous research results (Jiang et al., 2002). In addition, the LPnmax of P. elliottii E. seedlings showed a trend of first increasing and then decreasing with the increase of karst fissure thickness. The semi-soil and semi-stone habitat inhibited the photosynthesis of P. elliottii E. seedlings, while P. yunnanensis F. promoted it. This may be due to factors such as increased karst fissures and infiltration, leading to a water deficit in the soil niche. In this situation, the water transported to the leaves is also insufficient, causing stomatal closure and a decrease in CO₂ entering the leaves, both of which are raw materials for photosynthesis. When the leaves are dehydrated, starch hydrolysis is enhanced (Li et al., 2022), sugars continue to accumulate, and the output of photosynthetic products becomes slow, inhibiting the rate of photosynthesis. At the same time, due to water deficiency in the leaves, the electron transfer rate in chloroplasts decreases, coupled with the uncoupled photosynthetic phosphate, which affects the formation of the assimilation force (Ramírez et al., 2016). In addition, water deficiency can also affect the growth of leaves, inhibit the expansion of photosynthetic area, and reduce the absorption of light energy by leaves, all of which can lead to a decrease in photosynthetic rate. However, due to factors such as thin soil, mineral nutrients are deficient in the soil niche. For example, when the oxygen content in the soil is insufficient, it can hinder aerobic respiration in the roots, and the roots are one of the mechanisms for absorbing mineral elements, i.e., active uptake is carried out through the energy provided by the metabolism of root cells. The aerobic respiration of roots is inhibited, and the function of absorbing mineral elements is also inhibited. The lack of mineral elements can have an impact on plant photosynthesis in many aspects. Figure 4 also shows a decrease in mineral element content by increasing karst thickness. When lacking certain mineral elements, chlorophyll synthesis is hindered, and the collection, transmission, and conversion of light energy by plants are inhibited, resulting in a decrease in photosynthetic capacity. If severe hypoxia occurs, toxic substances such as alcohol produced by anaerobic respiration in roots can also lead to root rot, preventing it from performing its function of absorbing and transporting water and nutrients. Therefore, hindering the activity of the root system will indirectly hurt photosynthesis, so that, increasing the thickness of the karst will reduce the photosynthetic rate of P. elliottii E. seedlings. The LCP, Rd, and AQY can all reflect the plant's ability to utilize weak light. In this study, except for the S1/4 and S1/2 P. elliottii E. seedlings under I9d rainfall, the AQY of two coniferous seedlings in the other treatments was relatively low, lower than the general plant level of 0.03 ≤ AQY ≤ 0.05 (Chen et al., 2008), indicating that the light energy utilization efficiency of two coniferous seedlings is relatively low under weak light. The CO₂ response curve reflects the response characteristics of leaf photosynthetic capacity to different CO₂ concentration changes. The higher the CSP of plants, the stronger their adaptability to high CO₂ concentration environments, while plants with low CCP have the characteristics of high Pn and fast growth (Lai et al., 2020). The P. yunnanensis F. seedlings treated with I_6dS_1/2 and the P. elliottii E. seedlings treated with I_3dS₀ have larger CSP, lower CCP, and the lowest photorespiration, indicating that this treatment enhances the CO₂ utilization ability of P. yunnanensis F. and P. elliottii E. needles.

In summary, the physiological growth and photosynthetic characteristics of two types of coniferous seedlings in different soil niches are affected by the interval time of rainfall, and the photosynthetic physiological growth during the interval time of rainfall is also constrained by the soil niche. Under the same rainfall duration, with the increase of karst thickness, the growth of seedling height and ground diameter, the biomass of various organs, K⁺, Ca²⁺, Na⁺, and Mg²⁺accumulation of P. yunnanensis F. seedlings showed a significant change pattern of first increasing and then decreasing (P < 0.05), while P. elliottii E. seedlings showed a gradually decreasing trend (except for Ca²⁺). The biomass of each organ in two types of coniferous seedlings showed leaf > stem > root, while K⁺, Ca²⁺, and Mg²⁺ in each organ of P. yunnanensis F. seedlings showed leaf > root > stem, and Na⁺ showed root > leaf > stem. The accumulation of mineral elements in various organs of P. elliottii E. seedlings is as follows: roots > stems > leaves, and the accumulation of mineral elements in two types of coniferous seedlings is as follows: Ca²⁺>Mg²⁺>K⁺>Na⁺. The root characteristics (root length, root volume, root surface area, root diameter), SOD, POD, SP, photosynthetic pigment content, fluorescence parameters, and gas exchange parameters of P. yunnanensis F. seedlings gradually increase with the increase of karst thickness (except for 9 days of rainfall), indicating that a certain karst thickness can improve the activity of antioxidant enzymes and plant photosynthesis in P. yunnanensis F. seedlings. The root characteristics, antioxidant enzyme activity, and photosynthetic indicators of P. elliottii E. seedlings gradually decrease, indicating that increasing karst thickness inhibits the formation of photosynthesis and antioxidant enzyme activity in P. elliottii E. seedlings.

