Globally, cotton is the most widely produced and utilized natural fiber, with an annual economic value of around 600 billion USD (Khan et al., 2020). Among different cotton species, Upland cotton (Gossypium hirsutum L.) cultivars are the most widely cultivated, constituting 97% of the US production (USDA-ERS, 2024). In 2024, global cotton production reached 25 million tons, with the US contributing about 2.6 million tons from 11.2 million acres (MacDonald, 2024), accounting for 11% of total global production after China (24%), India (22%), and Brazil (13%) (USDA-FAS, 2024a). However, the US leads the cotton export market with an estimated export value of 5 billion USD (USDA-FAS, 2024b) holding a key role in the global cotton market. In 2024, the US cotton yield reduced by 7% compared to the previous year (USDA-NASS, 2024). This is attributed to the crop’s sensitivity to environmental cues like increased occurrences of heatwaves, prolonged drought, and other extreme weather events during crop growth and development (Amin et al., 2017). Increased carbon dioxide (CO₂) emissions from fossil fuel consumption and land use changes have elevated the atmospheric CO₂ concentration (|CO₂|) to about 425 ppm in 2024 (Lindsey, 2024). The rate of increase in CO₂ is currently around 2.5 ppm per year, and the consequent increase in absorbed heat energy in the atmosphere drives the rise in global temperatures.

According to NASA’s global air temperature monitoring and assessments, the average global annual temperature in 2024 was up by 1.28°C from its average during 1951 to 1980 (NASA, 2024). Notably, 2024 marked the warmest year on record since recordkeeping began in 1880. Since the 1970s, unusually hot summer days with both high day and nighttime temperatures have become more common over the last few decades in the United States (EPA, 2016). More specifically, the nighttime temperature in most locations is rising faster than the daytime temperature (Xia et al., 2014), contributing to a reduction in the diurnal temperature range (Easterling et al., 2000). In line with this trend, the global daily mean minimum temperature increased by 0.8°C per century, compared to a daily maximum temperature increase of 0.4 °C (NCEI, 2024). Plants exhibit modifications in physiological and growth characteristics under these changing temperature conditions (Impa et al., 2019). The rate of plant growth is dependent on the temperature, and the critical temperature for the crop (Hatfield and Prueger, 2015; Soni et al., 2025). Quantifying these modifications in plant growth and yield provides a rationale for exploring key traits that will help breed heat-stress-resilient cotton in the future.

The thermal kinetic window of cotton for optimum metabolic activity with maximum photosynthesis is at about 26-28°C (Burke et al., 1985). Higher than optimum temperatures promote growth and early maturation in cotton; however, they prevent plants from fully achieving their genetic potential (Reddy and Zhao, 2005; Saini et al., 2023). The response of plants to high-temperature conditions also depends on the growing conditions. Few field-based studies have reported that plant height, number of nodes, and internodal length decline under high-temperature conditions where the maximum growing temperature exceeds 40°C (Abro et al., 2021; Yousaf et al., 2023). At the same time, studies conducted under control conditions with optimum water and nutrients observed an increase in plant height and number of nodes with increasing growing temperatures (Reddy et al., 1992, 1998). However, all these studies agreed that high temperatures have an adverse effect on boll retention and cottonseed and lint yield. It can be explained by the imbalance between the source and sink, which adversely affects the yield of cotton (Nie et al., 2020; Qin et al., 2023). Similar to most crops, cotton is susceptible to heat stress during the reproductive phase (Salman et al., 2019; Farooq et al., 2023). It has been reported that high temperature stress deforms the reproductive organs, disrupting pollen maturation, germination, and ovule development, ultimately resulting in lower seed set and crop yield (Snider et al., 2009; Hatfield et al., 2018; Raja et al., 2019; Masoomi-Aladizgeh et al., 2021). The high night temperature (HNT) of 29-31°C is reported to have a disruptive effect on pollen development, leading to male sterility, mainly due to the limited supply of storage lipids and fatty acids as a result of increased respiration (Khan et al., 2020). These modifications have contributed to the increased square and flower abscission observed under high day and night conditions (Song et al., 2015; Loka and Oosterhuis, 2016; Bista et al., 2025).

