Globally, it is forecasted that by the end of the twenty-first century, the air temperature will ascend by 0.2°C per decade, resulting in inflate of 1.8–4.0°C temperature more than the existing level (IPCC, 2014). Global warming has led to climate change, causing adverse effects on plant growth, as high temperatures have detrimental results on crop production worldwide (Ohama et al., 2017). Heat stress is known to alter several morphological and physicochemical processes in plants, reducing yield and disrupting cellular homeostasis (Bita and Gerats, 2013). Heat stress can disturb cellular homeostasis due to the excess production of reactive oxygen species (ROS) (Buttar et al., 2020). Under stress conditions, a substantial increase in the concentration of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) manifests high membrane permeability, which eventually ends up with enhanced lipid peroxidation (Bi et al., 2016; Irshad et al., 2021). Fortunately, to cope with ROS, plants have triggered specific mechanisms to reverse the decline in growth caused by heat stress, which include: (i) enzymatic and non-enzymatic antioxidant defense system and (ii) osmoregulation by producing compatible solutes (Hasanuzzaman et al., 2013). The antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) quench, and detoxify ROS from the plant cell (Hu et al., 2012; Sharma et al., 2012). When plants are exposed to stress conditions, antioxidant enzymes perform a vital role in maintaining plant growth and development and various studies have revealed a direct association between plant thermotolerance and ROS scavenging (Hameed et al., 2012). Different environmental conditions accompany different growth stages of plants. “The suitable sowing date” is one of the most imperative factors to obtain optimum yield. Plant growth is modified to a specific environmental condition at various growth stages; thus, a suitable selection of sowing dates inflates the photosynthetic efficiency (Ali et al., 2021). In Kharif season crops, during early growth stages, the temperature reaches more than 40°C. It caused a significant reduction in seed yield due to frequent flower drops, lack of fertilization, non-viability of pollen, and catalytic deactivation of rubisco activase, leading to complete enzyme failure (Perdomo et al., 2017). Heat stress harms the retention of flowering and pod formation. The primary flower shedding occurred up to 40°C temperature in mungbean and flower drop has been reported more than 79% (Kaur et al., 2015). The reproductive stage is the most sensitive stage to heat stress, resulting in loss of flower buds and seed yield (Kaushal et al., 2013; Sharma et al., 2016). Heat stress causes degradation of chlorophyll, disturbance in electron flow, disruption of photosystem II, and attenuation in carbon fixation that collectively inhibit the process of photosynthesis (Hussain et al., 2021; Li et al., 2021).

For the stability of proteins and biological membranes, Zn plays a vital role. It also facilitates maintaining ROS assembly and scavenging, as it acts as an SOD cofactor. Zn, being a part of Cu/Zn-SOD, is present within the cytosolic and chloroplast enzymes that have a crucial role against oxidative stress (Khan et al., 2021). Zn, a necessary micronutrient for all plants, takes part in numerous physiological activities, i.e., pollen performance, biosynthesis of chlorophyll, enzymes, proteins, and metabolic processes (Singh et al., 2018). It is a key component of the enzyme carbonic anhydrase, which catalyzes the exchange of CO₂ and HCO₃ in the mechanism of C4 photosynthesis (Faizan et al., 2021). Recent advances in nanotechnology have revolutionized agriculture and offer immense potential in enhancing abiotic stress tolerance, mainly through upregulating plant antioxidant activities (Rana et al., 2021). Nanoparticles (NPs) are atomic or molecular aggregates having at least one dimension between 1 and 100 nm that can significantly alter their physicochemical characteristics compared to the bulk material (Rastogi et al., 2017). NPs present a proficient way in an organized fashion to spray fertilizers and pesticides with the high site-specificity due to their small particles and large ratio of surface area to volume (Prasad et al., 2012; Kah et al., 2018). NPs play a pivotal role in stressed plants, which could help the plants to tolerate the abiotic stresses (Martínez-Fernández et al., 2015; Aghdam et al., 2016; Dimkpa et al., 2017). Nano-ZnO has recently captured the vast research struggles due to its distinctive quality and multiple applications in numerous fields (Sturikova et al., 2018). It has been documented that nano-ZnO is far more efficient in augmenting the productivity and absorption of Zn because of its high surface area to volume ratio (Khan et al., 2021). Under stress, NPs increase the potential of an antioxidant defense system for scavenging the free radicals by changing the microRNA expression and regulating the numerous morphological, physiological, and metabolic processes of the plant (Siddiqi and Husen, 2016; Kambe et al., 2021).

