Quinoa (Chenopodium quinoa Willd., Amaranthaceae) is an underutilized protein-rich crop (Andreotti et al., 2022). Native to the Andean highlands and the lowlands of Central Chile in South America, this pseudo cereal is widely cultivated for its edible grains, primarily for human consumption (Bazile et al., 2016a, b). Quinoa is recognized for its resilience to climate stress and its capacity to thrive under harsh and unfavorable growing conditions (Choukr-Allah et al., 2016; Pulvento and Bazile, 2023). Quinoa grains possess a notably higher protein content than staple cereal crops such as barley, wheat, corn, and rice (Scanlin and Lewis, 2017; García-Parra et al., 2020). Its exceptional nutritional profile stems from a complete set of all nine essential amino acids (Pereira et al., 2019; Angeli et al., 2020). Moreover, it is gluten-free, making it suitable for individuals with celiac disease (Ceyhun Sezgin and Sanlier, 2019; Dakhili et al., 2019). Quinoa is therefore often marketed as a “functional food” or “superfood, “enhancing its global commercial appeal (Noulas et al., 2017). Consequently, quinoa cultivation has expanded significantly in recent years, with the crop now grown commercially in numerous regions beyond its native South America (Jacobsen, 2017; Alandia et al., 2020).

Young vegetative quinoa (YVQ), an unconventional leafy green vegetable, has recently gained scientific attention (Rubinovich et al., 2023, 2025; Huang et al., 2024). Resembling spinach in both texture and culinary versatility, the tender stalks and green leaves of the quinoa plant are highly nutritious and can be consumed either fresh or cooked (Adamczewska-Sowińska et al., 2021; Pathan and Siddiqui, 2022). YVQ can be cultivated year-round in open fields, greenhouses, or high tunnels (Pathan et al., 2023). Like quinoa grains, the leaves contain all nine essential amino acids; however, their protein content, expressed as a percentage of dry matter (DM), is significantly higher, ranging from 25% to 37%, compared to 9.1% to 15.7% in the grains (Pathan et al., 2019; Vazquez-Luna et al., 2019; Pathan and Siddiqui, 2022; Gómez et al., 2024). Furthermore, quinoa leaves have a higher protein content than other commonly consumed leafy greens such as spinach, chard, and broccoli (Vazquez-Luna et al., 2019). In addition to their superior protein profile, quinoa leaves are rich in copper, manganese, and potassium, and contain moderate levels of salt, calcium, phosphorus, and zinc (Pathan et al., 2019). Studies assessing the nutraceutical potential of YVQ have identified substantial levels of bioactive polyphenols in quinoa leaf extracts, which have been associated with inhibitory effects on the proliferation of prostate cancer cells (Gawlik-Dziki et al., 2013) and anti-inflammatory effects in macrophage cells (Khattib et al., 2025). Another notable advantage of YVQ is its lower concentration of saponins—bitter, antinutritive triterpenoid glycosides—compared to quinoa grains (Lim et al., 2020; Stoleru et al., 2022). YVQ is typically harvested between 30 and 62 days after sowing (DAS) (Pathan et al., 2023; Rubinovich et al., 2023), whereas detectable levels of sapogenins—the triterpenoid aglycone backbone of saponins—are only observed from 82 DAS onward (Dick Mastebroek et al., 2000; Abd El-Samad et al., 2018). Moreover, vegetative quinoa shoots at 60 DAS contain lower saponin levels than those at the reproductive stage (100 DAS) (Bernardo Solíz-Guerrero et al., 2002). These parameters, along with its high yield potential (Abd El-Samad et al., 2018; Adamczewska-Sowińska et al., 2021; Rubinovich et al., 2023, 2025), underscore the potential of cultivated YVQ as a novel, sustainable, and protein-rich leafy crop.

