59 bmcps BMC Plant Biology BMC Plant Biol BMC PMC13059601 13059601 13059601 41772431 10.1186/s12870-026-08417-w Glutathione-gold nanoclusters enhance sweet potato thermotolerance through improved photosynthesis, redox homeostasis, and cellular integrity Kumar Sunjeet 1 2 # Gurmendar 1 2 # Yu Rui 1 2 Ikram Muhammad 1 2 Kou Jingjing 1 2 Khan Muhammad Abbas 1 2 Wang Mengzhao 1 2 ✉ Zhu Guopeng 1 2 ✉ 1 School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya, 572025 China 2 Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, School of Tropical Agriculture and Forestry, Hainan University, Haikou, 570228 China ✉ Corresponding author. # Contributed equally. 2 3 2026 26 627 627 9 4 2026 © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . Abstract Background Global warming severely challenges sweet potato cultivation by disrupting physiological, biochemical, and structural processes. The potential of glutathione-gold nanoclusters (GSH-Au NCs) in enhancing thermotolerance remains largely unexplored in vegetable sweet potato, a key industrial and food security crop. We hypothesized that foliar application of GSH-Au NCs would mitigate heat-induced disruptions in photosynthesis, redox homeostasis, and cellular structures by enhancing antioxidant defenses and enzymatic activities. Results Under controlled heat stress (42 °C day/35°C night for 7 days), heat stress diminished shoot biomass, leaf area, RWC, and root biomass, while impairing photosynthesis and elevating oxidative damage (H 2 O 2 : 276.7% and MDA: 481.5%) and electrolyte leakage by 85.7%. GSH-Au NCs (2 mg L − 1 ) reversed these effects, enhancing growth, shoot and root biomass (45.3% and 28.3%), and RWC (7.9%). These improvements were associated with enhanced photosynthetic efficiency through elevated chlorophyll content (72.6%), Rubisco activity (26.4%), gas exchange parameters (Pn: 86.1% and Gs: 389%), and chlorophyll fluorescence (Fv/Fm: 16.0% and ETR: 48.2%). Mechanistically, GSH-Au NCs correlated with upregulation of ascorbate-glutathione (AsA-GSH) cycle, boosting enzymatic (SOD: 30.1%, APX: 33.6%, and GR: 20.9%) and non-enzymatic antioxidants (AsA/DHA: 33.8% and GSH/GSSG: 29.1%), while lowering oxidative markers: H 2 O 2 (37.8%), MDA (51.7%), and EL (26.8%), compared with heat stressed plants without NCs treatment. Transcriptional upregulation of SOD , APX , GR , DHAR , and MDHAR genes supported these effects. Additionally, GSH-Au NCs enhanced glyoxalase activity (Gly I: 36.9% and Gly II: 35.0%), reducing toxic methylglyoxal (29.3%). Higher proline (51.7%) and secondary metabolites (polyphenols: 38.3% and flavonoids: 62.9%) further strengthen stress resilience. Moreover, GSH-Au NCs restored stomatal behavior and preserved chloroplast and mitochondrial structure. Conclusions These findings highlight GSH-Au NCs (2 mg L − 1 ) as a sustainable, cost-effective, eco-friendly nanobiotechnological strategy for mitigating heat stress in sweet potato and promoting climate-resilient horticulture, advancing beyond prior studies on individual GSH or Au nanomaterials by demonstrating the synergetic effects of their combined nanocluster form for the first time in plant species. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-026-08417-w. Keywords: Thermotolerance, GSH-Au NCs, Redox balance, Photosynthetic efficiency, AsA-GSH cycle, Ultrastructure status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2025 Dec 29; Accepted 2026 Feb 17; Collection date 2026. Introduction Climate change-driven global warming poses a severe threat to plant health and agricultural productivity, as increasing temperatures and frequent heatwaves negatively affect crop production [ 1 , 2 ]. Heat stress is characterized by temperatures exceeding a plant’s optimal growth range, disrupting cellular homeostasis, impairing key physiological processes, and reducing crop yields, with long-term consequences for vegetation [ 2 , 3 ]. Studies predict a continued rise in global temperatures, potentially decreasing crop yields by 3–8% for every 1 °C increase, particularly in tropical and subtropical regions where 70% of the global population resides [ 4 , 5 ]. As a leading agricultural producer, China faces a growing threat from rising heatwaves, which endanger food security by damaging critical processes in crops, such as photosynthesis, water regulation, and cellular integrity [ 6 – 8 ]. Heat stress profoundly impacts plant physiology, particularly compromising photosynthetic efficiency [ 9 ]. Elevated temperatures induce stomatal closure, thereby restricting CO 2 uptake and diminishing Rubisco carboxylation efficiency, which simultaneously promotes photorespiration [ 10 ]. While stomatal closure helps conserve water, it also exacerbates CO 2 limitation, further reducing photosynthetic efficiency. The inactivation of key photosynthetic enzymes like Rubisco, chlorophyll degradation, and structural damage to chloroplasts intensifies these effects. Additionally, excessive electron flow in the photosynthetic transport chain, particularly between photosystem II (PSII) and PSI, increases generations of reactive oxygen species (ROS), including superoxide (O 2 •− ), hydrogen peroxide (H 2 O 2 ), and methylglyoxal (MG). Elevated ROS levels cause oxidative stress, lipid peroxidation, and membrane damage [ 6 , 10 , 11 ]. Malondialdehyde (MDA) is a marker of lipid peroxidation, increases under heat stress, indicating damage to cellular structures such as chloroplasts and mitochondria. Furthermore, restricted root respiration and phloem transport hinder sugar translocation, causing carbohydrate accumulation in leaves and further suppressing CO 2 assimilation [ 8 , 10 ]. Plants counteract heat stress conditions through defense systems, comprising enzymatic scavengers, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX), along with non-enzymatic compounds like glutathione (GSH) and proline. The ascorbate-glutathione (AsA-GSH) cycle is critical for ROS detoxification, neutralizing H 2 O 2 using AsA as an electron donor, producing monodehydroascorbate (MDHAR) and dehydroascorbate (DHAR). GR regenerates GSH from GSSG using NADPH, sustaining the cycle’s efficiency [ 12 , 13 ]. Additionally, the toxic compound MG is detoxified through the coordinated action of glyoxalase (Gly I and II) [ 11 ]. These defense mechanisms enable heat-tolerant cultivars to maintain cellular redox balance and protect their photosynthetic machinery, while sensitive cultivars often experience severe cellular damage and accelerated premature leaf senescence [ 3 ]. Prolonged stress exhausts the antioxidant defense system, rendering it ineffective. Applying exogenous growth regulators can then improve stress tolerance and recovery. Conventional methods to mitigate heat stress, such as chemical treatments and agronomic practices, often present limitations such as environmental toxicity, high costs, and risks to soil microbial communities [ 14 ]. In contrast, nanotechnology has emerged as a promising eco-friendly solution, which provides improved nutrient delivery, enhanced stress tolerance, and sustainable crop protection [ 15 , 16 ]. Various nanomaterials (NMs) like nanoclusters (NCs) and nanoparticles (NPs), including Au, SiO 2 , CeO 2 , and MT-Se, improve plant stress tolerance by enhancing biomass accumulation, nutrient uptake, photosynthetic efficiency, and ROS scavenging [ 17 – 20 ]. These nanomaterials influence cellular structure, physiological processes, and gene expression related to aquaporins, photosynthesis, and antioxidant defense [ 21 , 22 ]. Glutathione (GSH), a key regulator of detoxification, cell division, and stress tolerance, enhances antioxidant enzyme activity and protects photosynthetic processes under heat and other abiotic stress conditions [ 23 , 24 ]. While, gold (Au) nanomaterials and GSH supplementation have individually shown promise in enhancing plant stress tolerance, the combined application of glutathione-gold nanoclusters (GSH-Au NCs) has not yet been reported in any plant species. GSH-Au NCs were selected for their potential synergetic effects, where GSH’s antioxidant and detoxification roles such as ROS scavenging and AsA-GSH cycle regulation are enhanced by Au nanoclusters’ biophysical properties, such as high surface area for efficient delivery, free radical scavenging, and upregulation of stress response genes, as observed in previous Au NP studies [ 25 – 27 ]. This combination aims to provide a more targeted and stable internation compared to individual components. This study addresses key gaps in prior nano-enabled heat-stress mitigation research, which has focused on individual nanomaterials such as Au NPs and GSH alone in different crops [ 17 – 20 ], but lacks combined GSH-Au NC formulations, applications in vegetable sweet potato, and detailed mechanistic insights into ultrastructural, transcriptional, and glyoxalase responses. Sweet potato ( Ipomoea batatas ) was selected due to its high sensitivity to heat stress such as reduced biomass and yield at temperature more than 35 °C [ 13 , 28 , 29 ]. Sweet potato has agronomical importance as the world’s 