Under different treatments, our study showed that the maximum saturated light intensity and minimum light intensity of P. yunnanensis F. seedlings were 1624.530 and 21.395µmol·m^− 2·s^− 1, respectively, while the values for P. elliottii E. seedlings were 1081.100 and 27.148µmol·m^− 2·s^− 1, respectively. The CO₂ saturation points of P. yunnanensis F. and P. elliottii E. are 2171.070 and 1444.840µmol·m^− 1, respectively. Fuzzy membership analysis showed that under different treatments, P. yunnanensis F. seedlings showed that I_6dS_1/4>I_6dS_1/2>I_3dS_1/4>I_3dS_1/2>I_6dS₀>I_9dS_1/4>I_3dS₀>I_9dS₀>I_9dS_1/2. The P. elliottii E. seedlings showed that I_3dS₀ > I_3dS_1/4>I_6dS₀>I_3dS_1/2>I_6dS_1/4>I_9dS₀>I_6dS_1/2>I_9dS_1/4>I_9dS_1/2. In conclusion, prolonging rainfall duration has an inhibitory effect on the growth of two types of coniferous seedlings. Reducing rainfall duration promotes the growth and development of P. elliottii E. seedlings while increasing karst thickness promotes the growth and development of P. yunnanensis F. seedlings, which is better than P. elliottii E. seedlings. However, it has an inhibitory effect on the growth of P. elliottii E. seedlings. Therefore, P. yunnanensis F. is preferred as a tree species for vegetation restoration or rocky desertification governance in karst areas at different degrees of desertification.

Declaration of competing interest

The authors report no declarations of interest.

Funding

This research was supported by the National Natural Science Foundation of China Project (31260191), the Yunnan Provincial Department of Education Scientific Research Fund Project (2021J0166), and the Yunnan Provincial “Three Districts” Science and Technology Talent Support Program Fund (990023236).

Author Contribution

Shaojie Zheng designed this work and wrote this manuscript. Lin Wang participated in the experiment and organized the charts. Qiong Dong provided critical revisions and final approval of the article. Huiping Zeng, Xingze Li, Lian Li, Qian Hua, Yutong Wu, Jiumei Yang, and Fuying Yang participated in the experiment and data processing. All authors contributed to the final version of the manuscript.

Acknowledgments

We are very grateful to Southwest Mountain Forest Resources Conservation and Utilization of the Ministry of Education, Kunming, China for providing me with an experimental platform. Thank you to Professor Dong for providing guidance on this article.

Data availability

Data will be made available on request.

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Photosynthetic characteristics and antioxidant protective substances of two coniferous plants in karst fissures and under rainfall distribution

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Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Research Site and Planting Materials

Experimental Design

Sample determination and methods

Plant sampling

Growth rate

Root morphology characteristics

Physiological indicators

Photosynthetic pigments and fluorescence parameters

Photosynthetic gas exchange and response curve

Statistical analysis

Results

Growth and biomass

Growth rate

Biomass

Root system characteristics

K+、Ca2+、Na+、Mg2+

Antioxidant system

Photosynthetic pigments and fluorescence parameters

Photosynthetic pigment content

Chlorophyll fluorescence parameters

Photosynthetic characteristics

Gas exchange parameters

Pn-PAR and Pn-CO2 curves

Structural Equation Modeling

Fuzzy comprehensive analysis

Discussion

Growth and root characteristics

Physiological growth and antioxidant system

Photosynthetic pigments

Fluorescence and gas exchange parameters

Conclusion

Declarations

Declaration of competing interest

Funding

Author Contribution

Acknowledgments

Data availability

References

Additional Declarations

Status:

Journal Publication

Version 1

K⁺、Ca²⁺、Na⁺、Mg²⁺

Pn-PAR and Pn-CO₂ curves