High temperatures reduce the yield and yield-related parameters such as boll and seed number and lint yield in cotton (Abro et al., 2021; Yousaf et al., 2023). Research indicates that the impact of high-temperature stress becomes more severe when both day and night temperatures rise together, compared to an increase in either one alone (Zafar et al., 2021; Saini et al., 2023; Parkash et al., 2024). As high as a 50-60% reduction in seed and lint yield was observed under a high day and night temperature of 37/26 °C (Mishra et al., 2017). Similarly, the quality of cotton fiber is affected by the growing temperature conditions. A single seed generates around 10,000 to 20,000 fibers under ideal temperature conditions, which is around 26-28°C (Reddy et al., 1997). Temperatures above 35 °C can negatively impact the quality properties of the fiber, affecting its marketable value (Manan et al., 2022). A lower rate of photosynthesis, coupled with the disruption of enzymes involved in sucrose metabolism and cellulose synthesis, is the primary cause of reduced fiber quality under high-temperature conditions (Chen et al., 2017; Ding et al., 2021).

Although studies have been conducted to investigate the impact of high day and night temperatures on cotton yield and quality, a systematic study on the sole and combined effects of high day and nighttime temperatures on reproductive and yield-related traits under ambient and elevated CO₂ concentrations has not been attempted before. The study aimed to (i) understand the boll production and retention over time under various combinations of high day and/or night temperatures and CO₂, (ii) evaluate the effects of high day and/or night temperature stress on flower characteristics under different CO₂ levels, and (iii) estimate the impact of these temperature and CO₂ combinations on seed and lint yields and fiber quality, all under optimum water and nutrient conditions.

The cotton cultivar exhibited significant responses to temperature differences during flowering and boll development periods. All measured parameters were significantly different (p < 0.001 and p < 0.05) due to temperature variations, except for fiber uniformity and fiber strength ( Table 1 ). However, the eCO₂ during the cotton reproductive stage only altered the boll maturation period (p <0.001). The flower characteristics and fiber micronaire were influenced by the interaction of temperature and CO₂ treatments (p < 0.01 to p < 0.05).

The study highlights the decline in reproductive performance and yield potential of cotton plants grown under increased daytime and/or nighttime temperatures in both ambient and elevated CO₂ environments. The study specifically focuses on the boll production and retention efficiency of cotton when exposed to temperature fluctuations during the reproductive stage. It reports the temporal changes in boll production during the crop season. We observed a drastic increase in boll production under HT and HT+HNT conditions towards the end of the season, with approximately 50 bolls produced under HT and 70 bolls under HT+HNT conditions, averaged across the CO₂ environments. However, the produced bolls were affected by temperature, resulting in boll abscission. Significant changes in plant growth, flower characteristics, and seed yield were recorded in the study.

The plant height of cotton is a geometric trait that contributes to the biomass and harvest index (Jiang et al., 2016). The height of plants grown under HT and HT+HNT conditions increased by 28 and 37%, respectively, compared to OT, reaching almost 2 meters tall at HT+HNT and eCO₂ conditions. In our study, we observed an increase in flower abscission towards the end of the experiment, resulting in greater availability of carbon sources in the plant under high temperatures. Temperatures damaging to fruit production may alter the balance between vegetative and reproductive growth, leading to increased growth of both the main stem and branches (Hatfield and Prueger, 2015; Allen et al., 2018). Cotton, being plastic in nature with an indeterminate growth habit, it translocated the photosynthates to the growing meristematic cells, adding more nodes and internodes, leading to increased plant height (Emara and El-Sayed, 2021; Naveed et al., 2023). This is manifested by an increased number of nodes observed under HT and HT+HNT conditions in both CO₂ environments, resulting in 33 nodes under HT+HNT compared to 26 nodes under OT. It is also important to note that the plants under eCO₂ had slightly higher plant height and a non-significant increase in the number of nodes, which is attributed to the increased rate of photosynthesis under CO₂ fertilization (Zhang et al., 2017; Wang et al., 2020; Gong et al., 2025). These changes in plant height and nodes contributed to an increased number of leaves and a higher leaf area index (Li et al., 2015), which in turn contributed to a higher rate of light interception and a subsequent increase in carbon fixation. In turn, this contributes to the availability of a carbon source for further addition of nodes and internodes. The plant height and number of nodes grown under OT+HNT conditions were similar to those in plants under OT.