7th most economically important starch crop (global production of 93.5 million metric tons annually, and China producing 55% of global output alone) FAO, 2023, https://www.fao.org/faostat/en/#data/QCL/visualize ). Previous studies on sweet potato under heat stress have primarily focused on gene expression, storage root development, and genetic transformation, with limited research on vegetable sweet potatoes [ 28 – 31 ], and also the absence of previous nanomaterial-based stress mitigation studies in this species under heat stress. Therefore, we hypothesize that foliar application of GSH-Au NCs will enhance thermotolerance in sweet potato by (i) improving photosynthetic efficiency through increased pigment content, Rubisco activity, and gas exchange, (ii) strengthening redox balance via activation of the AsA-GSH cycle, glyoxalase system, antioxidant defenses, and related gene expression, and (iii) preserving structural integrity, including stomatal behavior and chloroplast ultrastructure. The findings aim to establish GSH-Au NCs as a cost-effective, eco-friendly, and sustainable approach to improve sweet potato growth and tolerance under heat stress, offering novel insights for climate-resilient agriculture. Materials and methods Characterization of purchased nanoclusters Analytical-grade GSH-Au NCs were obtained from XFNANO Materials Technology Co., Ltd. (Jiangsu, China) with a purity of higher than 99.5%. GSH-Au NCs were characterized using HR-TEM (S-4800, Hitachi, Tokyo, Japan). The nanomaterial suspension was ultrasonicated for 30 min in the dark, after which zeta potential, UV-visible absorption spectra, and particle size distribution were calculated using a Xetasizer Nano ZS90 (Malvern Instruments, Marburg, UK) [ 6 ]. GSH-Au NCs exhibited spherical morphology with an average < 3 nm (HR-TEM), positive zeta potential (22.7 ± 1.2 mV, confirming colloidal stability), and a UV-visible absorption peak at 418 nm (Fig. 1 ; S1). Fig. 1 Characterization of glutathione-gold nanoclusters (GSH-Au NCs) and growth parameters under normal and heat stress conditions. A HR-TEM image of GSH-Au NCs (scale bar: 2 nm), B zeta potential of GSH-Au NCs, C representative phenotypes of vegetable sweet potato under different treatments (CK: control, GA: 2 mg L − 1 GSH-Au NCs, HS: heat stress, GAHS: 2 mg L − 1 GSH-Au NCs + heat stress). and D-K) quantitative growth parameters: D plant height (cm plant − 1 ), E leaf area (cm 2 ), F number of leaves plant − 1 , G relative water content, H shoot fresh weight (g plant − 1 ), I shoot dry weight (g plant − 1 ), J root fresh weight (g plant − 1 ), and ( K ) root dry weight (g plant − 1 ). Data are mean ± SE from biological triplicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) Preparation of plant materials and experimental design The experiments used the sweet potato cultivar ‘HD7791’, which was selected and evaluated by our research group from the cultivated population in Hainan Province, China. Planting material (stem cuttings) for this study was sourced from our institutional stock. The cultivar’s identity was formally confirmed by our research team, and a resource is maintained at Hainan University. Stem cuttings were disinfected with fungicide and grown in soil-filled pots at 25–27 °C with a 16-h photoperiod. After a 7-day acclimatization period, the growth chamber was set to 42 °C (light) and 35 °C (dark), with 4,000 lx light intensity (80 µmol m −2 s −1 PPFD, based on LED conversion factors), a 16/8-h photoperiod, and 75% humidity. This moderate light level was selected to maintain consistent vegetative conditions during the 7-day acute heat stress exposure, allowing clear detection of heat-induced physiological disruptions (photosynthesis decline and oxidative damage) without confounding by high-light photoinhibition. This regime mimics extreme heatwaves in tropical and subtropical sweet potato growing regions, where summer temperatures often exceed 42 °C, causing 20–40% yield losses [ 4 , 5 , 28 ]. Foliar applications were initiated at a defined early vegetative stage (7 days post-acclimatization), when initial heat stress phenotypes such as mild wilting emerged uniformly across plants in pilot trials, ensuing consistent timing and minimizing bias. The selected concentration of GSH-Au NCs (0, 0.5, 1, 2, and 4 mg L −1 ) was based on preliminary optimization and concentrations previously reported as physiologically effective for nanomaterial-mediated stress mitigation without phytotoxic effects. The experimental design included the following treatments: (1) CK; Control, (2) GA1; 0.5 mg L −1 GSH-Au NCs, (3) GA2; 1 mg L −1 GSH-Au NCs, (4) GA3; 2 mg L −1 GSH-Au NCs, (5) GA4; 4 mg L −1 GSH-Au NCs, (6) HS; heat stress 42 °C, (7) GAHS1; 0.5 mg L −1 GSH-Au NCs + heat stress, (8) GAHS2; 1 mg L −1 GSH-Au NCs + heat stress, (9) GAHS3; 2 mg L −1 GSH-Au NCs + heat stress, and (10) GAHS4; 4 mg L −1 GSH-Au NCs + heat stress. Phenotypic and physiological data were recorded after 7 days of NC treatment. Determination of plant growth parameters Growth parameters of sweet potato seedlings, including height, leaf number, leaf area, and fresh and dry weights of roots and shoots, were evaluated. Leaf area was determined with a portable laser leaf area meter (CI-202, CID Bio-Science, USA). Fresh weights (FW) were reordered, followed by drying at 80 °C for 72 h to determine dry weights (DW). Relative water content (RWC) was calculated using established methods [ 24 ]. After determining the FW of the leaves, we immersed them in ddH 2 O for 4 h and then determined their turgor weight (TW). The leaves were then desiccated at 70 °C for 24 h. Finally, the RWC of the leaves was measured using the following formula; RWC (%) = [(FW-DW)/(TW-DW)] × 100 After washing, fresh roots were scanned using an Epson Expression 11000XL root scanner to determine root morphology. Images were analyzed using WinRHIZO 2003a software, a validated and reliable tool for root traits in recent plant studies [ 32 ]. Determination of photosynthesis-related parameters Leaf gas exchange parameters were measured using a portable photosynthesis system. Chlorophyll and carotenoid contents were determined by homogenizing 0.1 g of fresh leaves in 80% acetone, followed by centrifugation and absorbance measurement at 662, 645, and 470 nm. Chlorophyll and carotenoid visualization was performed using a TCS SP2 laser confocal microscope (Leica, Germany). A portable chlorophyll fluorometer was used to quantify ETR, qP, qN, Y(II), and Fv/Fm [ 32 , 33 ]. Electron microscopic analysis For scanning electron microscopy (SEM), fresh leaves were rinsed with distilled water, fixed in glutaraldehyde, and dehydrated in 80% ethanol. Samples were then dried using critical point drying, fixed on SEM stubs with conductive tape, and coated with platinum through sputtering for 10 min before SEM imaging [ 34 ]. For transmission electron microscopy (TEM), fresh leaf tissues were dissected into small pieces and fixed in 2.5% glutaraldehyde at 4 °C for 12 h. Samples were rinsed three times in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 15 min each, post-fixed in 1% osmium tetroxide for 1–2 h, and washed again three times in PBS for 15 min each. Dehydration was performed using a graded ethanol series (30%, 50%, 70%, and 90% for 15 min each, followed by 100% ethanol twice for 20 min each) and two changes of 100% acetone for 20 min each. Infiltration was carried out with acetone: embedding resin mixtures (1:1 at 37 °C for 3 h and 1:3 at 37 °C for 4 h), followed by pure embedding resin overnight at 37 °C. Samples were subsequently embedded in fresh resin molds and polymerized at 70 °C for 12–48 h. Ultrathin sections were cut, mounted on copper grids, stained with uranyl acetate for 8–15 min followed by lead citrate for 5–10 min, air-dried, and examined under a HITACHI HT7800 TEM [ 24 ]. Oxidative damage detection by histochemical localization H 2 O 2 and O 2 •− were localized using DAB and NBT staining, respectively. MDA was detected by staining fresh leaves with 10% Schiff’s reagent for 120 min, followed by washing in potassium metabisulfite solution to remove residual stain [ 35 ]. Determination of oxidative stress markers, antioxidants, and antioxidant enzymes Electrolyte leakage (EL) was measured using a previously established protocol [ 36 ]. Levels of oxidative stress markers (H 2 O 2 , O 2 •− , and MDA), antioxidants (AsA, DHA, GSH, and GSSG), Rubisco activity, and antioxidant enzyme activities (SOD, CAT, APX, GR, DHAR, MDHAR, POD, and GST) were quantified using commercially available kits from Nanjing Jiancheng Bioengineering and Solarbio. Leaf samples were homogenized, centrifuged, and the supernatants were analyzed following kit instructions [ 13 , 24 ]. MG content and activities of Gly I and Gly II were measured using a previously described protocol [ 36 ]. Determination of osmolytes and secondary metabolite content Soluble sugars were extracted by homogenizing leaf samples in double-distilled water, heating at 95 °C for 15 min, and centrifuging at 8,000 g for 12 min. Absorbance was measured at 620 nm using a