Climate change, associated with global warming, poses a significant threat to global food-production systems (Ramírez-Gil et al., 2019; Zandalinas et al., 2021). Projections indicate that climate change will lead to more frequent and intense heatwaves, with global mean annual temperatures expected to rise by up to 3°C by 2050 and 4.8°C by 2100 (Jagadish et al., 2020; Grüter et al., 2022). Elevated temperatures are perceived by plants as heat stress (Zandalinas et al., 2021), which can severely impair growth through physiological and developmental processes, ultimately leading to substantial yield losses (Fahad et al., 2017; Hassan et al., 2021). Heat stress disrupts key physiological processes in plants, including photosynthesis, respiration, stomatal conductance (g_s), and the regulation of leaf water potential (Sharma and Manjeet, 2020; Shapira et al., 2021). Elevated temperatures can impair net carbon assimilation through multiple mechanisms, such as enhanced photorespiration and mitochondrial respiration, inactivation of Rubisco, and reduced activity of photosystem II (PSII) (Teskey et al., 2015; Slattery and Ort, 2019). Responses of g_s to heat stress are species- and context-dependent, with some plants showing increased conductance and others, a decline (Haworth et al., 2018; Eustis et al., 2020). At the cellular level, heat stress can cause direct damage, including increased thylakoid membrane fluidity, elevated production of reactive oxygen species (ROS), enzyme inactivation, loss of membrane integrity, impaired protein regulation, and protein degradation (Balfagón et al., 2019). These physiological and biochemical disruptions can lead to substantial tissue damage, often evident in twigs and leaves through visible symptoms such as sunburn, leaf senescence, inhibited growth, and discoloration. The severity of these effects depends on the intensity, duration, and developmental stage at which the heat stress occurs (Filewod and Thomas, 2014; Fahad et al., 2017).

Quinoa exhibits considerable variation in heat tolerance among cultivars, with differences reported in photosynthetic performance and reproductive parameters under heat stress. Physiological indices commonly used to evaluate quinoa’s heat tolerance include chlorophyll fluorescence, gas-exchange parameters such as CO₂ assimilation and stomatal conductance, electrolyte leakage as a measure of membrane integrity, water use efficiency, and chlorophyll degradation (Eustis et al., 2020; Abbas et al., 2023). At the reproductive stage, pollen viability, grain and straw yield and composition are strong determinants of heat sensitivity (Hinojosa et al., 2019; Alvar-Beltrán et al., 2020; Tovar et al., 2020; Matías et al., 2021a, 2021b). While the effects of heat stress on quinoa grain and straw production have been previously studied (Hinojosa et al., 2019; Alvar-Beltrán et al., 2020; Tovar et al., 2020; Matías et al., 2021a, 2021b, 2022), the physiological responses of YVQ to high temperatures remain largely unknown. Given the increasing frequency of extreme heat events in recent years (Alon et al., 2022) and projected future extremes under climate-change scenarios approaching or exceeding 45-50 °C in quinoa-growing regions (Jagadish et al., 2020; Grüter et al., 2022), this study investigated the physiological responses and recovery capacity of YVQ plants following exposure to high and extreme temperatures of up to 55 °C under controlled conditions. While several studies have investigated the effects of such extreme temperatures in other plant species (Turhan et al., 2021; Aydogan and Turhan, 2022; Aydogan et al., 2025), to our knowledge, the present work represents the first investigation of YVQ under such conditions.

This study offers comprehensive insights into the physiological responses of YVQ leaves to various high-temperature gradients under controlled conditions. Our findings reveal a remarkable degree of resilience in YVQ plants, which maintained intact leaf morphology with no visible damage, even after exposure to several days of peak extreme temperatures of up to 55°C (Figure 2). Despite this apparent tolerance, closer examination of photosynthetic performance, chlorophyll content, membrane stability, and protein content indicated that YVQ undergoes distinct adjustments in its response to high temperatures, some of which may underlie its ability to sustain these harsh conditions.

Chlorophyll a fluorescence is a reliable and precise method for detecting damage to PSII and is widely regarded as one of the most effective approaches for studying heat tolerance in plants in vivo (Sharma et al., 2015). A reduction in Fv/Fm values relative to non-stressed conditions generally reflects impaired electron transport in the photosynthetic apparatus, potentially leading to photoinhibition (Baker, 2008). For example, Fv/Fm values decreased significantly in cotton plants exposed to 40°C compared to 30°C, and in water spinach exposed to 42°C compared to 25°C, indicating damage to the plants’ photosynthetic apparatus (Guo et al., 2020; van der Westhuizen et al., 2020). Young heat-sensitive tomato plants also exhibited lower Fv/Fm values compared to heat-tolerant genotypes following exposure to high temperatures under controlled conditions (Poudyal et al., 2019). An interesting outcome of the current study was the stability of the Fv/Fm values in the quinoa leaves, which remained unaffected across all treatments (Figure 4). Moreover, there was no significant (P > 0.05) correlation between temperature and Fv/Fm (Figure 8), and the linear mixed model showed that it was not significantly (P > 0.05) affected by temperature (Table 1). Thus, the high temperatures did not induce any damage leading to chronic photoinhibition in ‘Peppermint’ quinoa leaves, which would otherwise manifest as a sustained reduction in Fv/Fm values (Maxwell and Johnson, 2000; Shapira et al., 2021). These findings align with a previous study showing that heat-tolerant quinoa accessions maintained stable Fv/Fm values when young potted plants were exposed to high temperatures of 45°C (Eustis et al., 2020).

