Introduction

Climate change is impacting global food and nutritional security1. As a result of the unpredictability of rising temperatures, agricultural yields have dropped sharply as the number of hot days and nights increases while the number of cold days and nights decreases2,3,4,5. Based on their genetic structure, plants thrive in a preferred temperature range, with exposure above this range causing heat stress (HS)6. Changes in the degree and duration of high temperatures affect the entire plant life cycle, including morphological, reproductive, and developmental processes, owing to considerable breakdown of the cellular machinery. Heat stress during the reproductive and seed filling stages limits grain legume production7. High temperatures damage membrane thermostability8, reduce the relative leaf water content9, chlorophyll10 and photosynthetic efficiency11 to impair cellular functions.

Seed filling, which involves carbohydrate, lipid, and protein synthesis and the mobilization as well as accumulation of various components in developing seeds, is a crucial stage in the life cycle of all leguminous crops12,13. Heat stress during seed development affects this process, significantly reducing the seed yield in cereals14, legumes15,16,17,18,19, and oilseed crops20,21. Seed filling rate and seed weight are commonly used to assess the impact of heat stress and tolerance22. Reduced seed filling duration decreases seed size in pea, soybean, white lupin23, and cowpea24. In chickpea25 and lentil7, high temperatures increase the seed filling rate and decrease seed size. In rice26, wheat27, and maize28, heat stress during seed filling decreases seed size and weight. In addition to yield and yield-related traits, seed quality traits (seed starch accumulation, storage protein, and minerals) are negatively affected by heat stress in various crops, including several grain legumes3,7,13,29,30. Heat stress inhibits starch accumulation in seeds of cereals31, such as wheat32, rice33, and maize34, which could be a major factor affecting seed size. High temperatures affect protein accumulation in pea35 and soybeans5,36, lentil29, lupin37, chickpea30, and the oil content in oliferous crops38.

Chickpea performs well when the reproductive stage coincides with the optimum temperature (20–28 °C)39. As a heat-sensitive crop, chickpea is particularly vulnerable to elevated temperatures during critical stages of its growth and development. Heat stress can substantially affect chickpea plant growth and development. This can adversely influence the seed germination and seedling emergence40. Heat stress in the late vegetative stage of chickpea plants causes noticeable symptoms, such as leaf scorching, burning of stems and leaves, leaf rolling, leaf tip damage, leaf drying, and senescence41,42. In addition, heat stress during the reproductive stage frequently induces flower abscission or shedding, decreasing in the number of flowers14,21,41,43. Heat stress can reduce pollen grain and stigma viability, disrupt fertilization, and impede the growth of developing flowers and pods25,39,41,43. Consequently, pod abortion increases, reducing pod formation37,42,44. Thus, due to heat stress, chickpea plants produce fewer seeds with lower weight39,41,42,44. Heat stress can have a wide range of detrimental effects on various aspects of chickpea plants, including physiological and biochemical traits25,44. Chickpea is generally grown as a rain-fed crop during the winter season (November to April) in northern India and often experiences high-temperature (> 32 °C) stress during the reproductive and seed-filling phases (mid-February to April), especially in a late-sown environment40, adversely affecting seed development30.

Thus, the present study was specifically designed to investigate the effects of heat stress at the time of seed development on seed-filling processes and the consequent effects on nutritional quality in chickpeas, which have not been reported previously. This study involved contrasting genotypes for heat sensitivity, as identified in our previous study, to identify the target sites of heat stress and the probable mechanisms of heat tolerance. It was hypothesised that the heat-tolerant genotypes of chickpea would have less impact on seed development characteristics, including seed weight, seed size, and seed yield, compared to the heat-sensitive genotypes when exposed to heat stress.