commercial test kit from Nanjing Jiancheng Bioengineering, China. Total proteins and proline concentrations were quantified using commercial kits (A0452 and A107-1-1) following homogenization and centrifugation [ 13 , 24 ]. The Folin-Ciocalteu method was used for the determination of total polyphenols, and the aluminum chloride method was used for flavonoid content [ 24 , 37 ]. qRT-PCR analysis Total RNA was extracted from frozen leaf samples using a Trizol kit (Invitrogen, Santa Cruz, CA, USA). Reverse transcription was performed using a SuperScript III reverse transcriptase kit. The Actin gene (accession: EU250003 ) was used as the internal reference for normalization, selected based on its stable expression across treatments. Gene-specific primers were designed using Primer3 software (primer sequences listed in Table S2). qRT-PCR analysis was conducted using a Bio-Rad Mx3000P qPCR system with three biological replicates ( n = 3 independent plants per treatment). Expression levels were calculated using the 2 –∆∆ Ct method [ 38 , 39 ]. Statistical analysis All experimental measurements were conducted in biological triplicate ( n = 3 independent plants per treatment. Data were expressed as mean ± standard error (SE). Statistical differences among treatments were analyzed using one-way ANOVA followed by Duncan’s multiple range test at P < 0.05 in SPSS 25.0 (IBM, Chicago, USA). Duncan’s test was selected for its sensitivity in detecting differences in multi-treatment agricultural experiments, as is commonly used in plant stress studies. To minimize the risk of type I error inflation associated with Duncan’s test, all key results were cross-verified with HSD test ( P < 0.05), which showed no discrepancies in significance patterns. Statistical power was confirmed post-hoc for major parameters (power > 0.8). Pearson’s correlation analysis was performed using R software (Package ggcor). Results Plant growth variables Different concentrations were tested under both normal and heat stress conditions to investigate the biological impact of GSH-Au NCs on sweet potato plant. Under non-stress conditions, GSH-Au NCs (GA1 to GA4) lead to significant enhancements in growth and biomass accumulation compared to control plants ( P < 0.05), with the GA3 (2 mg L − 1 ) treatment significantly promoted growth, increasing shoot and root fresh biomass by 13.5% and 12.1% and dry biomass by 7.2% and 9.2% and photosynthetic pigments by 3.6–7.2% (Table S1; Fig. 2 ), indicating general growth-promoting effects in addition to stress mitigation. Fig. 2 Auto-fluorescence, chlorophyll and carotenoid content in the leaves of sweet potato under normal and heat stress conditions. A Auto-fluorescence of chlorophyll and carotenoids, and ( B - E ) chlorophyll and carotenoid content (mg g − 1 FW) under normal and heat stress conditions. Treatments: CK= control, GA = 2 mg L − 1 GSH-Au NCs, HS= heat stress, and GAHS = 2 mg L − 1 GSH-Au NCs + heat stress. Data are means ± SE from n = 3 biological replicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) However, plants subjected to heat stress severely impaired plant development, reducing plant height (24.1%), shoot FW and DW (41.6 and 43.1%), leaf number (56.5%), leaf area (66.7%), RWC (15.1%), root biomass (40.4% FW and 31.5% DW) compared to the control plants (Fig. 1 , Table S1). Remarkably, GSH-Au NCs mitigated these adverse effects, with the 2 mg L − 1 (GAHS3) treatment demonstrating the highest efficacy by enhancing plant height (18.9%), shoot biomass (45.3% FW, 35.2% DW), leaf traits (100% increase in leaf number and 95.7% in leaf area), RWC (7.9%), and root biomass (28.3% FW and 25.6% DW) relative to heat-stressed plants (Fig. 1 , Table S1). Based on these results, the 2 mg L − 1 GSH-Au NCs concentration was identified as the optimal treatment and was selected for further analysis. Root system architecture was also severely affected by heat stress ( P < 0.05; Fig. S1). However, sweet potato plants treated with 2 mg L − 1 GSH-Au NCs demonstrated notable recovery in root traits, including significant increases in root length (27.9%), surface area (68.1%), diameter (12.7%), root volume (59.9%), projected area, tips, forks, and crossings compared to heat-stressed plants (Fig. S2). Photosynthetic pigments, rubisco activity, and photosynthetic efficiency Under normal conditions, GSH-Au NCs slightly enhanced photosynthetic pigment levels and efficiency (Figs. 2 and 3 ). However, exposure to heat stress drastically decreased the level of T. Chl, Chl a, Chl b, and Car by 63.7%, 53.5%, 74.5%, and 46.6%, respectively (Fig. 2 ). While GSH-Au NCs (2 mg L − 1 ) significantly increased pigment concentrations under heat stress, with increases of 72.6% (T. Chl), 69.2% (Chl a), 79.1% (Chl b), and 28.2% (Car) compared to heat-stressed plants (Fig. 2 B-E). Confocal microscopy revealed uniform and dense red (chlorophyll) and green (carotenoids) fluorescence in both control and NC-treated plants under normal conditions. However, heat-stressed leaves exhibited a sharp decline in chlorophyll and carotenoid fluorescence. The application of GSH-Au NCs restored fluorescence intensity, aligning with the observed pigment recovery (Fig. 2 A). Fig. 3 Chlorophyll fluorescence, gas exchange parameters, and Rubisco activity of vegetable sweet potato under normal and heat stress conditions. A - E Fv/Fm, Y(II), qP, and ETR. F - I Pn, Gs, Ci, and Tr. J Rubisco activity under normal and heat stress conditions. Treatments: CK= control, GA = 2 mg L − 1 GSH-Au NCs, HS= heat stress, and GAHS = 2 mg L − 1 GSH-Au NCs + heat stress. Data are means ± SE from n = 3 biological replicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) The crucial carbon-fixing enzyme Rubisco showed 38.3% reduced activity under heat stress, creating a major constraint in photosynthetic efficiency. However, GSH-Au NCs significantly alleviated heat-induced inhibition of Rubisco, restoring activity by 26.4% compared to stressed plants (Fig. 3 J). This enhancement directly correlated with improved carbon assimilation rates, as evidenced by gas exchange. Chlorophyll fluorescence parameters, which reflect the functional status of photosystem II (PSII), were also severely compromised by heat stress. Fv/Fm, Y(II), qP, and ETR were reduced by 25.8%, 58.2%, 56.3%, and 55.7%, respectively, indicating impaired light energy conversion (Fig. 3 A-E). GSH-Au NCs treatment countered these negative effects, enhancing Fv/Fm by 16.0%, Y(II) by 93.6%, qP by 71.3%, and ETR by 48.2% (Fig. 3 A-E), thereby improving all photosynthetic efficiency under stress. Gas exchange measurements further supported these findings. Heat stress led to substantial reductions in Pn (57.1%), Gs (87.7%), Ci (47.0%), and Tr (66.1%) compared to control plants. However, GSH-Au NC-treated plants exhibited significant recovery, with Pn increasing by 86.1%, GS by 389.0%, Ci by 47.3%, and Tr by 124.3% (Fig. 3 F–I). These results revealed that GSH-Au NCs not only preserve photosynthetic machinery under thermal stress but also enhance stomatal functions and carbon assimilation. Stomatal parameters Heat stress severely affected stomatal characteristics in sweet potato leaves, reducing density (Fig. 4 A) and dimensions, including length (61.1%), width (75.3%), and pore length (61.0%). GSH-Au NCs treatment alleviated these morphological changes, increasing stomatal length by 90.7%, width by 161.4%, and pore length by 60.9% compared to heat-stressed plants (Fig. 4 B-E). GSH-Au NCs promoted stomatal opening and increased stomatal density, suggesting improved gas exchange capacity under stress conditions. Fig. 4 Stomatal characteristics in vegetable sweet potato under normal and heat stress conditions. A Stomatal density under 250 × magnification and scale bar of 200 μm, B stomata under 3000 × magnification and scale bar of 10 μm, C stomatal length, D stomatal width, and ( E ) stomatal pore length under normal and heat stress conditions. Treatments: CK= control, GA = 2 mg L − 1 GSH-Au NCs, HS= heat stress, and GAHS = 2 mg L − 1 GSH-Au NCs + heat stress. Data are means ± SE from n = 3 biological replicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) Chloroplast ultrastructure analysis TEM revealed distinct structural differences in sweet potato mesophyll cells under normal and heat-stressed conditions. In normal conditions, both control and GSH-Au NCs-treated mesophyll cells maintained uniform shapes, with chloroplasts exhibiting oval structures and intact thylakoid membranes. Stroma appeared clearly defined, cell walls remained thin with sharp edges, and grana lamellae were tightly stacked with well-organized grana and stroma lamellae. Mitochondria maintained their structural integrity, indicating normal respiration (Fig. 5 ). Fig. 5 Ultrastructural changes in sweet potato vegetable under normal and heat stress conditions. CK= control, GA = GSH-Au NCs, HS= heat stress, GAHS= heat stress + GSH-Au NCs. Abbreviations: CW= Cell Wall, Chl= Chloroplast, M= Mitochondria, C= Cytoplasm, S= Starch, SL= Stroma Lamellae, G= Grana, PG= Plastoglobules. Mesophyll cell has 4000 × magnification