Starch morphological, structural, pasting, and thermal properties of waxy maize under different heat stress durations at grain formation stage

Abstract

Heat stress (HS) is a major environmental constraint in sustaining global waxy maize production under future climate condition. Starch physicochemical properties under 5‐day (HS5) and 10‐day (HS10) postsilking HS were investigated using two waxy maize hybrids (Suyunuo5 and Yunuo7) as materials. Ambient and high temperatures were 28°C/20℃ and 35°C/27℃, respectively. Starch granules were enlarged and exhibited additional pitting or cellular structures, increased amylopectin chain length, increased relative crystallinity, and increased 1047/1022 cm⁻¹ ratio under HS. This finding indicated the stable and ordered structure of starch granules, especially under HS10. Pasting viscosities of Yunuo7 reduced by HS, and the change was the same under both HS5 and HS10. Peak viscosity of Suyunuo5 was unaffected and breakdown viscosity increased with HS5 although both characteristics decreased with HS10. HS increased gelatinization temperatures and retrogradation percentage. Results showed that HS enlarges starch granules with stable structure, reduces starch viscosities, and increases retrograde tendency, with severe detrimental effect under long HS durations.

INTRODUCTION

Crop growth and development are strongly affected by immediate environment. Among adverse environmental conditions, high temperature exerts detrimental effects on grain yield and quality (Tao et al., 2016; Wang & Frei, 2011). The 2018 Intergovernmental Panel on Climate Change predicted that the global mean temperature will increase by 1.5°C in the 2050s and generate severe effects on food security (Tollefson, 2018). The increased frequency of extreme high temperature events will inevitably affect crop physiological metabolism and grain development and generally reduce crop yield and quality (Tiwari & Yadav, 2019).

Starch is the predominant component (approximately 70%) of cereal grains and a key contributor to cereal crop yield and quality. Starch formation is very sensitive to adverse environment that close to pollination (Thitisaksakul et al., 2012). Starch formation and quality are affected by heat stress (HS) during grain filling (Thitisaksakul et al., 2012; Wang & Frei, 2011). Starch consists of two types of molecules, namely, amylose (linear α-1,4-polyglucan) and amylopectin (α-1,6-branched polyglucans). Content and structural changes of starch under HS induce the change of physicochemical characteristics and affect its utilization. Studies on wheat reported that postanthesis HS increases the amylose content but decreases amylopectin and total starch contents (Liu, Ma, et al., 2017; Liu, Zhao, et al., 2017) mainly due to the downregulated expression of all starch synthetic genes (except ISA2) under HS (Yang et al., 2019).

Wheat starch A granules increased in number, decreased in size, and exhibited additional pits; B granules decreased in both number and size; and proportionally increased protein matrix in endosperm cells of the grain contributed to variations in flour quality under HS (Hurkman & Wood, 2011). The proportion of small B-granule decreased and that of large B-granule increased, while A granules were unaffected by HS in barley starch. In addition, the decreased abundance of degree of polymerization (DP) 7–11 and increased abundance DP 20–37 resulted in high gelatinization temperatures (Cuesta-Seijo et al., 2019). The normal maize starch granule size decreased and gelatinization temperatures increased while branch-chain fractions were different between two inbred strains in response to HS (Lu et al., 1996). Loosely arranged starch granules of rice exposed to postanthesis short-term HS increased the appearance of single starch granules that resulted in the deterioration of rice eating quality (Chen et al., 2017). Additionally, the surface of starch granules under HS exhibited additional pits and poles and increased its susceptibility to enzymatic hydrolysis (Li et al., 2017). The decreased DP 6–15 and increased DP > 18 under HS increased starch gelatinization temperatures, while the gelatinization enthalpy of rice starch remained unaffected (Kato et al., 2019). HS increased the starch with medium–long chains in normal and waxy rice, thereby resulting in high gelatinization characteristics and trough and final viscosities (Fan et al., 2019). HS during pollen filling stages increased the starch accumulation and enlarged the granule size that resulted in low apparent amylose content, high ordered degree, and high pasting viscosities and gelatinization temperatures (Lin et al., 2020; Liu, Ma, et al., 2017; Liu, Zhao, et al., 2017). A field study reported that rice pasting property can be improved by decreasing starch granules (Yao et al., 2020). However, postheading HS shortened grain filling periods, induced poor grain development, and resulted in decreased pasting viscosities in rice, especially when HS occurred in the second week (Siddik et al., 2019).