Mungbean seems to be a promising crop because it is nutritionally sound, fixes nitrogen, overgrows, and requires less water (Samson et al., 2020). As a critical pulse crop, mungbean is anticipated to be a vital component of ameliorating and diversifying cropping systems and worldwide diets. It provides vital vitamins, minerals, and amino acids (Schafleitner et al., 2015; Vittal et al., 2018). Being an imperative leguminous summer-season crop, it is much responsive to biotic and abiotic stresses. In April and May, mungbean cultivation faces temperature >30°C, which can even go up to 45°C, particularly at the time of pod filling and flowers initiation to significantly influence the yield (Hanumantharao et al., 2016; Malaviarachchi et al., 2016; Kumari et al., 2021). Foliar application of nano-ZnO against sowing time-mediated heat stress was rarely studied in previous times. Therefore, it was hypothesized that Zn as NPs is an intelligent delivery system with a larger surface area, high sorption capacity, and slow-release kinetics to target areas, which can mitigate the deleterious effects of heat activating the physiochemical mechanisms of plants against oxidative stress. One of the suitable ways to study the effect of heat stress in field conditions is to extend the sowing dates that ensure high temperature (>40/25°C) during the reproductive stage. This also provides a fair insight to study the sensitivity of a crop against heat stress. Thus, this proposed study was planned in field conditions by adjusting different sowing dates to impose heat stress at the reproductive stage. The foliar-applied nano-ZnO was applied to study the physicochemical processes and yield attributes of mungbean crops under different sowing regimes generated heat stress.

Heat stress is an excellent debacle for the growth and productivity of crop plants. Mungbean genotype, which is used in this study, is recommended to sow on 1 March (S1); to inflict heat stress (>40/25°C) at reproductive stage, this genotype was also late sown with 3 weeks interval as S2 and S3. Subsequently, the delayed sown plants encountered the issue of heat stress at the flowering stage. The temperature was 4–6°C higher than the optimal sowing, which seriously affected the biochemical attributes performance, particularly pod set reduction, PPP, SPP, and seed yield, implying heat sensitivity of mungbean (Figure 1). Our observations on the decline in seed yield were inlined with some previous studies, which demonstrated the inhibitive effects of heat stress on mungbean crops (Sanat et al., 2006; Malaviarachchi et al., 2016).

To combat abiotic stresses, plants deploy an antioxidant defensive mechanism to ameliorate the negative influence of heat stress-generated ROS (Buttar et al., 2020). In our findings, depressed antioxidant activities were observed in heat-stressed environments S2 and S3 relative to no heat environment S1. As the level of oxidative damage is raised, the efficiency of photosynthesis decreases, which influences the growth of mungbean (Bansal et al., 2014). A direct relationship between heat stress and nano-ZnO doses was observed. Zn plays a central role in the stability of biomembranes and protein because it is a cofactor in the SOD enzyme (Faizan et al., 2020). Therefore, as the level of heat stress gets increased, the activities of SOD, POD, CAT, and TPC also get increased, at various concentrations of nano-ZnO, relative to control (Figure 3). Cu/Zn-SOD production may increase due to foliar application of nano-ZnO, which favored the defensive mechanism in scavenging of ROS, as reported by Mansoor (2013). In this study, escalation in the total phenolic activity was also examined due to the function of zinc, which coincided with the finding of Yuan et al. (2016). A strong negative correlation between antioxidants activities and MDA and H₂O₂ confirmed the positive role of nano-ZnO application against ROS under heat stress (Figure 7).