Materials and methods

Raising plants

The seeds of four chickpea genotypes, two heat-tolerant (ICCV07110 and ICCV92944) and two heat-sensitive (ICC14183 and ICC5912), procured from the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India, were raised in pots (8 kg capacity) filled with a mixture of air-dried soil (available N, P, and K of 54, 43, and 158 kg ha−1, respectively; loam; pH 7.1), sand, and farmyard manure (2:1:1 (v/v) ratio). The seeds were treated with a suitable Mesorhizobium ciceri strain (2.0 g kg−1 seed) before being sown in pots on November 1, 2018, at Panjab University, Chandigarh, India (30°45′38.2″ N,76°45′55.4″ E). Three seeds per pot were planted and thinned to two healthy plants after establishment. The plants were grown in a natural outdoor environment with an average temperature of 23.5/9.9 °C (day/night; Fig. 1), relative humidity of 63.4/39.5%, and light intensity of ~ 1,300–1,510 μmol m−2 s−1 until the beginning of pod set (107–110 d after sowing). At this stage, the pots were transferred to controlled-environment growth chambers, with half (five pots in three replicates; 5 × 3 = 15 pots per genotype) maintained at 25/15 °C (control) and the other half exposed to 32/20 °C (heat stress) until maturity.

Figure 1
figure 1

Temperature profile (Max., Min., Average) for plants grown in outdoor environment average temperature as 23.5/9.9 °C; day/night) prior to exposing them to heat stress under controlled environment (32/20 °C).

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Leaf function evaluation

Membrane damage

Fresh leaves, collected from the top branches of the control and stressed plants 10 days after initiation of pod filling (DAP), were tested for membrane damage using the electrolyte leakage method45. The leaves were washed with deionized water to eliminate any surface contaminants, placed in glass vials with deionized water (10 mL), and incubated at 25 °C for 24 h on a rotary shaker. Thereafter, electrical conductivity was measured using a conductivity meter.

Relative leaf water content

Leaf water status was measured by recording the fresh, turgid, and dry weights of the same leaves, collected at 10 DAP, according to the method described by Barrs and Weatherley46. Leaf stomatal conductance was recorded on the same leaves using a leaf porometer as described previously47.

Chlorophyll concentration and chlorophyll fluorescence

Chlorophyll (Chl) concentration was determined in fresh leaves collected from the top branches at 10 DAP and extracted in 80% acetone. The extracts were centrifuged at 10,000 rpm, and the absorbance was measured spectrophotometrically at 645 and 663 nm using the method described by Arnon et al.48. Photochemical efficiency, indicating PSII function, was measured as Chl fluorescence in the fresh leaves of the top branches at 11:00 using a modulated chlorophyll fluorometer (OS1-FL, Opti-Sciences, Tyngsboro, MA, USA), as detailed elsewhere25.

RuBisCo activity

RuBisCo activity was assayed in fresh leaves collected from top branches at 10 DAP. The leaves were homogenized in a pre-cooled mortar and pestle with a buffer solution containing 50 mM 1,3-bis tris (hydroxymethyl) methylamino propane (pH = 7.0), 10 mM NaHCO3, 10 mM MgCl2, 1 mM EDTA,10 mM DTT, 1 mM benzamidine, 1.5% polyvinyl polypyrrolidone, 1 mM phenylmethyl-sulfonyl fluoride, and 3 mM 3-methylbut-2-ene-1-thiol49. The extract was centrifuged at 10,000 rpm for 40 min, the supernatant was desalted and assayed according to the method described by Racker50and elaborated elsewhere47.

Sucrose synthase activity

Fresh leaves collected at 10 DAP, were homogenized in an ice-cold extraction medium containing HEPES/KOH buffer (200 mM; pH 7.8), 3 mM magnesium acetate,10 mM dithiothreitol (DTT), and 3 mM EDTANa2.2H2O. The homogenate was centrifuged at 10,000 rpm for 20 min at 4 °C, and the supernatant was collected for enzyme activity analysis, as per the method described by Xu et al.51.

Sucrose

Leaf sucrose was measured from fresh leaves extracted in 80% ethanol three times at 80 °C for 1.5 h. The extracts were evaporated at 40 °C in an air-circulating oven. Sucrose was measured using the method described by Jones et al.52, as detailed earlier53.

