Water is a necessary condition for plant growth. Water deficiency in the environment has a serious impact on the growth and survival of C. oleifera. Plant leaves are the most exposed organs of seedlings in the ground environment, and are also the main organs for photosynthesis and transpiration of plants. Drought can cause a series of changes in photosynthesis, physiology, biochemistry and leaf morphology of C. oleifera. We found that soil water potential decreased under drought stress, leading to leaf upward curl, shrinkage, focal spot, dry death and falling symptoms, and even serious whole plant death (Fig. 1). These results are consistent with previous studies [20, 21]. In August, the climate was hot and less rainy, and the transpiration of C. oleifera leaves was strengthened. The water lost by leaf transpiration was greater than that absorbed by roots, resulting in the lack of water in seedlings[19]. The stomata of leaves were forced to close, and the surface of leaves was dehydrated, so that the lower leaves of plants would contract upward. The seedlings of C. oleifera were rewatered, and the seedlings gradually recovered, which could absorb water normally. The leaves were reopened, and the color gradually became green.
Drought stress can lead to the production of a large number of reactive oxygen species in plant cells, and CAT in plants can effectively remove various reactive oxygen groups to prevent the damage of these groups to the cell membrane system [22, 23]. Our results were consistent with previous studies on wheat varieties, and it was found that CAT activity was enhanced under drought stress [24, 25]. This indicated that C. oleifera could increase its stress resistance by improving CAT activity (Fig. 2B). And compared with container seedlings, bareroot seedlings could mobilize the substances in vivo to resist this stress to a greater extent. Therefore, bareroot seedlings were more drought-resistant than container seedlings. However, Liu et al. and Zhou et al. studied Platycladus orientalis and Ormosia hosiei [26, 27], and found that container seedlings had stronger drought resistance than bareroot seedlings. This is contrary to the conclusion of this experiment, which may be because the main advantage of container seedling afforestation over bareroot seedlings is poor site conditions, especially in the environment with poor soil conditions and nutrients [3, 4]. During the drought, we found that the CAT activity of ‘HS-SC’ decreased in the late stage of drought, which was consistent with the results of Galinsoga parviflora [28], because the CAT activity exceeded the tolerance limit. SS is an ideal osmotic adjustment substance in plant tissues [29]. Under drought stress, plant cells accumulate SS to reduce the water potential in cells to maintain expansion pressure, thereby maintaining normal metabolic activities to adapt to stress environment [30, 31]. Feng et al. found that the soluble sugar content of Liquidambar formosana was the highest under mild drought stress, but decreased under severe drought stress [32]. This study found that with the increase of drought intensity, the content of SS in C. oleifera increased continuously, and there was no peak, which may be related to the difference of adaptive metabolic regulation of different plant leaves to drought stress (Fig. 2C). Moreover, we found that the SS growth rate of bareroot seedlings was the fastest with the increase of drought degree. After rewatering treatment, CAT activity, SS and ABA content of four C. oleifera materials showed a downward trend, indicating that plants effectively scavenge reactive oxygen species, reduce membrane lipid peroxidation, increase osmotic potential and restore normal physiological and metabolic functions during rehydration.
Insufficient or excessive supply of soil moisture will affect the photosynthesis of plants. Drought stress had a great influence on Pn. Under the rapid water loss stress in the short term, the variation trend of Pn of container seedlings and bareroot seedlings was consistent(Fig. 3A). Pn did not decrease immediately with the decrease of leaf water potential, but maintained at the same level as the original. Pn did not drop sharply until the water potential reached a certain value, until Pn was negative, which was consistent with the results of this experiment [33]. In the late drought period, the decrease of Pn of container seedlings was greater than that of bareroot seedlings. Drought stress can significantly reduce the Pn, E, Gs, WUE and Ls of C. oleifera, which is consistent with the results of Diao et al. study [18]. The decrease of photosynthetic rate may be affected by both stomatal and non-stomatal effects [34, 35]. It was found that under mild drought stress, the root cause of the decrease of leaf photosynthetic rate was the decrease of stomatal conductance, resulting in the decrease of Ci, the increase of stomatal limitation, and the decrease of photosynthesis, namely stomatal limitation of photosynthesis [36, 37]. Under severe drought stress, the root cause of the decrease of photosynthetic rate is the disintegration of chlorophyll in photosynthetic organs, the decrease of photosystem II activity, and the inhibition of RuBP carboxylase activity, which leads to the increase of Ci and the decrease of stomatal limitation, that is, the non-stomatal limitation of photosynthesis [38, 39]. This is consistent with the results of this experiment. When C. oleifera is subjected to drought stress, ABA induces the flow of extracellular Ca2 + into cells by preventing the flow of Ca2 + in stomatal guard cells, thereby increasing the concentration of Ca2 + in the cytoplasm of guard cells, promoting stomatal closure, and enhancing the drought resistance of plants [40, 41]. Ls decreased to 0.21–0.43 after 5 DAT; after 10 DAT, Ls of four materials decreased to negative. WUE objectively reflects the utilization of water by plants, and its level directly reflects the adaptability of plants to the environment. WUE objectively reflects the utilization of water by plants, and its level directly reflects the adaptability of plants to the environment [42]. We found that the WUE of C. oleifera increased slightly in the early stage of stress. The main reason was that C. oleifera increased WUE by reducing E, which was also a self-protection mechanism obtained by long-term evolution of plants in adversity. However, under severe stress, WUE decreased significantly, resulting in leaf wilting. In this process, Ls and WUE of ‘HS-SC’ were significantly lower than other C. oleifera materials (Fig. 3E-F).