and scale bar of 2 μm. Chloroplast structure has 15,000 × magnification and scale bar of 1 μm Heat stress induced significant ultrastructural changes; mesophyll cells became swollen, chloroplasts exhibited abnormal swelling, disrupted thylakoids and irregular stroma, and increased autophagic vesicles suggested intense self-digestion. Cell wall development was impaired, showing blurred edges, while grana lamellae became separated and mitochondria appeared damaged (Fig. 5 ). In contrast, GSH-Au NCs-treated plants under heat stress showed partial restoration of cellular structure. Cell sizes approached control levels, chloroplasts exhibited reduced swelling with partially restored thylakoid structure, and autophagic activity decreased. Cell wall edges regained clarity, grana and stroma lamellae showed improved organization, and mitochondrial swelling was less prominent (Fig. 5 ). These changes highlight the protective role of GSH-Au NCs in maintaining chloroplast ultrastructure under heat stress conditions. Oxidative damage, osmolytes, and non-enzymatic antioxidants Under heat stress, sweet potato leaves exhibited significant oxidative damage, as seen by elevated levels of ROS such as H 2 O 2 and O 2 •− , along with increased lipid peroxidation (MDA) and EL. Histochemical staining revealed distinct visual markers, such as brownish spots for H 2 O 2 , blue spots for O 2 •− , and magenta coloration of MDA, demonstrating their accumulation under heat stress. However, treatment with GSH-Au NCs effectively mitigated these effects by reducing the intensity and number of spots (Fig. 6 A). Concentration determination also showed that heat-stressed plants have escalated the content of H 2 O 2 by 276.7%, O 2 •− by 143.1%, MDA by 481.5%, and EL by 85.7% compared to the control plants ( P < 0.05; Fig. 6 B-E). Notably, GSH-Au NCs application under heat stress significantly mitigated oxidative damages, as evidenced by a substantial reduction in H 2 O 2 by 37.8%, O 2 •− by 36.6%, MDA by 51.7%, and EL by 26.8% compared to heat-stressed plants (Fig. 6 B-E). Fig. 6 Oxidative damage, osmolytes, and non-enzymatic antioxidants in vegetable sweet potato under normal and heat stress conditions. A Histochemical localization. B - E H 2 O 2 , O 2 •− , MDA, and electrolyte leakage (EL), F proline, G soluble sugars, H total polyphenols, and I ) total flavonoids under normal and heat stress conditions. Treatments: CK= control, GA = 2 mg L − 1 GSH-Au NCs, HS= heat stress, and GAHS = 2 mg L − 1 GSH-Au NCs + heat stress. Data are means ± SE from n = 3 biological replicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) To overcome oxidative stress, sweet potato plants accumulate osmolytes (proline and soluble sugars) and non-enzymatic antioxidants (polyphenols and flavonoids). Heat stress triggered a significant increase, with proline rising by 157.4%, soluble sugars by 192.1%, polyphenols by 167.8%, and flavonoids by 239.4% compared to control plants (Fig. 6 F-I). GSH-Au NCs further enhanced this adaptive response by significantly boosting proline (51.7%), polyphenols (38.3%), and flavonoids (62.9%) compared to heat-stressed plants. Interestingly, GSH-Au NCs reduced soluble sugars by 20.1% under heat stress (Fig. 6 F-I). Ascorbate, glutathione and their ratios This study observed significant changes in AsA-GSH cycle in sweet potato plants under heat stress. Specifically, heat stress led to a marked increase in AsA levels by 57.3%, while DHA content decreased by 11.9%, resulting in a significant enhancement of the AsA/DHA ratio by 78.1% compared to the control conditions. When GSH-Au NCs were applied under heat stress, the AsA content increased further by 17.9% and DHA levels declined by an additional 11.2%. This contributed to a 33.8% rise in the AsA/DHA ratio (Fig. 7 A-C). Fig. 7 Content of ascorbate, glutathione, and their ratios. A AsA, B DHA, C AsA/DHA ratio, D GSH, E GSSG, and ( F ) GSH/GSSG ratio under normal and heat stress conditions. Treatments: CK= control, GA = 2 mg L − 1 GSH-Au NCs, HS= heat stress, and GAHS = 2 mg L − 1 GSH-Au NCs + heat stress. Data are means ± SE from n = 3 biological replicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) Likewise, the levels of GSH, GSSG, and their corresponding GSH/GSSG ratio were also significantly elevated in response to heat stress by 150.6%, 76.0%, and 43.0%, respectively, relative to control plants. The application of GSH-Au NCs under heat stress further amplified these effects, increasing GSH by 56.0%, GSSG by 20.9%, and the GSH/GSSG ratio by 29.1% (Fig. 7 D-F). Antioxidant enzymes (ASA-GSH cycle) Exposure to heat stress significantly heightened the activities of several key enzymes in sweet potato. SOD, CAT, APX, GR, DHAR, and MDHAR activities increased by 89.2%, 63.6%, 414.4%, 216.7%, 237.1%, and 126.8%, respectively, compared to control plants. The application of GSH-Au NCs under heat stress further elevated the activities of all these mentioned enzymes except CAT. Specifically, GSH-Au NCs treatment increased SOD by 30.1%, APX by 33.6%, GR by 20.9%, DHAR by 54.9%, and MDHAR by 46.6%. Surprisingly, CAT activity decreased by 20.5% following GSH-Au NCs treatment (Fig. 8 A-F). Fig. 8 Antioxidant enzyme activities under normal and heat stress conditions. A - F GSH-AsA cycle enzymes (SOD, CAT, APX, GR, DHAR, and MDHAR). G-H) POD and GST activities. Treatments: CK= control, GA = 2 mg L − 1 GSH-Au NCs, HS= heat stress, and GAHS = 2 mg L − 1 GSH-Au NCs + heat stress. Data are means ± SE from n = 3 biological replicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) POD and GST play crucial roles in scavenging H 2 O 2 and detoxifying lipid peroxides, respectively. Heat stress elevated POD and GST activities by 197.0% and 83.8%, respectively. However, GSH-Au NCs treatment further increased POD by 33.5% and GST by 53.6% under heat stress conditions (Fig. G-H). Transcriptional expression of genes Under heat stress conditions, the transcriptional levels of multiple antioxidant enzymes–related genes were significantly upregulated in sweet potato plants, with the exception of CAT , which showed downregulation. Notably, SOD , APX , GR , DHAR , and MDHAR showed pronounced rises in transcriptional activity (Fig. 9 A-F). Treatments with GSH-Au NCs further enhanced the expression of these upregulated genes in heat-stressed plants, while CAT expression remained suppressed (Fig. 9 A-F), consistent with the observed enzymatic activity pattern. Similarly, POD and GST expression levels increased under heat stress. However, GSH-Au NCs treatment resulted in a non-significant reduction in POD levels, while GST expression was further enhanced under heat stress conditions (Fig. 9 G). Fig. 9 Transcriptional expression of genes encoding antioxidant enzymes under heat stress conditions. A - F Genes encoding GSH-AsA cycle enzymes ( SOD , CAT , APX , GR , MDHAR , and DHAR ). G , H
POD and GST genes. Treatments: CK= control, HS= heat stress, and GAHS = 2 mg L − 1 GSH-Au NCs + heat stress. Data are means ± SE from n = 3 biological replicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) Methylglyoxal and glyoxalase enzyme activities Heat stress significantly increased the accumulation of MG in sweet potato plants, with MG levels rising by 137.5% compared to control plants. However, application of GSH-Au NCs under heat stress effectively reduced MG content by 29.3%. Conversely, heat stress suppressed the activities of Gly I and Gly II by 37.7% and 40.3%, respectively, relative to control plants. Application of GSH-Au NCs under heat stress conditions significantly restored their activities, with Gly I and Gly II increasing by 36.9% and 35.0%, respectively (Fig. 10 A-C). Fig. 10 Methylglyoxal and glyoxalase enzymes under normal and heat stress conditions. A Methylglyoxal (MG) content, B Glyoxalase I (Gly I), and ( C ) Gly II activity. Treatments: CK= control, GA = 2 mg L − 1 GSH-Au NCs, HS= heat stress, and GAHS = 2 mg L − 1 GSH-Au NCs + heat stress. Data are means ± SE from n = 3 biological replicates. Different letters indicate significant differences (Duncan’s test, P < 0.05) Pearson correlation analysis The Pearson correlation matrix displayed significant relationships among key physiological parameters in heat-stressed sweet potato plants. The results showed strong positive correlations between oxidative stress markers (MDA, H 2 O 2 , O 2 •− , and MG), EL, and soluble sugar accumulation under heat stress conditions. In contrast, GSH-Au NCs treatment revealed significant negative correlations with these stress indicators (Fig. 11 ; Table S3), suggesting its protective role against oxidative damage. Furthermore, GSH-Au NCs exhibited positive correlations with growth and physiological parameters, such as plant biomass, gas exchange parameters, chlorophyll and carotenoid content, chlorophyll fluorescence, Rubisco activity, and components of the antioxidant defense system (except CAT). Additionally, positive associations were observed between GSH-Au NCs and glyoxalase enzymes, secondary metabolites accumulation, proline content, and stomatal morphological traits (Fig. 11 ; Table S3). Together, these results highlight the efficacy of GSH-Au NCs in enhancing the antioxidant defense mechanisms and improving thermotolerance in sweet potato plants. Fig. 11 Pearson correlation analysis of growth, physio-biochemical parameters in sweet potato plants under heat stress with GSH-Au NCs treatments. The abbreviations of given parameters are the same as mentioned above; SFW (shoot fresh weight), SDW (shoot dry weight), RFW (root fresh weight), RWC (relative water content), and so on. In the correlation matrix, orange-colored lines indicate positive correlation, while light blue dotted lines indicate negative correlation Discussion Heat stress impaired sweet potato growth, biomass, RWC, and root morphology (Fig. 1 ; S2), likely due to disrupted cell division, metabolism, and photosynthesis. These changes hinder nutrient and water uptake, further intensifying stress, as observed in various plants under stress conditions [ 10 , 40 , 41 ]. Unlike intensive conventional agriculture, nanotechnology enables sustainable production using nanomaterials that may simultaneously support stress adaptation and nutrient uptake efficiency [ 15 , 42 ]. In the present study, foliar application of GSH-Au NCs (2 mg L − 1 ) was associated with substantial mitigation of heat stress effects, contributing to recovery in shoot and root biomass, plant height, leaf development, and root architecture in sweet potato plants (Fig. 1 ; S2). These observations are consistent with studies where nanomaterials (Se, Ag, and TiO 2 ) improved growth, biomass, and RWC in crops such as tomato, wheat, and Chrysanthemum morifolium under heat stress [ 43 – 46 ]. The observed recovery in root traits (Fig. S2) may have helped compensate for stress-induced photoassimilate imbalances, potentially supporting nutrient and water uptake, promoting plant growth through enhanced turgor and cell expansion [ 47 , 48 ]. GSH-Au NCs were also associated with elevated RWC, likely linked to osmolyte accumulation, particularly proline. This pattern is consistent with reports on carbon-NPs. AgNPs and ZnONPs, which contributed to improved cellular homeostasis, membrane integrity, and water retention under heat stress conditions [ 45 , 49 , 50 ]. These responses suggest that GSH-Au NCs may support stress mitigation across growth, water balance, photosynthetic performance, metabolism, and structural integrity. Photosynthesis is highly sensitive to elevated temperatures, which can impair biochemical and structural components of the photosynthesis apparatus [ 51 ]. In this study, heat stress drastically reduced gas exchange parameters (Fig. 3 F-I), likely limiting CO 2 uptake and evaporative cooling, as previously reported in tomato, sorghum, and Chrysanthemum morifolium [ 52 , 53 ]. Heat stress also suppressed chlorophyll fluorescence (Fig. 3 A-E), indicating possible photosystem II (PSII) damage and impaired electron transport, contributing to reduced Fv/Fm ratio and impeding antenna molecules. These changes may result from altered enzyme activity, guard cell dysfunction, and ETC inhibition [ 47 ]. Accordingly, reduced Rubisco activity, chlorophyll and carotenoid content, and intensity were observed in sweet potato leaves (Figs. 2 and 3 ). However, GSH-Au NCs was associated with improved Gs, Tr, Pn, and Ci, suggesting enhanced mesophyll conductance and carbon assimilation. GSH-Au NCs treatment also contributed to recovery in chlorophyll fluorescence parameters (Fv/Fm, Y(II), qP, and ETR), increased chlorophyll and carotenoid content, and pigment fluorescence intensity (Figs. 2 and 3 ), and reduced qN, indicating improved PSII efficiency and photoprotective capacity. These responses are consistent with reports on Au, TiO 2 , MnO, and Se nanomaterials, which have been associated with mitigation of photoinhibition, ROS scavenging and preservation of gas exchange in tomato, coriander, and wheat [ 47 , 54 – 57 ]. Enhanced Rubisco activity (Fig. 2 J) further supports improved regulation of the Calvin-Benson cycle, consistent with previous findings on TiO 2 and CeO 2 [ 58 , 59 ]. These improvements are consistent with reduced oxidative stress contributing to sustained photosynthetic performance and energy production in sweet potato under heat stress. Heat stress can damage stomata and ultrastructure, disrupting gas exchange and photosynthetic efficiency [ 10 ]. In this study, sweet potato leaves exhibited reduced stomatal density, length, width, and pore length (Fig. 4 ), along with disorganized mesophyll cells, chloroplasts, and impaired mitochondria (Fig. 5 ). These structural alterations may hinder light-dependent reactions and mitochondrial ATP synthesis [ 8 , 52 ]. GSH-Au NCs was associated with improved stomatal traits (density, length, width, and pore length) (Fig. 4 ), potentially facilitating gas exchange, evaporative cooling, and CO 2 uptake. These observations align with reports on CeO 2 , ZnO, and Zn-Si NPs enhancing stomatal opening in sorghum and pea [ 52 , 60 ]. Electron microscopy revealed that GSH-Au NCs were associated with preserved mesophyll integrity, chloroplast structure (thylakoids, stroma, and grana), and mitochondrial morphology (Fig. 5 ), likely reducing oxidative damage. Preserved chloroplasts may support photosynthesis, while intact mitochondria may ensure energy metabolism. GSH-Au NC treatment also appeared to increase plastoglobuli accumulation, which may contribute to lipid metabolism and stress resilience. These structural responses are consistent with previous studies on AuNPs and SiO 2 NPs in wheat and rice, and CeO 2 NPs under cobalt stress [ 57 , 61 , 62 ]. These findings suggest that GSH-Au NCs may contribute to maintaining stomatal function and ultrastructure, thereby supporting photosynthetic recovery. Heat stress triggers excessive ROS generation, leading to oxidative and carbonyl stress that can damage lipids, proteins, and DNA [ 8 , 11 ]. In this study, heat-stressed sweet potato plants showed elevated H 2 O 2 , O 2 •− , MDA, MG, and EL, confirmed by histochemical staining (H 2 O 2 , O 2 •− , and MDA; Fig. 6 A-E). These effects are likely linked to photosynthesis decline and excess electron leakage. Soluble sugar accumulation (Fig. 6 G) may reflect osmotic adjustment but could also indicate disrupted carbohydrate metabolism [ 63 ]. GSH-Au NCs treatment was associated with reduced oxidative markers (H 2 O 2 , O 2 •− , MDA, MG, and EL) and less intense histochemical staining (Fig. 6 A-E). These findings are consistent with previous studies on Se, S, CeO 2 , ZnO, and TiO 2 nanomaterials suppressing oxidative damage under abiotic stress [ 44 , 47 , 64 ]. GSH-Au NCs also appeared to reduce soluble sugar levels, suggesting restored photosynthetic activity and carbohydrate metabolism toward growth and energy production [ 63 , 65 ]. Furthermore, GSH-Au NCs were associated with increased glyoxalase I and II activity (Fig. 10 ), likely contributing to MG detoxification through the GSH-dependent glyoxalase pathway. This pattern aligns with previous reports on nanoparticle-enhanced glyoxalase activity in other plant systems [ 47 , 66 ]. Together, these coordinated responses suggest that GSH-Au NCs may strengthen redox homeostasis and thermotolerance through multiple antioxidant layers. The AsA-GSH cycle plays a central role in antioxidant defense by detoxifying ROS and maintaining redox balance under heat stress [ 67 ]. Our results show that GSH-Au NCs was associated with increased AsA and GSH levels, elevated AsA/DHA and GSH/GSSG ratios (Fig. 7 A-F), indicating enhanced antioxidant capacity. Treatment also upregulated both the enzymatic activity (SOD, APX, GR, DHAR, and MDHAR) and gene expression of AsA-GSH cycle (Figs. 8 and 9 ). These responses are consistent with reports on carbon nanotubes, Se, S, and MnO nanomaterials enhancing AsA-GSH components in in Paeonia ostii , wheat, and tomato [ 47 , 66 , 68 , 69 ]. Upregulation of these AsA-GSH cycle genes may involve HSF, MYB, and WRKY transcription factors, potentially activated by ROS or NP-induced MAPK signaling [ 50 , 70 ]. Interestingly, GSH-Au NCs were associated with reduced CAT activity (Figs. 8 B and 9 B), likely due to decreased H 2 O 2 load from enhanced AsA-GSH pathway activity, consistent with ZnO-NP studies [ 71 , 72 ]. In contrast, increased POD and GST activity (Fig. 8 G-H) may aid in peroxides and xenobiotics detoxification [ 63 , 66 ]. GSH-Au NCs was also associated with enhanced proline levels (Fig. 6 F), likely supporting osmotic balance, membrane stability, and ROS scavenging in sweet potato plants under heat stress. This is consistent with reports on various nanoparticles increasing proline accumulation under abiotic stress [ 54 , 70 ]. Additionally, GSH-Au NC treatment contributed to higher secondary metabolites (polyphenols and flavonoids) in sweet potato leaves under heat stress (Fig. 6 H-I), which may contribute to ROS scavenging and photosynthetic recovery through phenylpropanoid pathway [ 13 , 73 ]. These observations align with effects of FeNPs, SeNPs, Ag NPs, and SiNPs in various plants [ 49 , 63 , 66 , 74 ]. Overall, the