Seed composition

Starch, sugar, protein, and fat contents were measured in the mature seeds. For the extraction of starch and sugars, seeds were homogenized with 30% (v/v) perchloric acid and 95% (v/v) ethanol, respectively, and their contents were estimated according to the method described by Dubois et al.54, using glucose (Sigma D9434; Sigma, WI, USA) as a standard. Crude proteins, crude fats, and minerals were analyzed according to AOAC standard procedures55.

Storage proteins were sequentially fractionated using the method described by Triboi et al.56. Mature seeds were homogenized in wholemeal flour. At each extraction step, the seed samples were continuously stirred for 60 min using a magnetic stirrer. The samples were centrifuged at 10,000 rpm for 30 min at extraction temperature to separate the soluble and insoluble fractions. Albumins and globulins were extracted with 25 mL sodium phosphate buffer (0.05 M; pH 7.8) and NaCl (0.05 M), respectively, at 4 °C. The prolamins were extracted from the previous pellet at 20 °C using 25 mL of 70% (v/v) ethanol. Similarly, glutelins were extracted from the previous pellet at 20 °C with 25 mL of 20 gL−1 sodium dodecyl sulfate (SDS), 2% (v/v) 2-mercaptoethanol (2-SH), and 0.05 M tetraborate buffer (pH 8.5). These were obtained from the supernatant after centrifugation at 10,000 rpm. The protein concentration in each fraction was measured according to the method described by Lowry et al.57.

Seed carbohydrates

Carbohydrates, such as glucose, fructose, and sucrose, were measured as described by Hu et al.58. From the seed extract, 20 µL was placed in an oven at 50 °C for 40 min to evaporate ethanol. Subsequently, 20 µL of distilled water was added to each sample, followed by the addition of 100 μL glucose assay reagent (Sigma), mixed, which were then heated at 30 °C for 15 min. The absorbance was measured at 340 nm to calculate the glucose content. Thereafter, each sample was heated twice with 0.25 U phospho-glucose isomerase at 30 °C for 15 min and then 83 U invertase for 60 min. After each incubation step, the absorbance was measured at 340 nm to estimate fructose and sucrose levels.

Seed growth rate and seed-filling duration

To measure seed growth rate, five pods plant–1 were tagged at the initiation of pod filling (pod size: ~ 1 cm). Seed dry weight was measured seven days after the onset of pod filling and at physiological maturity. The difference between the seed dry weights at the two stages, divided by the number of days to reach physiological maturity, indicated the seed filling rate day–1. The seed-filling duration for tagged pods was calculated by recording the number of days from the onset of seed filling to physiological maturity.

Yield traits

Yield traits (seed weight, seed number plant–1, and individual seed weight) were recorded from ten plants genotype–1 treatment–1; replicate data were pooled and presented.

Statistical analysis

The experiment was conducted using randomised block design

Observations were replicated three times, and the data were analysed for means and standard errors. ANOVA was conducted, with the least significant difference (LSD) calculated (P < 0.05) using AGRISTAT software.

Research involving plants

The seeds of four chickpea genotypes, two heat-tolerant (ICCV07110 and ICCV92944) and two heat-sensitive (ICC14183 and ICC5912), were obtained from the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), gene bank, India following the international norms, legislation and guidelines.

Results

ANOVA (2-way) Tables 1 and 2 show the mean sum of squares and significance for the genotype × treatment interaction, respectively, for various traits.

Table 1 ANOVA values for mean sum of squares in leaf and seed traits across the four chickpea genotypes under heat stress.
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Table 2 Analysis of variance (ANOVA) showing statistical significance in various studied traits measured in 4 genotypes under non-stress and heat stress.
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Leaf membrane damage

Leaf membrane damage (expressed as % electrolyte leakage) in the control plants varied from 9.1 to 9.3% in the HT genotypes and 10.3–10.8% in the HS genotypes (Fig. 2A). Heat stress increased membrane damage more in the HS genotypes (29.4–30.2%) than in the HT genotypes (19.5–21.2%) relative to their corresponding controls (Tables 1, 2).