Osmolytes, such as LP, GB, and TSP, are low-molecular weight substances utilized by plant cells and tissues under adverse environmental conditions to keep a steady water potential by accumulating them as organic solutes to create a hypotonic environment (Yancey, 2005). Heat stress triggered the breakdown of chlorophyll that caused a reduction in sucrose synthesis, which decreased the biosynthesis of LP, GB, and TSP. Foliar applications of nano-ZnO might inflate the biosynthesis of tryptophan, which is involved in photosynthesis; hence, it increased the synthesis of sucrose required for the biosynthesis of LP, GB, and TSP (Supplementary Figure 1). Hereafter, the biosynthesis of LP, GB, and TSP was increased in S2 and S3 by exogenous foliar application of nano-ZnO under heat stress relative to control (Waraich et al., 2012). In our finding, osmolytes (LP, GB, and TSP) were found to be positively correlated with antioxidants and chlorophylls (Figure 7).

Peroxidation of membrane lipid is a significant damaging effect of ROS and its extent in plants is detected by measuring MDA (Garg and Manchanda, 2009; Hussain et al., 2016; Zafar et al., 2017). The curtailment in H₂O₂ with the escalation in foliar-applied nano-ZnO doses was noted because Zn is a part of the most abundantly found Cu/Zn-SOD in plant inflated antioxidant defense mechanism for the production of Cu/Zn-SOD to detoxify ROS (Figure 8). The findings of Disante et al. (2011) also supported the fact that under Zn deficiency, the production of Cu/Zn-SOD gets decreased, which leads to lipid peroxidation by increasing the ROS level. However, our findings showed that under S2 and S3 heat stress control conditions, the insidious effects of heat stress caused more significant lipid peroxidation by degrading biomembranes, which reduced the ability of plants to produce chlorophylls and osmolytes. Although diminution was studied in MDA contents under nano-ZnO application, Zn as a gene activator plays a crucial role in synthesizing protein and signaling. A strong negative correlation of MDA and H₂O₂ with antioxidants was also noted (Figure 7).

Chlorophyll is usually believed to be the most resistant pigment against heat stress. Higher temperatures eventually result in decreased photosynthesis by disrupting the function and structure of chloroplast (Faseela et al., 2020). Hence, similar findings were observed in this study, leading toward reduced chlorophyll contents (Zafar et al., 2017). Photosynthesis gets diminished due to stomatal closure as an early plant response toward heat stress, favoring better utilization of relative water contents (Tan et al., 2011). Heat stress-mediated decline in gs due to closing of stomata led to decreased Ci, which resulted in the reduction of Pn, E, pigment contents, and electron transportation to PSII (Pagter et al., 2005; Buttar et al., 2020). In this study, heat stress-mediated decrement in chlorophyll contents, Pn, E, gs, and Ci was significantly alleviated by the foliar application of nano-ZnO. As the size of applied ZnO-NPs was 20 ± 5 nm (Figure 2C), so they can easily find their way from stomatal pores to the phloem vessels via plasmodesmata (40 nm in diameter) and become readily available as observed by Kumar and Nagesh (2019). Hence, it was noted that nano-ZnO application potentially affected the gas exchange measures, which can be attributed to the upregulation of antioxidant defense mechanism and downregulation of ROS. Prasad et al. (2012) explicated that nano-ZnO was more effective to enhance the chlorophyll contents, flowering, and yield of pods in peanuts. Application of nano-ZnO increased the chlorophyll contents in the heat-stressed environment (S2 and S3) at specific concertation, as Zn is required for the production of chlorophyll that rebates the activity of chlorophyllase enzyme, which led to producing more chlorophyll contents in a leaf tissue as explained by Turan et al. (2009). Application of ZnO-NPs at low concentrations was found to be effective in alleviating various abiotic stresses and enhanced plant growth and development (Dhoke et al., 2011; Soliman et al., 2015). Therefore, at a higher concentration of nano-ZnO, degradation of chlorophyll led to a decrease in the overall performance of IRGA attributes. Although, the exogenous foliar applications of nano-ZnO at 30 mg l^–1 inflated the biosynthesis of tryptophan amino acid, which is involved in photosynthesis that eventually increased the chlorophyll and growth of the plant (Waraich et al., 2012). Our findings revealed that the increased chlorophyll contents by nano-ZnO sprayed plants under heat stress was positively correlated with antioxidant enzymatic activity and osmoprotectants supporting its role in stress amelioration (Figure 7).