present study demonstrated that GSH-Au NCs (2 mg L − 1 ) was associated with substantial alleviation of heat stress in sweet potato plants. Mechanistically, GSH-Au NCs likely modulates redox balance by direct ROS scavenging (evidenced by reduced H 2 O 2 , O 2 •− , MDA, and EL) and transcriptional upregulation of AsA-GSH genes ( SOD , APX , GR , MDHAR , and DHAR ), consistent with Au NP effects [ 25 – 27 ]. Speculatively, Au’s catalytic properties may enhance GSH stability and regeneration, though enzyme kinetics require further confirmation. Similar to Au NPs in wheat (enhanced ROS scavenging) [ 26 ], our results show reduced oxidative damage; however, the GSH-Au NC synergy uniquely elevated GSH/GSSG by 29.1%, a response not reported in separate applications, highlighting novelty while aligning with GSH’s role in tomato [ 23 ]. Additionally, increased glyoxalase activities (Gly I and Gly II) by GSH-Au NC treatment contributed to lower methylglyoxal levels, further supporting redox homeostasis. These coordinated responses collectively contributed to recovery in growth, biomass, photosynthetic efficiency, and cellular ultrastructure while minimizing oxidative damage through ROS scavenging and AsA-GSH cycle activation. These findings position GSH-Au NCs as a promising, sustainable solution for crop resilience under abiotic stress. However, limitations include the lack of direct confirmation of nanocluster uptake and distribution via ICP-MS or TEM, reliance on short-term greenhouse assessments that may not fully reflect field variability or long-term outcomes, and absence of direct toxicity profiling. Future studies should prioritize localization, extended field trials, and multi-season evaluations. Practically, GSH-Au NCs offers a low-cost and eco-friendly solution for high temperature regions, but these limitations necessitate cautious interpretation. Conclusions This study demonstrates the first comprehensive evidence that GSH-Au NCs (2 mg L − 1 ) was associated with substantial enhancement of thermotolerance in vegetable sweet potato, contributing to improved growth, biomass, photosynthetic efficiency, and structural integrity. These effects were linked to reduced oxidative and carbonyl stress through activation of the AsA-GSH cycle, upregulation of antioxidant enzyme activities and gene expression (SOD, APX, GR, DHAR, MDHAR, POD, and GST), enhanced glyoxalase system (Gly I and II), and osmolyte (proline) accumulation. GSH-Au NC treatment also contributed to recovery in stomatal function, chloroplast and mitochondria ultrastructure, and increased secondary metabolites (polyphenols and flavonoids), further supporting resilience. These findings highlight GSH-Au NCs as a sustainable, cost-effective, and eco-friendly strategy for mitigating heat stress in sweet potato and potentially other crops, advancing beyond prior work on individual GSH supplementation or Au nanomaterials by demonstrating their combined nanocluster synergy. However, limitations include lack of direct However, further research is needed to confirm direct uptake, elucidate molecular mechanisms, validate these findings under field conditions, and assess long-term environmental impact and safety of nanomaterials application in agricultural crop systems. Supplementary Information
Supplementary Material 1: Table S1. Impact of glutathione-gold nanoclusters (GSH-Au NCs) on the growth and biomass of vegetable sweet potato under normal and heat stress conditions. Table S2. List of the primers of genes encoding GSH-AsA cycle enzymes and antioxidant POD and GST enzymes. Table S3. Table of Pearson’s correlation analysis. Fig. S1. Characterization of GSH-Au nanoclusters (GSH-Au NCs). Fig. S2. Impact of glutathione-gold nanoclusters (GSH-Au NCs) on the root traits of vegetable sweet potato under normal and heat stress conditions.
Acknowledgements This research was supported by the Project of Sanya Yazhou Bay Science and Technology City “Yazhou Bay” Jingying Talent Project (SKJC-JYRC-2024-14; SKJC-JYRC-2024-25), the Scientific Research Start-up Funding Project of Hainan University (XJ2400007871), the earmarked fund for CARS-10-Sweetpotato, and the Innovation Platform for Academicians of Hainan Province (YSPTZX202206). We are also highly thankful to the National Tropical Plants Germplasm Resource Center. Authors’ contributions Sunjeet Kumar: Conceptualization, Project administration, Resources, Writing - original draft, Validation, Methods, Visualization. Gurmendar: Investigation, Formal analysis, Writing original draft. Rui Yu: Investigation. Muhammad Ikram: Software. Jingjing Kou: Writing review \& editing. Muhammad Abbas Khan: Writing - review \& editing. Mengzhao Wang: Visualization, Project administration, Data curation, Validation. Guopeng Zhu: Conceptualization, Resources, Supervision, Funding acquisition. Data availability Data will be made available on request. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Sunjeet Kumar and Gurmendar contributed equally to this work. Contributor Information Mengzhao Wang, Email: mzwang@hainanu.edu.cn. Guopeng Zhu, Email: zhuguopeng@hainanu.edu.cn. References 1. Shelake RM, Kadam US, Kumar R, Pramanik D, Singh AK, Kim J-Y. Engineering drought and salinity tolerance traits in crops through CRISPR-mediated genome editing: Targets, tools, challenges, and perspectives. Plant Commun. 2022;3:100417. 10.1016/j.xplc.2022.100417.
2. Teng Z, Chen C, He Y, Pan S, Liu D, Zhu L, et al. Melatonin confers thermotolerance and antioxidant capacity in Chinese cabbage. Plant Physiol Biochem. 2024;212:108736. 10.1016/j.plaphy.2024.108736.
3. Tan Y, Cao Y, Mou F, Liu B, Wu H, Zou S, et al. Transcriptome profiling of two Camellia japonica cultivars with different heat tolerance reveals heat stress response mechanisms. Plants. 2024;13:3089. 10.3390/plants13213089.
4. El-Ramady H, Prokisch J, El-Mahrouk ME, Bayoumi YA, Shalaby TA, Brevik EC, et al. Nano-food farming approaches to mitigate heat stress under ongoing climate change: A review. Agriculture. 2024;14:656. 10.3390/agriculture14050656. 5. Kumar A, Bhattacharya T, Mukherjee S, Sarkar B. A perspective on biochar for repairing damages in the soil–plant system caused by climate change-driven extreme weather events. Biochar. 2022;4:22. 10.1007/s42773-022-00148-z. 6. Guo S, Hu X, Yu F, Mu L. Heat waves coupled with nanoparticles induce yield and nutritional losses in rice by regulating stomatal closure. ACS Nano. 2024;18:14276–89. 10.1021/acsnano.3c13165.
7. Lee C-C, Zeng M, Luo K. How does climate change affect food security? Evidence from China. Environ Impact Assess Rev. 2024;104:107324. 10.1016/j.eiar.2023.107324. 8. Zhang L, Chang Q, Hou X, Wang J, Chen S, Zhang Q, et al. The effect of high-temperature stress on the physiological indexes, chloroplast ultrastructure, and photosystems of two herbaceous peony cultivars. J Plant Growth Regul. 2023;42:1631–46. 10.1007/s00344-022-10647-9. 9. Toprak S, Coşkun ÖF. Heat stress mitigation by zinc oxide nanoparticles in pepper and watermelon. BMC Agric. 2026;2:2. 10.1186/s44399-025-00024-8. 10. Hu D, Zhang X, Xue P, Nie Y, Liu J, Li Y, et al. Exogenous melatonin ameliorates heat damages by regulating growth, photosynthetic efficiency and leaf ultrastructure of carnation. Plant Physiol Biochem. 2023;198:107698. 10.1016/j.plaphy.2023.107698.
11. Li Z-G, Xu Y, Bai L-K, Zhang S-Y, Wang Y. Melatonin enhances thermotolerance of maize seedlings ( Zea mays L.) by modulating antioxidant defense, methylglyoxal detoxification, and osmoregulation systems. Protoplasma. 2019;256:471–90. 10.1007/s00709-018-1311-4.
12. Sun C, Meng S, Wang B, Zhao S, Liu Y, Qi M, et al. Exogenous melatonin enhances tomato heat resistance by regulating photosynthetic electron flux and maintaining ROS homeostasis. Plant Physiol Biochem. 2023;196:197–209. 10.1016/j.plaphy.2023.01.043.
13. Kumar S, Yu R, Liu Y, Liu Y, Khan MN, Liu Y, et al. Exogenous melatonin enhances heat stress tolerance in sweetpotato by modulating antioxidant defense system, osmotic homeostasis and stomatal traits. Hortic Plant J. 2025;11:431–45. 10.1016/j.hpj.2023.12.006. 14. Chen F, Shen Z, Shi R, Zhang X, Zhang H, Li W, et al. Carbon dots-mediated plant adaptive responses to abiotic stress. Mater Res Bull. 2025;182. 10.1016/j.materresbull.2024.113137. October 2024:113137. 15. Ahmed T, Masood HA, Noman M, AL-Huqail AA, Alghanem SM, Khan MM, et al. Biogenic silicon nanoparticles mitigate cadmium (Cd) toxicity in rapeseed ( Brassica napus L.) by modulating the cellular oxidative stress metabolism and reducing Cd translocation. J Hazard Mater. 2023;459:132070. 10.1016/j.jhazmat.2023.132070.
16. Ghorbani A, Emamverdian A, Pehlivan N, Zargar M, Razavi SM, Chen M. Nano-enabled agrochemicals: mitigating heavy metal toxicity and enhancing crop adaptability for sustainable crop production. J Nanobiotechnol. 2024;22:91. 10.1186/s12951-024-02371-1. 17. Farooq MA, Islam F, Ayyaz A, Chen W, Noor Y, Hu W, et al. Mitigation effects of exogenous melatonin-selenium nanoparticles on arsenic-induced stress in Brassica napus. Environ Pollut. 2022;292:118473. 10.1016/j.envpol.2021.118473.
