Physiological response mechanism of Machilus faberi Hemsl under drought stress and rewatering

Plant materials and treatment

In May 2022, one-year-old seedlings of M. faberi with basically the same growth vigor were selected from the Germplasm Resources Garden of Hunan Botanical Garden Conservation Institute. The M. faberi seedlings was planted in a flowerpot (25 cm × 30 cm), using a cultivation substrate composed of garden soil, peat, perlite, and cow dung at a ratio of 8:6:3:1. The experimental site was located in the greenhouse of the flower base. In the greenhouse of the flower seedling base, all M. faberi seedlings were uniformly managed. The temperature was maintained at 25 °C during the day and 20 °C at night, with a relative humidity of 65% (temperature and humidity were measured using temperature and humidity sensors (DHT22, Shenzhen, China)). The light conditions were set to a 14-hour light/10-hour dark cycle with a light intensity of 500 µmol m⁻² s⁻¹.

After one month of pre-culture, different drought treatments were carried out using pot water control method (Wang et al., 2024). Water control treatments were implemented using a gravimetric method, wherein soil moisture content was monitored and maintained by regularly weighing of soil samples. The soil moisture content (%) was calculated as: $\text{Soil moisture content}=\frac{\text{Wet Weight}-\text{Dry Weight}}{\text{Dry Weight}}\times 100\text{\%}$. This calculation reflects the proportion of water in the soil relative to its dry weight. Four distinct soil moisture regimes were established to simulate different drought intensities: control (CK, soil moisture content 80%, representing well-watered conditions), mild drought (LD, soil moisture content 50%–60%), moderate drought (MD, soil moisture content 40%–50%), and severe drought (SD, soil moisture content 20%–30%). Soil moisture levels were adjusted and maintained daily by adding the required volume of water to compensate for losses due to evapotranspiration, ensuring consistent treatment conditions throughout the experimental period. Each replicate consisted of five pots, and the entire experiment was repeated three times for consistency. After the drought treatment, the plants were watered daily for seven days and this process was repeated three times to simulate the recovery phase. Rewatering was applied with a controlled amount of water to ensure consistent soil moisture. Data were collected on days 7, 14, and 21 after rewatering (WT7, WT14, and WT21). For each sampling date, leaf samples were taken from different plants to avoid potential confounding effects of leaf removal on the measured parameters.

The entire experiment comprised two phases: drought stress treatment (T0, T7, T14, T28, T35, T42, T49 and T56) and rewatering (WT7, WT14 and WT21). At 10:00 a.m. on the corresponding day of each treatment group, five mature leaves were collected from each plant and immediately transported to the laboratory for subsequent analysis, including the determination of chlorophyll content, malondialdehyde (MDA) concentration and antioxidant enzyme activity.

Morphological data measurement

The morphological characteristics measured included plant height, shoot growth length and shoot number. Plant height was measured using a tape measure from the soil surface of the potted plant to the top of the main stem. The shoots refer to the new growth emerging from the main stem after pruning. The length of all new shoots on each branch was measured and the average length was used as a replicate.

Chlorophyll content measurement

Mature leaves were collected at 10:00 a.m. on sunny days from the top of the plant and washed with deionized water. The samples were dried and sectioned on transparent paper. Then, 0.2 g of fresh leaf samples were weighed and soaked in 10 ml 95% ethanol for 24 h under dark conditions. Absorbance at 470 nm, 649 nm, and 665 nm was determined using an ultraviolet spectrophotometer (UNICO, Shanghai, China) (Dai et al., 2023). This process was repeated during drought stress treatment (T0, T7, T14, T21, T35, T42, T49 and T56) and rewatering (WT7, WT14 and WT21) to monitor changes in chlorophyll content.

Determination of photosynthetic parameters

Photosynthetic parameters were measured using a portable photosynthesis system (LI-6400, LI-COR Biosciences, Lincoln, NE, USA) under controlled conditions. Measurements were conducted between 9:00 and 11:30 a.m. on sunny days to minimize diurnal fluctuations in photosynthetic activity. Five mature leaves were selected randomly from each treatment group, ensuring uniformity in developmental stage and exposure. The maximum net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO₂ concentration (Ci) were recorded at a controlled light intensity of 500 µmol m⁻² s⁻¹, which is commonly used in such studies to represent moderate light conditions. This light intensity avoids potential light saturation or photoinhibition effects and ensure an accurate assessment of photosynthetic response. The assays were taken from a six cm² area of a leaf from a one-year-old M. faberi seedling, and the leaves were allowed to stabilize under the set conditions for approximately 2 min before data recording. Each measurement was repeated three times to ensure consistency and reliability, following standard protocols.