In contrast, earlier studies evaluating the effect of heat stress on mungbean plants grown in an outdoor environment at different intervals of time documented early shifting of phonological stages (Malaviarachchi et al., 2016). There was a phonological variation due to other sowing times in this study (Figure 1), which substantially decreased the flowering and podding duration, resulting in fewer flowers, pods, and seeds being produced. Pod set usually influenced pod and seed number, which is sensitive to heat stress in many crop species, including cotton (Snider et al., 2011), chickpea (Fang et al., 2010; Kumar et al., 2013), tomato (Li et al., 2013), and mungbean (Kaur et al., 2015).

Heat stress causes hindrance in the production of sucrose in leaves and creates an obstacle in its transport to reproductive organs (Prasad and Djanaguiraman, 2011; Li et al., 2012; Kaushal et al., 2013). Heat stress manifested significant diminution in PPP, SPP, and GY can be attributed to decreased chlorophyll contents, antioxidant activities, and osmolytes, leading to increased lipid peroxidation. The reduction in PPP and SPP caused by heat stress was reported in chickpea and soybean (Djanaguiraman et al., 2013). Many pods were aborted in heat-stressed mungbean plants of S2 and S3 as a result of limited sucrose import from the leaves. Hence, it causes a significant decrement in the number of filled pods as well as an increment in the number of infertile pods. Rainey and Griffiths (2005) demonstrated that heat stress in summers reduced yield because of pods and flowers abortion. Pod and seed diameters (data not given) were also significantly lower in S2 and S3 than in S1, indicating reduced sucrose translocation for developing sinks in heat-stressed S2 and S3 plants, as observed in chickpea (Awasthi et al., 2014) and tomato (Li et al., 2013). Although, under heat stress plants of S2 and S3, foliar-applied nano-ZnO increased PPP, SPP, and GY than control. This might be because Zn being an NPs, easily gets transported from the application site to the site of action through microcellular spaces, likely via plasmodesmata (Etxeberria et al., 2016). The entered nano-ZnO enhanced antioxidants competency against ROS in the plant system and produced more chlorophylls contents that ameliorate pollen performance, pollen germinability, and viability. This caused more fruit setting and eventually grew more PPP and SPP (Thakur et al., 2010; Prasad et al., 2012). Thus, nano-ZnO application in S2 and S3 ameliorated the functional aspects of reproductive organs and enhanced the antioxidant and osmolytes activities. This may be a primary cause of less pod abortion and, consequently, more pods and seeds per plant led toward more GY. Moreover, yield components (GY, SPP, and PPP) strongly correlated with chlorophyll contents, antioxidant activities, and osmoprotectants (Figure 7).

Among abiotic stresses, heat stress-mediated damage resulted in fluctuation and shrinkage of crop growth, development, and overall yield. Conclusively, under the umbrella of above-discussed facts and analysis, attenuation in antioxidants and osmolytes and escalation in MDA and H₂O₂ were witnessed during finely tuned sowing time generated heat stress imposition in S2 and S3. Antioxidant performance, osmolytes activities, and gas exchange measures improve with foliar application of nano-ZnO in all the sowing dates, which were obvious as reduced ROS accumulation. Upregulated antioxidants and osmoprotectants prevented oxidative damage justified the beneficial role of nano-ZnO supplementations to ameliorate tolerance mechanism against heat stress and, consequently, improved yield of mungbean crop. Particularly, 30 mg l^–1 dose of nano-ZnO performed better in all the sowing dates and better ameliorate the negative impact of heat stress. Further study is needed to be studied at the molecular level to unleash nano-ZnO-associated pathways and gene response about tolerance mechanism of heat-stressed mungbean plant.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

HAK conceived the original research plans and wrote the original draft of the manuscript. MFS and QW designed and advised the research and agreed to serve as the author responsible for contact and ensure communication. HAK and JN performed the experiments. ZG assisted in microscopic experiments. SAR, SS, and SA analyzed the data and made a figure. MHS, SAR, and RK directed the experiments and revised the manuscript. NG, SW, SAR, and QW contributed to the editing and reviewing of the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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