18. Khan MN, Li Y, Khan Z, Chen L, Liu J, Hu J, et al. Nanoceria seed priming enhanced salt tolerance in rapeseed through modulating ROS homeostasis and α-amylase activities. J Nanobiotechnol. 2021;19:276. 10.1186/s12951-021-01026-9. 19. Ulhassan Z, Yang S, He D, Khan AR, Salam A, Azhar W, et al. Seed priming with nano-silica effectively ameliorates chromium toxicity in Brassica napus . J Hazard Mater. 2023;458:131906. 10.1016/j.jhazmat.2023.131906.
20. Wang C, Liu X, Li J, Yue L, Yang H, Zou H, et al. Copper nanoclusters promote tomato ( Solanum lycopersicum L.) yield and quality through improving photosynthesis and roots growth. Environ Pollut. 2021;289:117912. 10.1016/j.envpol.2021.117912.
21. Gao M, Chang J, Wang Z, Zhang H, Wang T. Advances in transport and toxicity of nanoparticles in plants. J Nanobiotechnol. 2023;21:75. 10.1186/s12951-023-01830-5. 22. Mahakham W, Sarmah AK, Maensiri S, Theerakulpisut P. Nanopriming technology for enhancing germination and starch metabolism of aged rice seeds using phytosynthesized silver nanoparticles. Sci Rep. 2017;7:1–21. 10.1038/s41598-017-08669-5.
23. Nahar K, Hasanuzzaman M, Alam MM, Fujita M. Exogenous glutathione confers high temperature stress tolerance in mung bean ( Vigna radiata L.) by modulating antioxidant defense and methylglyoxal detoxification system. Environ Exp Bot. 2015;112:44–54. 10.1016/j.envexpbot.2014.12.001. 24. Kumar S, Wang S, Wang M, Zeb S, Khan MN, Chen Y, et al. Enhancement of sweetpotato tolerance to chromium stress through melatonin and glutathione: Insights into photosynthetic efficiency, oxidative defense, and growth parameters. Plant Physiol Biochem. 2024;208:108509. 10.1016/j.plaphy.2024.108509.
25. Feichtmeier NS, Walther P, Leopold K. Uptake, effects, and regeneration of barley plants exposed to gold nanoparticles. Environ Sci Pollut Res. 2015;22:8549–58. 10.1007/s11356-014-4015-0. 26. Ferrari E, Barbero F, Busquets-Fité M, Franz-Wachtel M, Köhler H-R, Puntes V, et al. Growth-promoting gold nanoparticles decrease stress responses in Arabidopsis seedlings. Nanomaterials. 2021;11:3161. 10.3390/nano11123161.
27. Venzhik Y, Deryabin A, Naraikina N, Zhukova K, Dykman L. The influence of Au-based nanoparticles on some physiological, biochemical and molecular characteristics of wheat plants during low temperature hardening. Plant Physiol Biochem. 2024;213:108837. 10.1016/j.plaphy.2024.108837.
28. Ji CY, Jin R, Xu Z, Kim HS, Lee C-J, Kang L, et al. Overexpression of Arabidopsis P3B increases heat and low temperature stress tolerance in transgenic sweetpotato. BMC Plant Biol. 2017;17:139. 10.1186/s12870-017-1087-2.
29. Yu J, Su D, Yang D, Dong T, Tang Z, Li H, et al. Chilling and heat stress-induced physiological changes and microRNA-related mechanism in sweetpotato ( Ipomoea batatas L). Front Plant Sci. 2020;11:687. 10.3389/fpls.2020.00687.
30. Kang L, Kim HS, Kwon YS, Ke Q, Ji CY, Park S-C, et al. IbOr regulates photosynthesis under heat stress by stabilizing IbPsbP in sweetpotato. Front Plant Sci. 2017;8:00989. 10.3389/fpls.2017.00989. 31. Wijewardana C, Reddy KR, Shankle MW, Meyers S, Gao W. Low and high-temperature effects on sweetpotato storage root initiation and early transplant establishment. Sci Hortic. 2018;240:38–48. 10.1016/j.scienta.2018.05.052. 32. Kumar S, Liu Y, Wang M, Khan MN, Wang S, Li Y, et al. Alleviating sweetpotato salt tolerance through exogenous glutathione and melatonin: A profound mechanism for active oxygen detoxification and preservation of photosynthetic organs. Chemosphere. 2024;350:141120. 10.1016/j.chemosphere.2024.141120.
33. Mumtaz MA, Hao Y, Mehmood S, Shu H, Zhou Y, Jin W, et al. Physiological and transcriptomic analysis provide molecular insight into 24-epibrassinolide mediated Cr(VI)-toxicity tolerance in pepper plants. Environ Pollut. 2022;306:119375. 10.1016/j.envpol.2022.119375.
34. Khan MA, Kumar S, Wang Q, Wang M, Fahad S, Nizamani MM, et al. Influence of polyvinyl chloride microplastic on chromium uptake and toxicity in sweet potato. Ecotoxicol Environ Saf. 2023;251:114526. 10.1016/j.ecoenv.2023.114526.
35. Altaf MA, Hao Y, Shu H, Mumtaz MA, Cheng S, Alyemeni MN, et al. Melatonin enhanced the heavy metal-stress tolerance of pepper by mitigating the oxidative damage and reducing the heavy metal accumulation. J Hazard Mater. 2023;454:131468. 10.1016/j.jhazmat.2023.131468.
36. Kaya C, Ashraf M, Alyemeni MN, Rinklebe J, Ahmad P. Alleviation of arsenic toxicity in pepper plants by aminolevulinic acid and heme through modulating its sequestration and distribution within cell organelles. Environ Pollut. 2023;330:121747. 10.1016/j.envpol.2023.121747.
37. Mohammadi H, Abdollahi-Bastam S, Aghaee A, Ghorbanpour M. Foliar-applied silicate potassium modulates growth, phytochemical, and physiological traits in Cichorium intybus L. under salinity stress. BMC Plant Biol. 2024;24:288. 10.1186/s12870-024-05015-6.
38. Kumar S, Huang X, Li G, Ji Q, Zhou K, Zhu G, et al. Comparative transcriptomic analysis provides novel insights into the blanched stem of Oenanthe javanica . Plants. 2021;10:2484. 10.3390/plants10112484.
39. Kumar S, Huang X, Ji Q, Qayyum A, Zhou K, Ke W, et al. Influence of blanching on the gene expression profile of phenylpropanoid, flavonoid and vitamin biosynthesis, and their accumulation in Oenanthe javanica . Antioxidants. 2022;11:470. 10.3390/antiox11030470.
40. Ullah I, Toor MD, Yerlikaya BA, Mohamed HI, Yerlikaya S, Basit A, et al. High-temperature stress in strawberry: understanding physiological, biochemical and molecular responses. Planta. 2024;260:118. 10.1007/s00425-024-04544-6.
41. Xing X, Ding Y, Jin J, Song A, Chen S, Chen F, et al. Physiological and transcripts analyses reveal the mechanism by which melatonin alleviates heat stress in chrysanthemum seedlings. Front Plant Sci. 2021;12:673236. 10.3389/fpls.2021.673236.
42. Yao Y, Yue L, Cao X, Chen F, Li J, Cheng B, et al. Carbon dots embedded in nanoporous SiO 2 nanoparticles for enhancing photosynthesis in agricultural crops. ACS Appl Nano Mater. 2023;6:110–8. 10.1021/acsanm.2c03843. 43. El-Saadony MT, Saad AM, Najjar AA, Alzahrani SO, Alkhatib FM, Shafi ME, et al. The use of biological selenium nanoparticles to suppress Triticum aestivum L. crown and root rot diseases induced by Fusarium species and improve yield under drought and heat stress. Saudi J Biol Sci. 2021;28:4461–71. 10.1016/j.sjbs.2021.04.043.
44. El-Saadony MT, Saad AM, Soliman SM, Salem HM, Desoky E-SM, Babalghith AO, et al. Role of nanoparticles in enhancing crop tolerance to abiotic stress: A comprehensive review. Front Plant Sci. 2022;13:946717. 10.3389/fpls.2022.946717.
45. Haghighi M, Abolghasemi R, Teixeira da Silva JA. Low and high temperature stress affect the growth characteristics of tomato in hydroponic culture with Se and nano-Se amendment. Sci Hortic. 2014;178:231–40. 10.1016/j.scienta.2014.09.006. 46. Seliem MK, Hafez Y, El-Ramady H. Using of nano-selenium in reducing the negative effects of high temperature stress on Chrysanthemum morifolium Ramat. J Sustainable Agricultural Sci. 2020;46:47–59. 10.21608/jsas.2020.23905.1203. 47. Babzada SA, Raja V, Bhat AH, Qadir SU, Radhakrishnan A, Kumar N, et al. Alleviating lanthanum stress in tomato plants using MnO nanoparticles and triacontanol: Impacts on growth, photosynthesis, and antioxidant defense. J Hazard Mater. 2025;491:137746. 10.1016/j.jhazmat.2025.137746.