Determination of relative conductivity and MDA

The plant leaves of the same size were selected and washed three times with distilled water. The surface water was removed using filter paper. The leaves were then cut into strips of suitable length (avoiding the main vein), and 0.1 g fresh samples were weighed in triplicate and placed in a centrifuge tube containing 10 ml of deionized water. The samples were soaked for 12 h at room temperature, and then the electrical conductivity of the extract (R1) was measured using a conductivity meter (DDS-11A, Shanghai, China). After that, the samples were heated in a boiling water bath for 30 min, cooled to room temperature and shaken, and then the electrical conductivity of the extract (R2) was measured again (Chen et al., 2010). Relative conductivity was calculated as the ratio of R1 to R2 (R1/R2).

For MDA assay, 0.1 g sample was added to a solution containing 0.1% trichloroacetic acid, centrifuged at 10,000 g for 10 min, and 500 µl of supernatant was collected. Then 500 µL of 20% trichloroacetic acid solution containing 0.5% thiobarbituric acid (TBA) was added and heated at 90 °C for 20 min. After ice bath, the solution was centrifuged at 10,000 rpm/min for 5 min. The absorbance of the supernatant was measured at 532 nm and 600 nm, and the 20% trichloroacetic acid solution containing 0.5% TBA was used as a control (Zhang et al., 2016).

Detection of antioxidant system

The activity of SOD was determined by NBT reduction method, POD activity was determined by guaiacol colorimetric method, APX activity was determined by UV spectrophotometry (Luo, 2021), and three groups of repeated controls were set up for each determination.

Detection of osmotic adjustment system

The content of soluble sugar was determined by anthrone-sulfuric acid colorimetry. The content of SP was determined by coomassie brilliant blue method. The content of Pro was determined by acid ninhydrin method (Wang et al., 2023). Three replicates were set for each determination.

Observation of leaf anatomical structure

An inverted microscope was used to observe and photograph the leaves of M. faberi that had been made into paraffin sections, and Leica Application Suite V4 was used to measure the numerical differences of anatomical structures (upper epidermis, lower epidermis, palisade tissue, sponge tissue) of M. faberi leaves under different treatments.

Data analysis

All experiments were performed with three biological replicates, and the results are presented as the mean ± standard deviation of three independent experiments. The plant height, new branch number, new branch length, chlorophyll a, chlorophyll b, chlorophyll a+b, chlorophyll a/b, Pn, Ci, Tr, RC, MDA content, SOD activity, POD activity, APX activity, SP content, SS content and Pro content were analyzed using one way analysis of variance (ANOVA) with SPSS software. Duncan’s multiple range test was employed to determine significant differences between means when the ANOVA results were significant. A significance level of p < 0.05 was used to define statistically significant differences.

FactoMineR and ggplot2 packages of R was used to do principal component analysis and correlation analysis of plant height, new branch number, new branch length, chlorophyll a, chlorophyll b, chlorophyll a+b, chlorophyll a/b, Pn, Ci,Tr, RC, MDA content, SOD activity, POD activity, APX activity, SP content, SS content and Pro content. All the data were obtained from different time points under drought stress, and each treatment group carried out independent repeated experiments (three biological replicates in each group). To ensure the reliability of the correlation analysis, all data were normalized relative to the control plants at each corresponding time point before statistical analysis. Specifically, the normalization process involved subtracting the mean value of the CK group at the corresponding time point from the treatment group data, followed by scaling based on the standard deviation of the CK group. The Pearson correlation coefficients were calculated using the mean values of three independent biological replicates per group. Additionally, variance analysis was performed within the CK group to confirm that fluctuations were within acceptable experimental limits and did not introduce artefacts into the correlation results.

