This study investigated how progressive high-temperature exposure affects the physiology and protein expression of Paris polyphylla var. yunnanensis. Plants were acclimated and then exposed to stepwise temperature treatments (25°C, 30°C, 35°C, 40°C, 45°C). High temperature caused direct impairment of photosynthesis, decreased chlorophyll content and net photosynthetic rate (Pn), increased proline accumulation and relative electrical conductivity, and disrupted metabolic homeostasis. Quantitative proteomics identified 893 differentially expressed proteins (DEPs), with marked changes in proteins involved in protein processing and synthesis, the circadian pathway, and the glutathione antioxidant pathway. Dynamic expression changes were observed in endocytosis and autophagy-related proteins, notably ATG3 and ATG8C, suggesting a protective role for autophagy under heat stress.
Paris polyphylla var. yunnanensis is a traditional medicinal plant with an optimal growth temperature of 15–25°C and documented sensitivity to elevated temperatures. Global warming poses a threat to both wild distribution and cultivated production. High temperatures can negatively affect plant physiology through reduced photosynthetic efficiency, accelerated senescence, impaired root function, and disrupted water relations. Plants typically respond with morphological, osmotic, antioxidant, and molecular defenses, including increased levels of osmoprotectants such as proline, upregulation of antioxidant systems like glutathione, and activation of heat-responsive molecular machinery such as heat shock proteins and transcriptional networks.
Three-year-old rhizomes with buds were obtained from Midu County, Yunnan Province. To ensure genetic uniformity, all rhizomes came from the same vegetative propagation batch. Seedlings were planted in humus:perlite (2:1 v/v) pots, acclimated for 60 days under controlled greenhouse conditions (20–25°C, 70–80% relative humidity, 75% shading). Sixty uniform healthy plants were selected; for each temperature treatment (25°C, 30°C, 35°C, 40°C, 45°C) there were three biological replicates, each replicate consisting of four individual plants.
Plants were maintained in a light incubator (12 h:12 h light-dark, 400 µmol·m-2·s-1, 75 ± 10% RH). A gradual acclimation scheme simulated progressive heat stress: the control group remained at 25°C for 48 h; the 30°C group was transferred from 25°C to 30°C for 48 h; the 35°C group experienced 30°C for 48 h followed by 35°C for 48 h; the 40°C group was sequentially acclimated through 30°C and 35°C to 40°C; and the 45°C group was acclimated through 30°C, 35°C, 40°C and finally 45°C, with each step lasting 48 h. Temperature increases were initiated at the start of the dark period. Samples were flash-frozen in liquid nitrogen and stored at −80°C after each treatment.
Net photosynthetic rate (Pn) and other gas-exchange parameters were measured with a portable photosynthesis system (Li-6400). Leaves with comparable baseline Pn were selected and marked. Measurements began on the second day after each temperature adjustment during the light period (9:00–12:00). Parameters recorded included Pn, stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr). Derived indices such as non-stomatal limitation (Ci/Gs) and stomatal limitation (Ls) were calculated according to provided formulas. For each treatment, at least five leaves per plant were measured across three plants.
Total chlorophyll content and chlorophyll a/b ratios were quantified using standard methods. Proline content was measured by the acidic ninhydrin reaction. Relative electrical conductivity (EL, %) — a proxy for membrane integrity — was determined by measuring initial conductivity (E1) at room temperature and total conductivity (E2) after boiling samples; EL was calculated from these values.
Proteomic profiling identified 893 differentially expressed proteins across the temperature treatments. Many DEPs were associated with protein processing and synthesis. Proteomic pathway analyses highlighted elevated expression of proteins related to the circadian pathway and the glutathione antioxidant pathway. Proteins implicated in endocytosis and autophagy, including ATG3 and ATG8C, exhibited dynamic expression changes as temperatures increased.
Progressive increases in temperature led to marked declines in photosynthetic performance and pigment content. The experiment reported reduced total chlorophyll and lowered Pn under higher-temperature treatments, consistent with impaired photosynthesis. Concurrent physiological markers indicated stress: proline accumulation increased, and relative electrical conductivity rose, indicating compromised membrane stability and disrupted cellular osmotic balance.
Proteomics revealed large-scale changes in protein expression under heat. A significant subset of DEPs related to protein processing and synthesis, suggesting that translational and post-translational responses are mobilized during heat stress. Upregulation in components of the circadian pathway and the glutathione pathway points to altered temporal regulation and enhanced antioxidant defenses under elevated temperature conditions.
Notably, proteins tied to endocytosis and autophagy showed dynamic modulation. ATG3 and ATG8C, canonical autophagy-related proteins, changed expression across treatments, supporting the view that autophagy and related trafficking processes may play a protective role in heat tolerance by facilitating removal or recycling of damaged cellular components.
The combined physiological and proteomic data indicate that high temperature compromises photosynthetic machinery and membrane integrity in P. polyphylla var. yunnanensis while activating molecular pathways for damage mitigation. Osmoprotectant accumulation (proline) and increased antioxidant pathway components (glutathione-related proteins) are consistent with general plant heat-response strategies. Changes in protein processing and synthesis machinery reflect a broad adjustment of cellular proteostasis. Dynamic regulation of autophagy-associated proteins suggests autophagic processes contribute to cellular protection under thermal stress.
This work delineates physiological signatures and proteomic changes associated with progressive heat stress in Paris polyphylla var. yunnanensis, including impaired photosynthesis, membrane disruption, osmoprotectant accumulation, and modulation of autophagy-related proteins. The dataset provides a foundation for future genetic or biotechnological approaches aimed at improving heat tolerance in this medicinal species.
All relevant data are reported in the manuscript and supporting information. The study was funded by the Xiamen Science and Technology Plan project and the Basic Research Project of the Fujian Institute of Subtropical Botany. The authors declared no competing interests.