Maize (Zea mays L.) is an important cereal crop that serves as food, feed, and industrial raw materials (Niu et al., 2019). Its economical and nutritional value mainly depends on the content and structure of starches. Among various types, waxy maize starch is distinct with starch composed of nearly pure amylopectin, which provides advantages of viscosity, stability, and retrogradation (Lu & Lu, 2012). Extremely high temperatures during summer generate a detrimental effect on starch physicochemical properties of waxy maize because this period often overlaps with the maize grain development stage in field wheat–maize rotation system. Our previous studies reported that short (5 days) (Gu et al., 2018), medium (15 days) (Lu et al., 2013, 2016), and long (entire grain filling stage) (Lu et al., 2014) HS durations at the grain filling stage affect starch structural and functional properties of waxy maize, with the most severe effect close to pollination. However, studies on the effect of HS durations on starch physicochemical properties are limited.

High temperatures are typical during summer. For example, average day/night temperatures from 21 July to 10 August in 2016-2021 were 35.4°C/27.6°C, 36.0°C/27.6°C, 35.0°C/26.9°C, 35.7°C/27.6°C, 30.9°C/27.6°C, and 32.1°C/25.1°C and continuous days with a maximum temperature higher than 35°C was 13, 19, 8, 12, 5, and 3 days in Yangzhou, China. We hypothesize that variable HS durations produce starch with different functional properties due to HS duration uncertainty in various years. Two waxy maize hybrids, namely, heat-sensitive Suyunuo5 (SYN5) and heat-tolerant Yunuo7 (YN7), were used in this study. The study aimed to clarify (1) the effect of 5- and 10-day HS durations at early grain formation stage on pasting and thermal properties and (2) reveal underlying mechanisms of induced changes in the starch granule morphology and size, amylopectin chain length, and crystalline structures. The results of this study may provide a reference for efficient utilization of waxy maize starch in zones frequently exposed to HS.

MATERIALS AND METHODS Experimental design

A pot trial was conducted at the Yangzhou University Experimental Farm in the spring of 2019 (sowing date: March 15) using Suyunuo5 (SYN5) and Yunuo7 (YN7) as materials. The pot with a height of 38 cm and a diameter of 43 cm was filled with 30 kg of sieved sandy loam soil (organic matter, 11.5 g/kg; total nitrogen, 1.02 g/kg; alkali hydrolysable nitrogen, 37.4 mg/kg; available phosphorus, 12.5 mg/kg; and exchangeable potassium 115.6 mg/kg). Plants per pot were provided with 10 g of compound fertilizer (N/P₂O₅/K₂O = 15%/15%/15%) at the transplantation stage and 6.6 g of urea (N = 46%) at the six-leaf stage.

Plants were grown in a field environment until the silking stage. Manual pollination was performed on the same day, and then, pots were moved to the greenhouse the next day for temperature treatments. Temperatures in the intelligent greenhouse (L × W × H = 4 m × 3 m; cooling/heating capacity, 115 kW; rated power input, 29 kW; Shanghai Yileng–Carrier Air Conditioning Equipment Co., Ltd., China) were 35°C/27°C (heat stress, HS) and 28°C/20°C (ambient temperature, AT). HS durations were 5 (2–6 days after pollination, HS5) and 10 (2–11 days after pollination, HS10) days. The temperature was reset to AT until maturity (approximately 40 days) after ceasing the HS. The soil moisture content was controlled at 70%–80% and then resupplied with water the next morning after weighing the day weight loss. Humidity was controlled at 70% throughout the grain filling stage. Each treatment included 40 pots.