Effects of leaf phenotype and growth under drought stress

The effects of different levels of drought stress on leaves showed evident differences (Fig. 1A). Under LD stress, leaves started to drop on the 56th day, characterized by a noticeable loss of turgor and partial curling. Under MD stress, leaves exhibited visible wilting by the 42nd day, accompanied by yellowing and the necrosis of older leaves. SD stress led to pronounced shrinkage and withering of leaves by the 36th day, with a rapid progression to the desiccation and abscission of older leaves. After rewatering, plants subjected to LD and MD stress demonstrated significant recovery, regaining turgor and chlorophyll integrity, whereas recovery in the SD group was slower and incomplete (Fig. 1B).

Moreover, different degrees of drought stress also significantly affected the overall growth of plants. During the whole drought treatment, the new shoot length and plant height of each treatment group increased slowly. After rewatering, these parameters increased rapidly, except for the plant height of the SD group, which decreased in WT14-WT21 (Figs. 1C–1D). Under drought stress, compared with CK group, the number of new shoots of M. faberi leaves in each treatment group decreased from T0 to T14, and then fluctuated (Fig. 1E).

Effects of photosynthetic pigments under drought stress and rewatering

On the 21st day of drought stress, the contents of chlorophyll a and chlorophyll b in M. faberi leaves increased significantly, reaching a peak. Specifically, under different drought treatments, the content of chlorophyll a ranges from 2.41 to 2.60 mg/g (Fig. S1A), while the content of chlorophyll b ranges from 1.01 to 1.15 mg/g. However, with the intensification of drought stress, chlorophyll content began to decline. After rewatering, chlorophyll a and chlorophyll b levels recovered significantly regardless of the drought stress level, and exceeded the CK group after three times of rewatering.

Under SD stress, the chlorophyll a/b ratio continued to decline until the 28th day. Under MD stress, the chlorophyll a/b ratio was relatively stable and reached the minimum on the 35th day. In contrast, under LD stress, the chlorophyll a/b ratio continued to rise until the 49th day, reaching a maximum of 2.47 mg/g (Fig. S1C). The chlorophyll a+b showed an upward trend on the 21st day of drought stress, followed by a decline until the 56th day. After rewatering treatment, the chlorophyll a+b increased again and exceeded the levels in the CK group (Fig. S1).

Effects of photosynthetic parameters under drought stress and rewatering

After drought treatment, the Ci of the leaves decreased and then increased. LD and MD groups reached the minimum on the 35th day, and SD group reached the minimum at T21 period. Then the Ci of each treatment group increased, but was lower than that of the control group. After rewatering, the Ci of LD and MD treatment groups began to increase significantly, while the SD treatment group was still in the mean stress period (Fig. 2A).

Under the whole drought treatment, the Pn of LD and MD groups showed a trend of increasing first and then decreasing under continuous stress, while the SD group showed a gradual downward trend (Fig. 2B). After rewatering, the Pn gradually increased. The recovery rates under different degrees of stress were as follows: LD > MD > SD, with all recovery levels significantly lower than that of the CK group.

Under different stress levels, the Gs of M. faberi leaves showed different patterns. The LD and MD groups showed a trend of increasing first and then decreasing under continuous stress, while the SD group showed a gradual downward trend. After rewatering, the Gs of LD and MD treatment groups increased, while that of SD group was lower than other group in WT7 period (Fig. 2C).

During drought stress, all groups showed lower Tr than To. During drought stress, there was no significant difference in Tr between CK and all stressed group until T14 (Fig. 2D). The differences compared to CK appeared at T21, T28 T35, T42, T49, T56, WT7 and WT14 for MD and SD groups, and exhibited at T35, T42 and T56 for LD group (Fig. 2D). After rewatering, all groups recovered well at WT21 (Fig. 2D).

Effects of relative conductivity and MDA content under drought stress and rewatering

The RC of M. faberi leaves showed significant changes across different treatment groups (Fig. 3A). Under LD treatment, the RC initially increased, then decreased on the 28th day, increased again to a peak on the 49th day, and finally decreased on the 56th day. Compared with CK group, LD group showed significant difference at T7, T42 and T56, MD group exhibited significant difference at T35, T42, T49 and WT7, while SD group displayed significant difference at T21, T42, T49, T56, WT7 and WT14 (Fig. 3A). After rewatering, all groups showed no significant difference at WT21 (Fig. 3A).