As the plant grows and adds more nodes, it contributes to the production of fruiting sites. This is evident from the increased production of bolls towards the end of the growing season under HT and HT+HNT conditions with less boll retention ( Figure 2 ). The final yield of seed and lint depends on the number of bolls produced and their retention. The plants allocated more carbon sources towards the first-formed bolls up to 35 days after treatment, retaining all the developed bolls. Upon increased exposure to HT and HT+HNT conditions, the plants were unable to support the bolls, resulting in abscission. The increased availability of carbon source contributed to the increased production of bolls under eCO₂ conditions in all the temperature environments (Reddy et al., 1995b; Jans et al., 2021; Gong et al., 2025). Under high temperatures, the number of fruiting sites on vegetative branches also increased, as observed in other studies (Reddy et al., 1995a; Abro et al., 2025). On average, the boll retention percent under normal conditions exceeds 80%, which is essential for achieving a good lint yield. However, under HT and HT+HNT conditions, most of the bolls failed to reach maturity and dropped within 7 to 10 days. As a result, boll retention was significantly reduced to only 28% under HT and 19% under HT+HNT conditions. The BMP is the time interval between flowering and boll opening. The study also observed a reduction in the BMP with an increase in temperature, whether during daytime or nighttime. The eCO₂ further reduced the BMP compared to aCO₂ at all temperature conditions above OT. Under increased temperatures (HT and HT+HNT) above the optimum, the plants attempt to shorten the life cycle of a boll to 40 days, compared to 47 days under optimal temperatures (OT). Even the BMP under OT+HNT was reduced by 2–3 days compared to OT.

The reduced seed and lint yield observed in the study can be attributed to multiple reasons. The number of male and female reproductive organs is a yield-determining factor as the number of seeds per boll depends on the number of ovules per locule, and successful fertilization depends on the pollen load and its quality (Song et al., 2015). The study observed a significant reduction in both crucial traits, including the number of anthers per flower ( Figure 4 ). Reproductive development is susceptible to high temperatures, both before and after anthesis (Lokhande and Reddy, 2014; Song et al., 2015; Bista et al., 2025). The number of ovules flower^-1 was reduced by 26% and 30% under OT+HNT and HT conditions, respectively, under aCO₂, compared to OT. Increased carbon availability under eCO₂ minimized the adverse effect of increased temperatures under OT+HNT and HT conditions, resulting in only 16 and 20% lower numbers of ovules, respectively. However, CO₂ fertilization did not help mitigate the adverse effect of increased day and night temperatures on cotton. As discussed earlier, cotton plants under high temperatures divert photosynthate to vegetative biomass rather than to reproductive structures, thereby limiting available resources for producing ovules (Lokhande and Reddy, 2014; Snider and Adegbenro, 2025). A similar observation was made for several pollen grains anther^-1, resulting in a reduction of 24% and 39% under HT and HT+HNT conditions, respectively, compared to OT. At OT, however, it did not enhance pollen production under increased temperatures above OT. The reduced availability of non-structural carbohydrates in the floral buds under high temperatures may have caused a reduction in male and female gametophyte development in cotton plants (Borghi et al., 2019).

The quality and integrity of pollen are crucial for successful fertilization of the ovule and thus the boll and seed set. Pollen development consists of two sequential stages: microsporogenesis and microgametogenesis, which are highly sensitive to temperature fluctuations (Masoomi-Aladizgeh et al., 2021; Lohani et al., 2025). The study observed significant structural alteration of pollen grains with increasing temperature, regardless of CO₂ availability. The shrinkage and deformation observed at temperatures above OT indicate a reduced availability of potential pollen grains for fertilization, resulting in insufficient pollen germination and pollen tube growth (Song et al., 2014; Qian et al., 2025). It is independent of carbon availability and is mainly influenced by temperature conditions. The release of pollen grains from the anther, termed anther dehiscence, is critical for successful fertilization as it ensures the transfer of viable pollen to the stigma, facilitating pollination and subsequent fruit development (Deng et al., 2021). Both growing conditions and genetic regulations influence this process (Wilson et al., 2011; Zheng et al., 2019; Miglani et al., 2025). We observed anther indehiscence under HT and HT+HNT conditions at both aCO₂ and eCO₂, highlighting the impacts of temperatures on cotton flowering and fertilization. This indehiscence could be due to a reduced number of pollen grains per anther and distorted pollen structure, leading to insufficient pressure for anther opening. Alternatively, it may result from temperature-induced disruption in gene expression required for anther dehiscence (Sakata and Higashitani, 2008; Lohani et al., 2025). The anther indehiscence was not observed under OT+HNT conditions, suggesting that a slight increase in night-time temperature (33/24 °C) compared to 33/21 °C does not affect the genes responsible for anther dehiscence. Additionally, our findings indicate that a minimum of 265 pollen grains of anther^-1 is sufficient for successful anther dehiscence. A positive correlation between anther sterility and high temperatures was reported 15 and 16 days prior to anthesis, indicating that microgametophyte development was susceptible to high temperatures immediately after meiosis of the microspore mother cells (Song et al., 2014). This may have resulted in the retention of early-formed flowers under high-temperature conditions.