Figure 2
figure 2

Membrane damage (as electrolyte leakage %; (a)), relative leaf water content (RLWC; (b)) and stomatal conductance (gS; (c)) in heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown under control (25/15 °C) and heat stress (32/20 °C) environment. LSD: (P < 0.05) (genotypes x treatment interaction): Chl: 2.65; RLWC: 9.76; gS: 32.8.

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Leaf water status

Leaf water status [(as relative leaf water content (RLWC; %)] in the control plants differed from 81.4 to 84.3% in the HT genotypes and 79.4–81.2% in the HS genotypes (Fig. 2B). Heat stress decreased RLWC in the HS genotypes more than that in the HT genotypes compared to their respective controls.

The stomatal conductance (gS) was in the range of 301–317 mmol m–2 s–1 in the HT genotypes and 326–342 mmol m–2 s–1 in the HS genotypes (Fig. 2C). Heat stress increased gS much more in the HT genotypes (54–58%) than in the HS genotypes (6–10%) relative to their corresponding controls. (Tables 1, 2).

Photosynthetic ability

Leaf Chl concentration (Fig. 3A), an indicator of the stay-green trait, in the control plants varied from 23.6 to 25.3 mg g–1 DW in the HT genotypes and 22.5–24.5 mg g–1DW in the HS genotypes. Under heat stress, Chl concentration decreased more significantly in the HS genotypes (49–61%) than in the HT genotypes (23–26%) relative to their respective control groups (Tables 1, 2).

Figure 3
figure 3

Chlorophyll content (Chl; a) and Chl fluorescence (ChlF; b) in heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown under control (25/15 °C) and heat stress (32/20 °C) environment. LSD: (P < 0.05) (genotypes x treatment interaction): Chl: 2.65; Chl F: 0.059.

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Photosynthetic efficiency, as indicated by chlorophyll fluorescence (Fig. 3B), was measured as the Fv/Fm ratio. In the control plants, in the HT genotypes, it was in the range of 0.75 to 0.76, while in the HS genotypes, it varied from 0.73 to 0.77. When exposed to heat stress, the Fv/Fm ratio decreased more prominently in the HS genotypes (44–56%) than in the HT genotypes (19.7–25.3%) relative to their respective control groups (Tables 1, 2).

RuBisCo activity (Fig. 4A)—an indicator of carbon fixation ability in control plants, exhibited values between 631 and 654 nmol CO2 min–1 mg–1 protein in HT genotypes and 609–674 nmol CO2 min–1 mg–1 protein in HS genotypes. Heat stress reduced RuBisCo activity more in the HS genotypes (29–46%) than in the HT genotypes (10–12%) relative to their corresponding controls (Tables 1, 2).

Figure 4
figure 4

Ribulose 1,5 bisphosphate carboxylase (RuBisCo; a), Sucrose synthase (SS; b) and sucrose (Suc; c) in heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown under control (25/15 °C) and heat stress (32/20 °C) environment. LSD: (P < 0.05) (genotypes x treatment interaction): RuBisCo: 36.8; SS: 6.8; Suc: 6.1.

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Sucrose synthase activity (Fig. 4B) in the control plants differed from 51 to 56 µmol min–1 mg–1 protein in the HT genotypes and 52–53 µmol min–1 mg–1 protein in the HS genotypes. Heat stress decreased sucrose synthase activity more in the HS genotypes (54–59%) than in the HT genotypes (24–26.9%) relative to their corresponding controls (Tables 1, 2).

Leaf sucrose concentrations (Fig. 4C) in the control plants varied from 43.4 to 51.3 µmol g–1 DW in the HT genotypes and 40.1–42.3 µmol g–1 DW in the HS genotypes. Heat stress reduced leaf sucrose concentration more in the HS genotypes (49.6–54.1%) than in the HT genotypes (23–30%) relative to their respective controls (Tables 1, 2).

Yield and yield-related traits

Yield traits ((Tables 1, 2, 3) included total plant dry weight (aboveground) plant–1, seed weight plant–1, average seed weight, average seed size, pod number plant–1, and seed number per 100 pods.

Table 3 Yield traits, seed growth rate, and seed filling rate in heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown in control (25/15 °C) and heat stress (32/20 °C) environments.
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In relation to their respective control groups, following impacts were observed due to heat stress.