The MDA content in all treatment groups initially increased and then decreased as drought stress duration extended, but the MDA content differed significantly across treatments. Under LD stress, it peaked at 36.37 nmol/g FW on the 21st day, significantly higher than the CK group at T7, T21, T56 and WT7, but significantly lower than the CK group at WT14 and WT21 (Fig. 3B). Compared with CK group, MD group showed significant difference at T7, T14, T21, T28, T35, T56, WT7 and WT14, while SD group exhibited significant difference at T7, T14, T21, T35, T42, T56, WT14 and WT21 (Fig. 3B).

Effects of drought stress and rewatering on antioxidant system

Under continuous drought stress, POD activity all stressed groups were increased than To. Compared with CK group, LD group showed significant higher POD activity at T21, T28, T35, T42, T49 and T56, MD group exhibited significant higher POD activity at T7, T21, T28, T35, T42, T49 and T56, and SD group displayed significant higher POD activity at T7, T21, T28, T35, T42, T49 and T56 (Fig. 4A). LD and MD groups exhibited a gradual increase, reaching maximum values of 37,495.80 (×10³ U/min/g FW) and 34,167.07 (×10³ U/min/g FW) on the 56th day of stress, respectively, and they already started to differ from CK on T21. After rewatering, LD and MD groups showed higher POD activity than CK group at WT7, while LD, MD and SD groups exhibited significant lower POD activity than CK group at WT 14 and WT21 (Fig. 4A).

SOD activity also increased under drought stress, reaching a peak level of 202.10, 205.17, and 213.68 U/g FW on the 56th day in the LD, MD, and SD groups. Compared with CK group, LD group displayed significant difference at T7, T42, T49 and T56, MD group showed significant difference at T7, T42, T49, T56, WT14 and WT21, while SD group exhibited significant difference at T7, T35, T42, T49, T56, WT14 and WT21 (Fig. 4B). After rewatering, SOD activity in all groups gradually decreased.

APX activity demonstrated an initial increase followed by a decrease under drought stress (Fig. 4C). The LD group peaked at 162.29 U/g FW on the 28th day, the MD group reached its maximum on the 21st day, and the SD group peaked at 200.48 U/g FW on the 35th day, respectively (Fig. 4C). In comparison to CK group, LD group displayed significant difference at T7, T14, T35, T42, T49 and T56, MD group showed significant difference at T7, T14, T21, T28, T35, T42, T49 and T56, and SD group exhibited significant difference at T7, T14, T28, T35, T42, T49 and T56 (Fig. 4C). After rewatering, APX activity decreased at WT7 and then increased at WT14 and WT 21 in CK, LD and MD groups, while APX activity decreased at WT14 and WT21 in the SD group (Fig. 4C). All stress treatment groups (LD, MD and SD) showed significant difference compared with CK group at WT7, MD displayed significantly higher APX activity than CK, while MD and SD exhibited significantly lower APX activity than CK and LD at WT21 (Fig. 4C).

Effects of drought stress and rewatering on osmotic adjustment substances

The content of SS exhibited a general downward-upward-downward trend. During the stress period, the SS content of LD and MD treatment groups reached the maximum on the 28th day, which were 57.99 mg/g and 58.30 mg/g, respectively. The SD treatment group reached a maximum of 52.65 mg/g on the 7th day, significantly higher than the CK group (Fig. 5A). After rewatering, the SS content of each treatment group showed an increasing trend, reaching the highest value on the 7th day of rewatering, with significantly higher level at both WT7 and WT14 than CK group. At WT21, the SS content of LD and MD group was significantly lower than CK group, while SD group showed no significant difference with the CK group (Fig. 5A).

The results showed that the SP content of M. faberi increased gradually. Under LD stress, the SP content reached the highest value of 7.17 mg/g on the 28th day, significantly higher than that of the CK group at T28. Under MD and SD stress, the SP content reached the maximum of 9.48 mg/g and 9.41 mg/g on the 49th day, respectively, significantly higher than that of the CK group at T49 (Fig. 5B). After the end of drought stress, the plants returned to normal water supply, and the SP content of each treatment group began to decrease slowly.