After RW, ABA content of four C. oleifera materials decreased, stomatal opening and Pn increased ( Fig. 2–3 ). In this study, it was found that the Pn levels of the four C. oleifera materials after RW were higher than those of the control group, which was caused by the compensation effect of plants [43]. The compensation effect is common in plants, usually produced after injury or stress [44], which is an important self-regulation mechanism for plants to resist environmental pressure or injury [45]. Studies have found that after rewatering under a certain range of drought stress, crops have a positive effect of compensation or overcompensation in physiological metabolism, growth and development in the short term to compensate for the damage and loss of crops caused by drought stress [46]. However, the compensation effect of rehydration after drought stress is conditional, severe stress, especially long-term severe stress will reduce the compensation effect and even produce damage effect [47] .
Chlorophyll fluorescence is a fast, accurate and non-destructive probe that can reflect the light energy absorption, utilization, transmission and dissipation processes of plant chloroplast PSI and PSII ( mainly PSII ). Fv/Fm is often used as a probe to reflect the stress degree of environmental factors [48], and its decrease is mainly used to reflect the photoinhibition damage of PS II complex [49]. Studies have shown that when the Fv/Fm value is less than 0.75, it indicates that the plant is in a stress state [50]. In this experiment, the Fv/Fm of four C. oleifera materials decreased to about 0.5 after severe drought, indicating that the PSII reaction center was closed under drought stress, and the damage degree of container seedlings was greater than that of bare root seedlings, therefore, bareroot seedlings were more drought resistant than container seedlings (Fig. 4C). PSII electron transfer is carried out after photochemical reaction, which leads to the splitting ( oxidation ) of water molecules. As a result, ETR is useful in a variety of plant stress studies. This study discovered that after drought stress, PSII and ETR of C. oleifera materials reduced dramatically (Fig. 4D-E), implying that the photochemical efficiency of PSII in leaves decreased under drought stress, either due to photochemical damage or photoprotection of the PSII reaction center [51]. In this experiment, the Fo increased as the drought time increased (Fig. 4A), indicating that the PS II reaction center closed under severe drought stress, preventing electron transfer and lowering photosynthetic apparatus activity [52, 53].
Chlorophyll fluorescence indexes restored to varied degrees after rewatering, and photosynthetic electron transfer was normal (Fig. 4). Among them, the recovery degree of multiple indexes of the four materials exceeded the control level, indicating that the compensation phenomenon was widespread in C. oleifera physiology, which was consistent with the experimental results of Diao et al.[18]. The Fv/Fm of ‘HJ- BS’ was significantly lower than other materials after rehydration.
Plant leaves are sensitive to water stress [54]. The thicker the leaves are, the stronger the drought resistance is. Therefore, the change of leaf anatomical structure is the basis for plant response and adaptation to environmental changes [55, 56]. The upper and lower epidermis and cuticle of plant leaves are located on the leaf surface, which together constitute the protective layer of leaves. The differentiation of palisade tissue and spongy tissue reflects the water content of the environment to a certain extent [57]. In this study, only SR values of the four C. oleifera materials showed an upward trend under drought stress, and all the indexes showed a downward trend (Table 1). This may be because the water deficit affected the normal growth and division of leaf cells, resulting in the obstruction of leaf growth, the decrease of palisade tissue thickness and spongy tissue thickness, and the change of tissue structure, which eventually led to the decrease of leaf thickness. Plant leaf thickness, palisade tissue thickness and CTR value are positively correlated with plant drought resistance, which are commonly used as important indicators to measure the drought resistance of plants. The larger the CTR is, the stronger the drought resistance is [58, 59]. Paraffin sections showed that ‘HS- BS’ had the largest leaf thickness and palisade tissue thickness. After drought stress, normal water supply, C. oleifera leaf cells returned to normal growth and division, and found that C. oleifera ‘Huashuo’ leaf thickness were higher than the control level, its recovery ability is strong.
As a photosynthetic organ, chloroplasts are easily damaged by stress. Early structural changes in chloroplasts occur when leaves are subjected to abiotic stresses [60]. Therefore, examining chloroplast ultrastructure is very important for studying the effects of photosynthesis and abiotic stress on photosynthetic apparatus [61]. In this study, we compared the chloroplast ultrastructure changes of four C. oleifera materials after drought and rehydration. We found that drought treatment significantly damaged chloroplast structure, loosened thylakoid until disintegration, and increased the number of liposomes in chloroplast, indicating that the photosynthetic apparatus of C. oleifera materials were destroyed after drought stress. Therefore, through the analysis of the above results, we can conclude that the decrease of photosynthetic rate in the late stage of drought stress is mainly due to the destruction of photosynthetic mechanism by non-stomatal factors. After rewatering, ‘HS-BS’ has a relatively complete lamellar matrix and less liposomes, except for the rupture of chloroplast membrane, which indicates that it has a strong recovery ability.