  1. a.

    Total plant dry weight: Heat stress caused a reduction of 60–62% in the HS genotypes and 25–27.4% in the HT genotypes.

  2. b.

    Pod number per plant: Heat stress resulted in a decrease of 55–57% in the HS genotypes and 29–30% in the HT genotypes (see Fig. 5).

  3. c.

    Seed number per 100 pods: Heat stress led to a reduction of 62–63% in the HS genotypes and 23–27% in the HT genotypes (see Fig. 5).

  4. d.

    Average seed weight: Heat stress caused a decrease of 62–66% in the HS genotypes and 26–32% in the HT genotypes (see Fig. 5).

  5. e.

    Average seed size: Heat stress resulted in a decrease of 60% in the HS genotypes and 24–27% in the HT genotypes.

  6. f.

    Seed yield per plant: Heat stress led to a decrease of 61–72% in the HS genotypes and 27–29% in the HT genotypes.

  7. g.

    Harvest index: Heat stress caused a reduction of 64–67% in the HS genotypes and 30–34% in the HT genotypes.

Figure 5
figure 5

Morphological effects of heat stress (HS) observed on chickpea plants: plant height with more number of pods; under control (a), reduced plant height with lower number of pods; under HS (b), healthy leaves; under control (c), leaf chlorosis under HS (d), leaves necrosis; under HS (e), leaf scorching/leaf bleaching of leaflets due to photooxidation under HS (f), healthy flower; under the control (g), aborted flower under HS (h), comparative pod size under control and HS (i) and comparative seed size under control and HS environment (j).

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Seed-filling rate and duration

Heat stress decreased the seed-filling rate by 59–63% and the seed-filling duration by 55–58% in the HS genotypes relative to their corresponding controls, with reductions of 25–31% and 25–28%, respectively, in the HT genotypes (Tables 1, 2, 3).

Seed components

The various seed components (Fig. 6, Tables 1, 2) generally decreased with heat stress exposure in both genotypes compared with their respective controls. Heat stress decreased the starch content by 47–54% in the HS genotypes and 15 -18% in the HT genotypes. Seed protein content decreased by 42.8–56.7% in the HS genotypes and 17.5–19.7% in the HT genotypes. The fat content decreased by about 69.7% in the HS genotypes and 24.3–25. 8% in HT genotypes. Crude fiber and ash contents also decreased more in the HS genotypes than in the HT genotypes. However, there was a significant increase in the soluble sugar content in the HT and HS genotypes.

Figure 6
figure 6

Seed composition in heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown under control (25/15 °C) and heat stress (32/20 °C) environment. LSD: (P < 0.05) (genotypes x treatment interaction): Starch: 3.65; Proteins: 2.14; Soluble sugars: 0.87; Fat: 0.24; Crude fiber: 0.38; Ash: 0.32.

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Seed storage proteins

Heat stress severely reduced various seed storage proteins (Fig. 7, Tables 1, 2), more so in the HS genotypes than in the HT genotypes, compared to their respective controls. Heat stress decreased albumins by 59.6–66.7% in the HS genotypes and 26.2–29.5% in the HT genotypes. The globulins decreased by 66.6–70.6% in the HS genotypes and 37.2–38.0% in the HT genotypes. Glutelins showed a 50.2–52.5% reduction in the HS genotypes and a 13.5–15.7% reduction in the HT genotypes. At the same time, prolamins exhibited a 62.57–69.5% reduction in the HS genotypes and a 25–33% reduction in the HT genotypes.

Figure 7
figure 7

Seed storage proteins in heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown under control (25/15 °C) and heat stress (32/20 °C) environment. LSD: (P < 0.05) (genotypes x treatment interaction): Albumins: 1.76, Globulins: 6.12; Glutelins: 2.13; Prolamins: 0.78.