The increase in CCI in the leaves of Control quinoa plants at the After 1d time point (Figure 4) likely reflects a natural developmental process (Eustis et al., 2020). A similar trend was observed in plants exposed to the 40–46°C treatments; interestingly, in these cases, CCI continued to increase during the recovery phase (After 14d), similar to findings in ‘Kaslaea’ and ‘17GR’ quinoa accessions following 45°C heat treatment (Eustis et al., 2020). These results contrast with the expected chlorophyll degradation under stress due to lipid peroxidation in membranes and chloroplasts (Fahad et al., 2017; Hassan et al., 2021), suggesting that quinoa leaves may upregulate chlorophyll synthesis or reduce its degradation under these high temperatures. The increase in CCI was delayed under the higher 49°C and 52°C treatments compared to the other treatments. The absence of a CCI increase under the 55°C treatment indicates a threshold beyond which chlorophyll synthesis may be partially impaired, supporting the view that exposure to 55°C can override protective responses.

Gas-exchange measurements revealed a more sensitive response to high temperatures (Figure 5). Both A and g_s were significantly affected by temperature, time point and their interaction (Table 1), consistent with the central role of photosynthetic gas exchange in plant responses to heat stress. In the Control treatment, gas-exchange parameters were not affected After 1d. In contrast, these parameters were negatively correlated with temperature at this time point (Figure 8A). After 1d, A and g_s declined significantly after exposure to the high temperatures of 43°C and >40°C, respectively, with the lowest values observed at 52– 55°C. A reduction in photosynthetic rate and g_s was also found in water spinach plants exposed to 35°C, 40°C, or 45°C, and wucai (Brassica campestris) plants exposed to 40°C (Zou et al., 2017; Wang et al., 2023). Interestingly, After 14d, both A and g_s were significantly reduced in the Control plants, possibly due to a natural developmental process. However, at this time point, neither parameter was correlated with temperature (Figure 8B). Moreover, A and g_s recovered to their Before values in the 43–49°C treatments and the 46–52°C treatments, respectively. This indicates that following exposure to these high temperatures, YVQ plants retain the capacity to restore photosynthetic activity once the heat subsides. A previous study reported similar results, showing elevated photosynthetic rate and g_s in several quinoa accessions following exposure to heat treatments of 40°C or 45°C (Hinojosa et al., 2019; Eustis et al., 2020). However, in those studies, the effects of higher temperatures were not investigated. The lack of recovery for both A (at 52–55°C) and g_s (at 55°C) suggests that these temperatures are the threshold beyond which photosynthesis becomes irreversibly impaired, reinforcing the notion that extreme heat can override protective mechanisms, as seen with the CCI measurements (Figure 4). The strong positive correlation between A and g_s observed here (Figure 8) underscores the central role of stomatal regulation in carbon assimilation under heat stress (Slattery and Ort, 2019).

At the cellular level, electrolyte leakage is a reliable method for assessing cell membrane integrity and is commonly used as an indicator of direct heat-induced injury (Demidchik et al., 2014). Indeed, this parameter was not affected in the control treatment (Figure 6). Electrolyte leakage showed a biphasic response to high temperature: following exposure to 43–46°C, there was an unexpected reduction in electrolyte leakage, whereas at 52°C and 55°C, leakage increased, particularly After 14d. Reduced electrolyte leakage at intermediate temperatures may reflect stress priming, whereby moderate heat induces protective adjustments that stabilize the membranes (Bäurle, 2016; Hassan et al., 2021). In contrast, the elevated electrolyte leakage at the extreme temperatures of 52°C and 55°C is consistent with heat-induced cellular damage (Gulen and Eris, 2004; Hassan et al., 2021). The significant correlations observed between electrolyte leakage, carbon assimilation, g_s, CCI, and protein content After 14d (Figure 8B) suggest that membrane stability is closely integrated with broader cellular responses to stress in quinoa leaves following the recovery period.

