Chlorophyll plays an important part in the assimilation, transfer and conversion of light energy during photosynthesis. Its content is therefore closely related to the carbon fixation efficiency of photosynthesis and, because photosynthate provides the energy source for metabolic responses, plays an important role in the drought resistance of plants. Chlorophyll fluorescence is often used to analyze plant photosynthesis and photosynthetic physiology under stress1. Fm is the fluorescence output when the reaction center of PSII is completely closed, and therefore reflects the maximum electron transfer through PSII41. Fv/Fm represents the energy conversion efficiency of PSII reactions, and can be used to measure the degree of external stress42.
The chlorophyll content and Fm of A. squarrosum first increased and then decreased under moderate and severe drought, indicating that A. squarrosum adjusted its energy capture during the early stage of drought, and because electron transfer was relatively stable, normal photosynthesis was maintained. As stress intensified during prolonged drought, chlorophyll degradation accelerated and electron transfer through PSII slowed, which was similar to the effect of drought stress on chlorophyll of A. halodendron1. On 1 August, when the drought treatments began, the leaves of A. squarrosum in the control became noticeably yellow and slightly wilted, and the chlorophyll content and Fm were lower than those in the drought treatments. After re-watering, the chlorophyll content and Fm of A. squarrosum decreased, but they increased with increasing drought intensity. There is a limit to plant demand for water, and both too much and too little water are not conducive to plant growth. As a pioneer species during vegetation succession in sandy land, A. squarrosum is a xerophyte33. The soil moisture content in the control was higher than its requirements, and its photosynthesis was obviously adversely affected by controlling the water content at a higher level than the plants required. For A. squarrosum, Fv/Fm decreased with increasing drought duration and intensity. This is because drought reduced the electron transfer capacity of PSII and photochemical activity, leading to excessive accumulation of excitation energy, and adversely affecting photosynthesis. Fv/Fm increased after re-watering on 8 August, when the reduction of stress slowed the inhibition of photosynthesis by drought, by decreasing the inhibition of photosynthesis.
For S. viridis, the chlorophyll content, Fm and Fv/Fm of S. viridis decreased with increasing drought duration and intensity, indicating that drought stress hindered the biosynthesis of chlorophyll, and that chlorophyll decomposition increased, leading to a decreased chlorophyll content. At the same time, the electron transfer via PSII slowed, thereby inhibiting photosynthesis. Fm and Fv/Fm of S. viridis increased after re-watering on 8 August, showing that rehydration relieved the drought stress. In addition, Fv/Fm increased and Fm decreased after re-watering on 14 August, suggesting that the damage to PSII was mitigated by rehydration, but the electron transfer in the PSII reaction center continued to be slower than normal. The chlorophyll content of S. viridis did not return to normal after re-watering, indicating that the leaves of S. viridis were damaged by both prolonged and severe drought stress and that chlorophyll synthesis was significantly affected1.
The cell membrane is both a dynamic barrier between the cell’s interior and its surroundings, and a channel for the exchange of substance and information with its environment43. In particular, it controls water transfers between the cell and its environment, leading to changes in RWC. RWC can be used to indicate the degree of dehydration of cells and assess the level of drought suffered by plants44. Under drought stress, the loss of water in plants is directly related to the stability of the cell membrane, and a stable cell membrane is the most basic requirement for maintaining sufficient water to support the cell’s physiological functions. ROS are produced in large quantities under stress, and this can trigger or exacerbate peroxidation of membrane lipids to produce malondialdehyde. Malondialdehyde can damage the membrane and functional molecules such as proteins and nucleic acids in cells, leading to damage or destruction of the membrane’s structure and functions. This, in turn, can increase the permeability of the membrane, leading to growth inhibition or even death. Therefore, changes in membrane permeability and the malondialdehyde content can reflect the degree of membrane lipid peroxidation and cell damage under stress1,3,34.