The seed and lint yield in cotton is a function of boll production and retention. The study showed that the reduction in boll retention, either due to unsuccessful fertilization and/or limited translocate allocation, contributed to the decline in seed and lint yield in cotton. This is also exacerbated by the shortened BMP, which reduces the time required to incorporate sufficient biomass into the developing bolls (Lokhande and Reddy, 2014). The highest seed cotton, lint, and seed weights were observed under OT conditions at eCO₂ ( Table 2 ). This is attributed to enhanced boll production, number of retained bolls, and the number of pollen grains in anther^-1 under CO₂ fertilization, due to increased availability of photosynthates. The increasing temperatures beyond 33/24 °C resulted in a drastic reduction, causing a decline of almost 90%, 88%, and 84% in seed cotton, lint, and seed weights compared to OT across the CO₂ treatments. The reproductive failure of flowers developed under high-temperature conditions, accompanied by limited biomass partitioning towards the bolls, has led to the observed results. It is also important to note that the lint yield of plants under OT+HNT was reduced by 18% compared to OT, indicating the sensitivity of lint development to HNT conditions. The increased respiration under HNT conditions and limited cellulose supply to the bolls resulted in reduced lint weight under these conditions (Xu et al., 2021; Yang et al., 2023). This also contributed to the reduced weight of seeds under OT+HNT and HT+HNT conditions.

Fiber is the main economic product of the cotton crop, and the market value depends on its quality. The slight reduction in fiber length under HT and HT+HNT conditions is possibly affected by the limited biomass allocation to the boll development (Lokhande and Reddy, 2014; Xu et al., 2017; Abro et al., 2025). At optimum temperatures, the cotton fiber elongates over 2000-3000-fold within approximately 20 days after anthesis (Ruan et al., 2005). High temperatures during this period adversely affect the fiber elongation, in turn, shortening the fiber length (Lokhande and Reddy, 2014; Ali et al., 2025). The micronaire is an indirect measure of fiber fitness and maturity. Premium-quality fiber requires a micronaire value between 3.8 and 4.9 (Bange et al., 2009). In the eCO₂ environment, fibers produced under HT and HT+HNT conditions had micronaire values of 2.95 and 4.65, respectively. The micronaire value below 3.8 indicates fiber immaturity, while values above 4.9 suggest coarser fibers with undesirable spinning characteristics (Luo et al., 2016). The temperature and eCO₂ had no notable effects on fiber uniformity and strength. These fiber properties are likely primarily determined by the plant’s genetics and are only minimally influenced by growing conditions.

This study highlights the adverse effects of high air temperatures on cotton reproduction and yield, both in ambient and elevated CO₂ conditions. Although boll production increased later in the season in response to high temperatures, high rates of abscission led to poor retention and ultimately reduced seed and lint yields. The observed increase in plant height and node numbers suggests a shift in resource allocation from reproductive growth to vegetative growth. High daytime and nighttime temperatures significantly hindered pollen production, anther dehiscence, and ovule formation, resulting in decreased fertilization success. While eCO₂ levels improved photosynthesis, they did not fully counteract the yield losses caused by reproductive failures due to high temperatures. Under these high temperature conditions, seed cotton, lint, and seed yields experienced a sharp decline. These findings emphasize cotton’s vulnerability to rising air temperatures and the limited ability of elevated CO₂ to mitigate these adverse effects. These findings should be further validated under field conditions to gain a more comprehensive understanding of plant behavior under current and future CO₂ and temperature conditions.