Although heat stress can upregulate specific proteins—such as heat-shock proteins, ROS-detoxifying enzymes, and other stress-related proteins—overall protein content is often reduced (Vierling, 1991; Gulen and Eris, 2004; Kotak et al., 2007). Here, in contrast, we found that total protein content is strongly influenced by temperature, exhibiting a positive correlation and reaching its highest level at 55°C, significantly higher than Control levels (Figures 7, 8B; Table 1). This pattern may reflect the accumulation of stress-related proteins. However, it is important to emphasize that the present study quantified total nitrogen-based protein without distinguishing between functional storage proteins and stress-induced proteins. Therefore, although quinoa leaves maintained high protein levels following severe heat stress, the nutritional quality and digestibility of these proteins remain to be determined.

The findings of this study have important agronomic implications. With the projected increase in the frequency and severity of heatwaves under climate change (Jagadish et al., 2020; Grüter et al., 2022), the ability of YVQ to withstand acute heat stress with no visible damage and while retaining high protein levels underscores its potential as a climate-resilient leafy crop. Since YVQ has a short growth cycle (typically harvested 30–62 DAS, depending on the growing season) (Rubinovich et al., 2023, 2025), its resilience to transient stress events makes it particularly suitable for Mediterranean and arid regions, where extreme heat events often coincide with key growth periods. Moreover, the retention of high protein concentrations even at extreme temperatures suggests that nutritional quality may be preserved, supporting its role as a sustainable alternative to conventional leafy greens. While this study provides important insights, experiments were conducted under controlled growth conditions and assessed short-term responses to heat exposure (5 days), which cannot fully replicate the complexity of field environments where heat stress often coincides with drought, high irradiance, or nutrient limitations (Cohen et al., 2021; Zandalinas and Mittler, 2022). Moreover, quinoa is known to exhibit substantial genotype-specific variation in heat tolerance (Eustis et al., 2020; Matías et al., 2021b). Therefore, the present findings should not be generalized to all quinoa genotypes. Future work should compare quinoa genotypes to identify genetic variation in heat tolerance.

Overall, these findings support the potential of YVQ as a novel, climate-resilient leafy crop, particularly in regions that are susceptible to increasing heat extremes. In contrast to grain-producing quinoa, for which high temperatures during flowering and seed filling have been linked to substantial yield reductions—a major barrier to its global expansion (Fuentes and Bhargava, 2011; Hinojosa et al., 2018, 2019; Alvar-Beltrán et al., 2020; Matías et al., 2021b), YVQ shows promise as a viable crop under warming and extreme climatic conditions. It can maintain nutritional quality under severe heat stress and serves as a promising model for investigating plant resilience to acute thermal events. Moderate heat exposure (40°C) did not appear to induce a meaningful adaptive response, as physiological parameters remained close to control levels with no evidence of acclimation. In contrast, exposure to 43–49°C elicited clear short-term stress responses, followed by partial recovery over time, suggesting the activation of protective and repair mechanisms typical of short-term heat adaptation. However, at the extreme temperatures of 52–55°C, the damage exceeded the plants’ compensatory capacity, resulting in irreversible impairment of carbon assimilation and membrane integrity. Notably, protein content reached its highest values with the 55°C treatment, suggesting the accumulation of stress-related proteins as part of the plant’s terminal response to severe heat stress. Although days with peak temperatures of 55°C are currently rare and confined to specific geographical regions, recent climate models predict an increase in such extreme events in the coming years (Jagadish et al., 2020; Grüter et al., 2022). Therefore, future studies should focus on investigating and assessing the resilience of different quinoa accessions, along with its wild relatives that are tolerant to extreme heat (Curti et al., 2022; Xu et al., 2025), under both controlled and field conditions, to better prepare for the anticipated impacts of global warming. Future work should also incorporate additional quantitative indices, such as chlorophyll extraction and leaf relative water content, to further complement the non-destructive measurements used in this present study.