48. Zhang K, Han X, Fu Y, Khan Z, Zhang B, Bi J, et al. Biochar coating promoted rice growth under drought stress through modulating photosynthetic apparatus, chloroplast ultrastructure, stomatal traits and ROS homeostasis. Plant Physiol Biochem. 2024;216. 10.1016/j.plaphy.2024.109145. September:109145. 49. Iqbal M, Raja NI, Mashwani Z, Wattoo FH, Hussain M, Ejaz M, et al. Assessment of AgNPs exposure on physiological and biochemical changes and antioxidative defence system in wheat ( Triticum aestivum L) under heat stress. IET Nanobiotechnol. 2019;13:230–6. 10.1049/iet-nbt.2018.5041.
50. Cao Y, Turk K, Bibi N, Ghafoor A, Ahmed N, Azmat M, et al. Nanoparticles as catalysts of agricultural revolution: Enhancing crop tolerance to abiotic stress: a review. Front Plant Sci. 2025;15:1510482. 10.3389/fpls.2024.1510482.
51. Teng L, Qing L, Shumei W, Xuepeng Z, Yuanquan C, Wangsheng G, et al. High temperature effects on maize photosynthesis during stress and recovery phase at the seed setting stage. BMC Plant Biol. 2025;25:454. 10.1186/s12870-025-06047-2.
52. Djanaguiraman M, Belliraj N, Bossmann SH, Prasad PVV. High-temperature stress alleviation by selenium nanoparticle treatment in grain sorghum. ACS Omega. 2018;3:2479–91. 10.1021/acsomega.7b01934.
53. Jahan MS, Hasan MM, Alotaibi FS, Alabdallah NM, Alharbi BM, Ramadan KMA, et al. Exogenous putrescine increases heat tolerance in tomato seedlings by regulating chlorophyll metabolism and enhancing antioxidant defense efficiency. Plants. 2022;11:1038. 10.3390/plants11081038.
54. Omar AA, Heikal YM, Zayed EM, Shamseldin SAM, Salama YE, Amer KE, et al. Conferring of drought and heat stress tolerance in wheat ( Triticum aestivum L.) genotypes and their response to selenium nanoparticles application. Nanomaterials. 2023;13:998. 10.3390/nano13060998.
55. Qi M, Liu Y, Li T. Nano-TiO2 improve the photosynthesis of tomato leaves under mild heat stress. Biol Trace Elem Res. 2013;156:323–8. 10.1007/s12011-013-9833-2.
56. Sardar R, Ahmed S, Yasin NA. Titanium dioxide nanoparticles mitigate cadmium toxicity in Coriandrum sativum L. through modulating antioxidant system, stress markers and reducing cadmium uptake. Environ Pollut. 2022;292:118373. 10.1016/j.envpol.2021.118373.
57. Venzhik Y, Deryabin A, Popov V, Dykman L, Moshkov I. Priming with gold nanoparticles leads to changes in the photosynthetic apparatus and improves the cold tolerance of wheat. Plant Physiol Biochem. 2022;190:145–55. 10.1016/j.plaphy.2022.09.006.
58. Cao Z, Stowers C, Rossi L, Zhang W, Lombardini L, Ma X. Physiological effects of cerium oxide nanoparticles on the photosynthesis and water use efficiency of soybean ( Glycine max (L.) Merr). Environ Sci Nano. 2017;4:1086–94. 10.1039/C7EN00015D. 59. Sidhu AK, Sharma M, Bhickchand Agrawal S, Pradip Bhavsar P, Samota MK. Nanomaterial strategies for enhancing plant resilience in the face of temperature stress. CABI Agric Bioscience. 2024;5:60. 10.1186/s43170-024-00255-w. 60. Elshoky HA, Yotsova E, Farghali MA, Farroh KY, El-Sayed K, Elzorkany HE, et al. Impact of foliar spray of zinc oxide nanoparticles on the photosynthesis of Pisum sativum L. under salt stress. Plant Physiol Biochem. 2021;167:607–18. 10.1016/j.plaphy.2021.08.039.
61. Muhammad S, Ulhassan Z, Munir R, Yasin MU, Islam F, Zhang K, et al. Nanosilica and salicylic acid synergistically regulate cadmium toxicity in rice. Environ Pollut. 2025;364:125331. 10.1016/j.envpol.2024.125331.
62. Salam A, Qi J, Fan X, Khan AR, Kah M, Zeeshan M, et al. Cerium oxide nanoparticle protects maize from cobalt stress: Insights from transcriptomics and oxidative stress response analysis. ACS Appl Mater Interfaces. 2025;17:36455–68. 10.1021/acsami.5c05835.
63. Shahzad R, Koerniati S, Harlina PW, Hastilestari BR, Djalovic I, Prasad PVV. Iron oxide nanoparticles enhance alkaline stress resilience in bell pepper by modulating photosynthetic capacity, membrane integrity, carbohydrate metabolism, and cellular antioxidant defense. BMC Plant Biol. 2025;25:170. 10.1186/s12870-025-06180-y.
64. Rehman A, Khan S, Sun F, Peng Z, Feng K, Wang N, et al. Exploring the nano-wonders: Unveiling the role of nanoparticles in enhancing salinity and drought tolerance in plants. Front Plant Sci. 2024;14:1324176. 10.3389/fpls.2023.1324176.
65. Dang K, Mu J, Tian H, Gao D, Zhou H, Guo L, et al. Zinc regulation of chlorophyll fluorescence and carbohydrate metabolism in saline-sodic stressed rice seedlings. BMC Plant Biol. 2024;24:464. 10.1186/s12870-024-05170-w.
66. Shah T, Khan Z, Alahmadi TA, Shah MA, Ahmad MZ, Rasool S, et al. Nanoselenium inhibits chromium toxicity in wheat plants by modifying the antioxidant defense system, ascorbate glutathione cycle, and glyoxalase system. Environ Exp Bot. 2024;220:105697. 10.1016/j.envexpbot.2024.105697. 67. Jahan MS, Shu S, Wang Y, Chen Z, He M, Tao M, et al. Melatonin alleviates heat-induced damage of tomato seedlings by balancing redox homeostasis and modulating polyamine and nitric oxide biosynthesis. BMC Plant Biol. 2019;19:414. 10.1186/s12870-019-1992-7.
68. Riaz M, Zhao S, Kamran M, Ur Rehman N, Mora-Poblete F, Maldonado C, et al. Effect of nano-silicon on the regulation of ascorbate-glutathione contents, antioxidant defense system and growth of copper stressed wheat ( Triticum aestivum L.) seedlings. Front Plant Sci. 2022;13:986991. 10.3389/fpls.2022.986991.
69. Zhao D, Wang X, Cheng Z, Tang Y, Tao J. Multi-walled carbon nanotubes prevent high temperature-induced damage by activating the ascorbate-glutathione cycle in Paeonia ostii T. Hong et J. X. Zhang. Ecotoxicol Environ Saf. 2021;227:112948. 10.1016/j.ecoenv.2021.112948.
70. Kumari A, Gupta AK, Sharma S, Jadon VS, Sharma V, Chun SC, et al. Nanoparticles as a tool for alleviating plant stress: Mechanisms, implications, and challenges. Plants. 2024;13:1528. 10.3390/plants13111528.
71. Priyanka N, Venkatachalam P. Biofabricated zinc oxide nanoparticles coated with phycomolecules as novel micronutrient catalysts for stimulating plant growth of cotton. Adv Nat Sci NanoSci NanoTechnol. 2016;7:045018. 10.1088/2043-6262/7/4/045018. 72. Rajput VD, Minkina T, Kumari A, Harish, Singh VK, Verma KK, et al. Coping with the challenges of abiotic stress in plants: New dimensions in the field application of nanoparticles. Plants. 2021;10:1221. 10.3390/plants10061221.
73. Liu Y, Cao X, Yue L, Wang C, Tao M, Wang Z, et al. Foliar-applied cerium oxide nanomaterials improve maize yield under salinity stress: Reactive oxygen species homeostasis and rhizobacteria regulation. Environ Pollut. 2022;299:118900. 10.1016/j.envpol.2022.118900.
74. Ulhassan Z, Ali S, Kaleem Z, Shahbaz H, He D, Khan AR, et al. Effects of Nanosilica priming on rapeseed ( Brassica napus ) tolerance to cadmium and arsenic stress by regulating cellular metabolism and antioxidant defense. J Agric Food Chem. 2025;73:4518–33. 10.1021/acs.jafc.4c08246.
Associated Data Supplementary Materials Supplementary Material 1: Table S1. Impact of glutathione-gold nanoclusters (GSH-Au NCs) on the growth and biomass of vegetable sweet potato under normal and heat stress conditions. Table S2. List of the primers of genes encoding GSH-AsA cycle enzymes and antioxidant POD and GST enzymes. Table S3. Table of Pearson’s correlation analysis. Fig. S1. Characterization of GSH-Au nanoclusters (GSH-Au NCs). Fig. S2. Impact of glutathione-gold nanoclusters (GSH-Au NCs) on the root traits of vegetable sweet potato under normal and heat stress conditions. Data Availability Statement Data will be made available on request.