In A. squarrosum, membrane permeability in the control on 1 August was significantly less than those under moderate and severe drought, but the malondialdehyde content did not differ among the treatments. The change of membrane permeability may have resulted from degreasing of membrane lipids and destruction of the membrane structure after phospholipid dissociation45. From 1 to 13 August, malondialdehyde content of A. squarrosum in the control first decreased and then increased, while membrane permeability increased continuously, indicating that membrane lipid peroxidation was significantly alleviated in wet soil after short-term drought. In contrast, the serious water deficit during the late stage of drought increased peroxidation of membrane lipids and malondialdehyde accumulation, suggesting that the cell membranes in the control had been damaged during the drought process. The malondialdehyde content and membrane permeability of A. squarrosum increased in the control after rehydration on 8 August, but decreased after rehydration on 14 August. This suggests that rehydration during the early stages of drought can exacerbate the peroxidation of membrane lipids and damage the cell membrane, but that rehydration during the late stages of drought mitigated the stress and eased the damage. Many studies showed that membrane permeability and the malondialdehyde content increased synchronously under stress1, but this contradicts our results for A. squarrosum in the control. This may be because the high soil moisture content in the control was not conducive to normal growth of this xerophyte. That is, long-term natural selection in the species’ arid sandy environment would lead to continuous adaptation to its environment, allowing A. squarrosum to become widely distributed in the mobile dunes of the Horqin sandy land46. With increasing drought duration, the malondialdehyde content and membrane permeability of A. squarrosum increased under both moderate and severe drought, indicating that the accumulation of malondialdehyde after drought stress damaged cell membrane and increased its permeability.
Setaria viridis is a late-successional species, and showed different responses to drought. With increasing drought duration and intensity, the RWC of S. viridis decreased, but its malondialdehyde content and membrane permeability increased. This suggests that the water deficit caused drought stress, leading to increasing peroxidation of membrane lipids and damage to cell membrane’s structure and function. The chlorophyll content, Fm and Fv/Fm of S. viridis decreased with increasing drought duration and severity, and Fv/Fm of S. viridis was significantly negatively correlated with membrane permeability, which increased with increasing drought stress. This indicated that membrane lipid peroxidation and the accumulation of ROS under drought stress damaged the membrane and inhibited photosynthesis. Re-hydration of S. viridis increased RWC on both dates and in all drought treatments. This was accompanied by decreased malondialdehyde content, particularly after the 14 August re-watering, and by decreased membrane permeability. Rehydration reduced membrane lipid peroxidation, but it did not return to the control level, showing that drought caused a certain degree of damage that may be permanent or that may take some time to be repaired3.
Stress can disrupt the balance of ROS metabolism in aerobic plants. When the concentrations of ROS are too high, peroxidation of membrane lipids and the equilibrium for exchanges of cell materials is also disrupted, resulting in a series of physiological and metabolic disorders. To counteract these disorders, plants have evolved protective enzymes during long-term evolution. The enzymes can eliminate O2-, H2O2, OH- and O- and reduce the damage they cause to the plant47. The changes in antioxidant enzyme activities of both species differed under drought stress. SOD played an active role during initial protection against membrane lipid peroxidation and its activity in A. squarrosum increased gradually during the drought. On 7 August, the peroxidase and catalase activities decreased in the control. Because ROS are a metabolism by-product of photorespiration, photosynthesis was inhibited by short-term drought, and the decreased accumulation of ROS caused by protective antioxidant enzymes reduced membrane lipid peroxidation by decreasing levels of malondialdehyde48. On 7 and 13 August, the activities of protective enzymes in A. squarrosum under moderate and severe drought were greater than that in the control. Drought stress led to the accumulation of ROS, and increased membrane lipid peroxidation, as reflected by the malondialdehyde content. At the same time, the accumulated ROS also stimulated the antioxidant enzyme protection system to continuously increase the activities of enzymes, so as to maintain balance of ROS49.
Setaria. viridis showed different responses. From 1 to 13 August, its peroxidase activity first decreased and then increased, but catalase activity showed the opposite pattern, and SOD activity increased gradually, indicating the existences of coordination among these enzymes under drought stress 50. When catalase activity weakened, SOD and peroxidase activities compensated for this weakness to scavenge more ROS and mitigate cell membrane damage. The catalase activity in S. viridis remained less than 50 U.g-1DW.min-1 throughout the study. After rehydration, catalase activity in the control was significantly greater than those under moderate and severe drought, which indicated that its catalase activity did not play an effective role in regulation of ROS under drought stress. Some of the antioxidant enzymes of both species did not recover after rehydration, which may be related to the possibility that in xerophytes, rehydration did not immediately improve physiological metabolism. It is possible that their antioxidant enzyme systems were so damaged that they would take longer than our study period to return to normal levels.