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Seed carbohydrate metabolism

The sucrose concentration (Fig. 8, Tables 1, 2) in the seeds of heat-stressed plants decreased more in the HS genotypes (53–55%) than in the HT genotypes (25–28.7%). In contrast, heat stress markedly increased glucose and fructose concentrations by 41–45% and 49–50% in the HT genotypes and 33–35% and 50–60% in the HS genotypes, respectively, relative to their corresponding controls.

Figure 8
figure 8

Seed carbohydrates in heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown under control (25/15 °C) and heat stress (32/20 °C) environment. LSD: (P < 0.05) (genotypes x treatment interaction): Sucrose: 1.13, Glucose: 0.41; Fructose: 0.39.

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Heat stress decreased activities of enzymes-sucrose synthase, soluble starch synthase, and acid invertases (Tables 1, 2, 4), more so in the HS genotypes (46.4–46.8%, 40–45%, and 42.5–45.4%, respectively) than in the HT genotypes (17.6–18.2%, 17–22%, and 19.3–22.6%, respectively) in relation to their respective controls.

Table 4 Enzymes related to carbohydrate metabolism in seeds of heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown in control (25/15 °C) and heat stress (32/20 °C) environment.
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Mineral nutrients

The heat stress treatments had a significant impact on the seed Ca, P, and Fe concentrations of both HS and HT genotypes compared to the controls, with decreases of 47–54% for Ca, 33–37% for P, and 59% for Fe in HS genotypes, and 21–25% for Ca, 21–25% for P, and 31–35% for Fe in HT genotypes (Table 1, 2, and 5).

Table 5 Seed minerals in heat-tolerant (HT1: ICCV07110; HT2: ICCV92944) and heat-sensitive (HS1: ICC14183; HS2: ICC5912) chickpea genotypes grown in control (25/15 °C) and heat stress (32/20 °C) environments.
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Discussion

Heat stress during the seed-filling stage had a significant negative impact on chickpea seed yield and quality, particularly in the HS genotypes compared to the HT genotypes. Reduced seed numbers under heat stress have been attributed to a decrease in pod numbers caused by fertilization failure, as previously reported for chickpea25,59. Heat stress also led to a substantial decline in single-seed weight owing to disruptions in the accumulation of various seed components, resulting in decreased seed weight per plant and harvest index. These findings align with those of previous studies highlighting the detrimental effects of high temperatures on yield-related traits in chickpea59,60 and other crops, such as wheat61 and maize62. Additionally, heat stress adversely affected seed nutritional components, including carbohydrates, proteins, and fats, leading to poor seed quality3,5,13,30,36.

Heat stress impairs vegetative growth by damaging leaf tissues, disrupting of various functions and eventually inhibiting seed development10. Membrane damage in leaves is a reliable indicator of thermotolerance in legumes63. High temperatures disrupt membrane organization or generate reactive oxygen species (ROS)64 that target the cell membranes. Heat stress resulted in reduced leaf chlorophyll content and necrosis, which is consistent with observations in bent grass65. A reduction in chlorophyll content can occur because of the inhibition of biosynthesis and/or photooxidation. Tissue death caused by heat stress is likely a direct consequence, as reported in other plant species, such as the common bean66. Moreover, heat stress caused a significant decrease in chlorophyll fluorescence, indicating impaired photosynthetic efficiency, similar to the findings in heat-stressed cotton67 and lentil7. Heat stress severely disrupted the carbon fixation ability, as measured by RuBisCo activity, in chickpea, which was associated with reduced stomatal conductance or inhibited RuBisCo activity68. Heat stress also decreased sucrose synthase activity, limiting carbon assimilation, and subsequently reducing leaf sucrose content8,68. Another study reported similar inhibition of photosynthesis, RuBisCo activity, and sucrose synthase activity in heat-stressed lentil plants69.

Leaf water status, indicated by relative leaf water content (RLWC), was significantly decreased in heat-stressed chickpea plants, suggesting the impact of intensified water stress on the aforementioned leaf traits under high temperatures70. A previous study also reported a reduction in RLWC in chickpea plants grown in a high-temperature environment. In our study, heat stress increased stomatal conductance in the HT genotypes but decreased it in the HS genotypes, which explains the higher RLWC values in the HT genotypes. Water loss from heat-stressed chickpea leaves emerged as a crucial factor affecting cellular metabolism and differentiating heat sensitivity among the contrasting genotypes. The HT chickpea genotypes exhibited higher RLWC and stomatal conductance under heat stress than the HS genotypes, resulting in greater photosynthetic activity. Previous studies have highlighted the effect of leaf water status on heat tolerance in mung bean71 and alfalfa plants9.

