Understanding the effect of heat stress during seed filling on nutritional composition and seed yield in chickpea (Cicer arietinum L.)

Increasing temperature affects all food crops, thereby reducing their yield potential. Chickpea is a cool-season food legume vital for its nutritive value, but it is sensitive to high temperatures (> 32/20 °C maximum/minimum) during its reproductive and seed-filling stages. This study evaluated the effects of heat stress on yield and qualitative traits of chickpea seeds in a controlled environment. Chickpea genotypes differing in heat sensitivity [two heat-tolerant (HT) and two heat-sensitive (HS)] were raised in pots, initially in an outdoor environment (average 23.5/9.9 °C maximum/minimum), until the beginning of pod set (107–110 days after sowing). At this stage, the plants were moved to a controlled environment in the growth chamber to impose heat stress (32/20 °C) at the seed-filling stage, while maintaining a set of control plants at 25/15 °C. The leaves of heat-stressed plants of the HT and HS genotypes showed considerable membrane damage, altered stomatal conductance, and reduced leaf water content, chlorophyll content, chlorophyll fluorescence, and photosynthetic ability (RuBisCo, sucrose phosphate synthase, and sucrose activities) relative to their corresponding controls. Seed filling duration and seed rate drastically decreased in heat-stressed plants of the HT and HS genotypes, severely reducing seed weight plant–1 and single seed weight, especially in the HS genotypes. Yield-related traits, such as pod number, seed number, and harvest index, noticeably decreased in heat-stressed plants and more so in the HS genotypes. Seed components, such as starch, proteins, fats, minerals (Ca, P, and Fe), and storage proteins (albumin, globulins, glutelin, and prolamins), drastically declined, resulting in poor-quality seeds, particularly in the HS genotypes. These findings revealed that heat stress significantly reduced leaf sucrose production, affecting the accumulation of various seed constituents, and leading to poor nutritional quality. The HT genotypes were less affected than the HS genotypes because of the greater stability of their leaf water status and photosynthetic ability, contributing to better yield and seed quality traits in a heat-stressed environment.


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.
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 method 45 .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 Weatherley 46 .Leaf stomatal conductance was recorded on the same leaves using a leaf porometer as described previously 47 .
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 elsewhere 25 .

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 EDTANa 2 .2H 2 O.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 earlier 53 .
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 procedures 55 .
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.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.

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 -1 DW 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).
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).www.nature.com/scientificreports/RuBisCo activity (Fig. 4A)-an indicator of carbon fixation ability in control plants, exhibited values between 631 and 654 nmol CO 2 min -1 mg -1 protein in HT genotypes and 609-674 nmol CO 2 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).
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 40.1-42.3µmol 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).
In relation to their respective control groups, following impacts were observed due to heat stress.
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.5).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).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).e.Average seed size: Heat stress resulted in a decrease of 60% in the HS genotypes and 24-27% in the HT genotypes.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.g.Harvest index: Heat stress caused a reduction of 64-67% in the HS genotypes and 30-34% in the HT genotypes.

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).

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 chickpea 25,59 .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 chickpea 59,60 and other crops, such as wheat 61 and maize 62 .Additionally, heat stress adversely affected seed nutritional components, including carbohydrates, proteins, and fats, leading to poor seed quality 3,5,13,30,36 .
Heat stress impairs vegetative growth by damaging leaf tissues, disrupting of various functions and eventually inhibiting seed development 10 .Membrane damage in leaves is a reliable indicator of thermotolerance in legumes 63 .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 grass 65 .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 bean 66 .Moreover, heat stress caused a significant decrease in chlorophyll fluorescence, indicating impaired photosynthetic efficiency, similar to the findings in heat-stressed cotton 67 and lentil 7 .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 activity 68 .Heat stress also decreased sucrose synthase activity, limiting carbon assimilation, and subsequently reducing leaf sucrose content 8,68 .Another study reported similar inhibition of photosynthesis, RuBisCo activity, and sucrose synthase activity in heat-stressed lentil plants 69 .
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 temperatures 70 .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 bean 71 and alfalfa plants 9 .
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 transporters 72 .We observed marked 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.LSD (P < < 0.05) for genotypes x treatment interaction: TDW (2.3), SY (1.5), SW (10.2),SS (1.3), PN (2.1), SN (7.6), HI (0.083), SGR (1.13), SFD (2.11).Values represent mean ± S.E.Different lower-case letters for each trait indicate significant variations (P < 0.05).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 73 , which is similar to the findings in maize 34,74 .Similarly, heat stress decreases the activities of sucrose synthase and starch branching enzymes in aromatic rice grains, impairing starch accumulation 75 .The decrease in proteins, including storage proteins, could be attributed to insufficient precursors and/or inhibited biosynthetic enzyme activities 76 , which is consistent with previous studies on maize 77 , lentil 7 , and soybean 36 grown in high-temperature environments.In contrast, maize seeds exhibit increased protein levels under heat stress because of the increased activity of associated enzymes 34 .Downregulation of seed storage  www.nature.com/scientificreports/protein-encoding genes under heat stress be another possible reason for the decline in protein in chickpea, as in soybean 36 .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 as reported in common bean 77 and canola 78 .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 leaves 79 , 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.www.nature.com/scientificreports/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 sucrose being available for export to seeds.Furthermore, HT genotypes demonstrated more 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.

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).