Pro content exhibited an upward–downward–upward trend under LD treatment, with a peak of 5.65 mg/g on the 21st day and a minimum of 0.34 mg/g on the 49th day. Under MD stress, Pro content fluctuated, ultimately reaching a maximum of 7.96 mg/g on the 56th day, significantly higher than the CK group. Notably, under SD stress, Pro content increased sharply at T49, T56 and WT7, significantly higher than the CK, LD and MD groups. After rewatering, Pro content decreased in all treatments, with higher levels in LD group at WT21, in MD group at WT7, and in SD group at WT7 and WT14 (Fig. 5C).

Effects of drought stress and rewatering on leaf anatomical structure

Anatomical structure of the leaves revealed that under LD stress, the thickness of the upper epidermis increased slowly, showing no significant overall upward trend compared to the CK group. Under MD and SD stress, the thickness of the upper epidermis showed an upward trend, but the MD stress showed a downward trend from the 49th day until the 14th day of rewatering, and the SD showed that the thickness of the epidermis continued to decrease after the stress (Fig. S2A). Under LD stress, the thickness of palisade tissue continued to rise to the 28th day, and reached the highest value of 0.093 µm at this time. Then, with the extension of stress time, it began to gradually decrease until the normal watering after the stress, and the thickness of palisade tissue began to rise. The changing trend of palisade tissue under MD stress was consistent with that of LD, and the difference was that MD began to decrease until the 35th day. Under SD stress, the palisade tissue of leaves reached the lowest value of 0.05 µm on the 35th day, and then began to rise (Fig. S2B). The variation trend of sponge tissue under different drought degrees was inconsistent (Fig. S2C). The observation of the anatomical structure of the lower epidermis showed that the thickness of the lower epidermis of the leaves under LD showed an overall upward trend. Under MD stress, the thickness of the lower epidermis showed a downward trend and then an upward trend. The thickness of the lower epidermis of SD showed an upward trend and continued to rise after rewatering (Fig. S2D).

Investigation of leaf tissue under drought stress showed that under LD and MD conditions, cell structures remained clear, with small intercellular spaces and tightly arranged cells, with minimal changes in cell water loss. In contrast, under SD stress, palisade tissue showed narrowing and significant water loss by the 35th day, continuing until the 56th day (Fig. S3). After rewatering, cells in the rehydrated tissues of LD- and MD-treated plants exhibited compact arrangement with minimal intercellular spaces and well-defined structural integrity, whereas SD-treated cells showed a slower recovery, characterized by large intercellular spaces and a more disorganized, loose cellular arrangement (Fig. S4).

Correlation analysis of each index of M. faberi under drought stress

There was a significant correlation between various physiological and biochemical indices of M. faberi under drought stress (Fig. 6). Gs was significantly positively correlated with Pn and Tr, and the correlation coefficient was above 0.85. There was also a significant positive correlation between Pn and Gs, and the correlation coefficient was above 0.90. There was a significant positive correlation between chlorophyll a/b and chlorophyll a, chlorophyll a+b, and the correlation coefficient was above 0.90. Chlorophyll a+b was significantly positively correlated with Pn, Tr and new shoot length. Chlorophyll a content was significantly positively correlated with Pn and Tr. Chlorophyll a/b content was positively correlated with Tr.

At the same time, we also found that chlorophyll a+b and chlorophyll a was significantly negatively correlated with Pro, SP and Ci content. There was a significant negative correlation between SS and SOD activity. There was a significant negative correlation between Pn and Pro.

Principal component analysis

Principal component analysis revealed that the first principal component had a variance contribution rate of 52.7%, primarily contributed by plant height, Ci, and RC. The second principal component, with a variance contribution rate of 16.9%, was mainly contributed by POD activity, Tr, and photosynthetic rate. The cumulative contribution rate of these two principal components reached 69.4% (Fig. S5). The first principal component highlighted the significant role of chlorophyll b, chlorophyll a, chlorophyll a+b, Ci, plant height, and RC in reflecting the response of M. faberi leaves to drought stress. Overall, as drought stress increased from group A to group D, the relative aggregation of each treatment group gradually became more discrete, indicating a positive correlation between the degree of stress and the growth indices of M. faberi.