Heat stress significantly affects seed filling in chickpea, leading to a marked reduction in seed-filling rate and duration, thereby inhibiting the accumulation of various constituents such as starch, proteins, and fats. The decrease in these seed components could result from disrupted import of precursors from leaves, decreased biosynthesis in seeds, or both. Sucrose import into seeds can be significantly inhibited in heat-stressed plants because of reduced availability in leaves and decreased expression of sucrose transporters72. We observed marked inhibition of sucrose synthase and soluble starch synthase activities in chickpea seeds, leading to reduced carbohydrate content and smaller seeds. In wheat, heat stress decreases the activities of sucrose synthase, soluble starch synthase, glucokinase, and ADP glucose pyrophosphorylase, resulting in poor starch and fat accumulation73, which is similar to the findings in maize34,74. Similarly, heat stress decreases the activities of sucrose synthase and starch branching enzymes in aromatic rice grains, impairing starch accumulation75.

The decrease in proteins, including storage proteins, could be attributed to insufficient precursors and/or inhibited biosynthetic enzyme activities76, which is consistent with previous studies on maize77, lentil7, and soybean36 grown in high-temperature environments. In contrast, maize seeds exhibit increased protein levels under heat stress because of the increased activity of associated enzymes34. Downregulation of seed storage protein-encoding genes under heat stress could be another possible reason for the decline in storage protein in chickpea, as evidenced in soybean36. The differing observations in this regard may be attributed to variations in heat stress treatments across the studies.

Fat accumulation decreased significantly in heat-stressed chickpea seeds, possibly because of a decline in acetyl-CoA content resulting from inhibited photosynthetic activity, as reported in common bean77 and canola78. Further research is required to understand the mechanisms that affect protein and fat synthesis in chickpea seeds at high temperatures.

Heat stress inhibits the accumulation of minerals (Ca, Fe, and P) in chickpea seeds, possibly because of impaired translocation from leaves79, which disrupts transport mechanisms in seeds and the mobilization of various molecules and ions. Further investigation is necessary to understand the transport mechanisms of these minerals in chickpea seeds developing under high-temperature environments.

Observations of contrasting chickpea genotypes under heat stress revealed that the heat-tolerant (HT) genotypes exhibited better leaf maintenance, less membrane damage to their photosynthetic activity, higher water retention and greater stomatal conductance. Consequently, heat stress affected leaf sucrose generation less in the HT genotypes than in the HS genotypes, resulting in more sucrose being available for export to seeds. Furthermore, the HT genotypes demonstrated more stable enzymes for sucrose and starch synthesis than the HS genotypes, leading to increased starch and sucrose contents. Similar implications can be speculated for other storage components such as proteins and fats, which require further investigation. Nonetheless, our observations of contrasting chickpea genotypes have provided valuable insights into the target sites of heat stress in the leaves and seeds, which can be leveraged in the development of heat-tolerant chickpea cultivars.

In conclusion, heat stress during the chickpea seed-filling stage negatively affected seed yield and quality, particularly in heat-sensitive genotypes. Heat stress reduced seed numbers by decreasing pod numbers because of fertilization failure. It also led to a smaller seed size and decreased the accumulation of carbohydrates, proteins, and fats. Heat stress disrupted leaf chlorophyll content, photosynthetic efficiency, and carbon fixation, causing a decrease in leaf water content and impaired seed filling, resulting in reduced starch, protein, and fat levels. Heat stress also inhibited mineral accumulation in the seeds. The contrasting genotypes revealed the importance of improved leaf maintenance and water retention in heat-tolerant genotypes. These findings highlight the need for heat-tolerant cultivar development and further research to understand its underlying mechanisms.