Starch isolation

Starch isolation was conducted following our earlier reported method and 100 g of grain steeping in 500 ml of 1 g/L NaHSO₃ solution at room temperature for 48 h (Lu & Lu, 2012). Samples were rinsed with distilled water and then ground with a blender for 2.5 min. Suspensions were passed through a 100-mesh sieve. Remaining materials on the screen were homogenized again for 1.5 min and then passed through the same sieve. The starch–protein slurry was collected in a 1000 ml wide-neck flask and allowed to stand for 4 h. The supernatant was removed and the settled starch layer was collected in 50-ml centrifuge tubes and then centrifuged at 3000 g for 10 min. The upper nonwhite layer was removed with a scoop. The white layer was resuspended in distilled water and then stirred for 30 min before centrifugation. Isolation procedures were repeated three times. Protein, lipid, and ash contents in the isolated starch were lower than 4, 2, and 2 mg/g, indicating that starch purity reach the Chinese National Standard of edible maize starch (GB/T 8885−2017).

Starch morphology

Starch granules were mounted on circular aluminum stubs with double-sided tape, coated with gold, examined under a scanning electron microscope (GeminiSEM 300; Carl Zeiss), and then photographed at an accelerating potential of 5 kV (Lu & Lu, 2012).

Granule size distribution

Size distributions of starch granules were determined using the method (Gu et al., 2018). Instrument accuracy was verified using Malvern standard glass particles. The instrument can measure sizes of 0.1 and 2000 μm. Size distribution was expressed in terms of the volume of equivalent spheres. The average granule size was defined as the volume weighted mean.

Amylopectin chain length distribution

Debranched amylopectin chain length distribution was determined via high-performance anion-exchange chromatography with pulsed amperometric detection (Dionex-ICS 3000; Dionex Corporation) for the degree of polymerization (DP) 6–90 after debranching the starch using isoamylase (EC 3.2.1.68 Sigma) dissolved in 50 mM of NaAc (Shi et al., 2018). Standard triple potential waveform was used. Periods and pulse potentials were T1 = 0.40 s with 0.20 s of sampling time, E1 = 0.05 V; T2 = 0.20 s, E2 = 0.75 V; and T3 = 0.40 s, E3 = 0.15 V. Data were collected using the Chromeleon software. The weight fraction of DP 6–12, 13–24, 25–36, and 37–76 was measured based on the area of peaks. Eluents were prepared in distilled deionized water with N2. Eluent A was 200 mM NaAc in 200 mM NaOH and eluent B was 200 mM NaOH. Linear components were separated on a Dionex CarboPac PA10 column with a gradient elution of 0 min, 10% eluent A; 10 min, 10% eluent A; 30 min, 60% eluent A; 50 min, 60% eluent A; and 60 min, 10% eluent A at a column temperature of 30°C and a flow rate of 0.4 ml/min. A CarboPac PA10 guard column was installed in front of the analytical column.

X-ray diffraction pattern

X-ray diffraction (XRD) patterns of starch were obtained with an X-ray diffractometer (D8 Advance; Bruker-AXS) following the detailed method (Shi et al., 2018). The diffractometer was operated at 200 mA and 40 kV. The scanning region of the diffraction angle (2θ) ranged from 3° to 40° at a step size of 0.04° and a count time of 0.6 s. Relative crystallinity (%) was calculated as the percentage of total crystalline peak areas to that of the total diffractogram (total crystalline and amorphous peak areas) using the MDI Jade 6 software.

Attenuated total reflectance Fourier Transform Infrared spectrometry

Starch (30 mg) was mixed with 25 μl of distilled water. The resulting slurry was placed on the sample platform of a Fourier transform infrared (FTIR) spectrometer (Cary610/670; Varian). FTIR spectra were recorded from 1200 cm⁻¹ to 800 cm⁻¹ and then deconvoluted with a resolution enhancement factor of 1.9 and a half width of 19 cm⁻¹ (Shi et al., 2018). Absorbance values at 1047 and 1022 cm⁻¹ were extracted from the spectra after water subtraction, baseline correction, and deconvolution.

Pasting properties

Pasting properties of starch (28 g total weight; 7% db, w/w) were estimated following a previously described method (Lu & Lu, 2012). The suspension was equilibrated at 50°C for 1 min, heated to 95°C at 12°C/min, maintained at 95°C for 2.5 min, cooled to 50°C at 12°C/min, and then maintained at 50°C for 1 min. Paddle speed was set to 960 rpm for the first 10 s and then decreased to 160 rpm for the remainder of the analysis.

Thermal properties

Starch thermal characteristics were explored using differential scanning calorimetry (DSC, Model 200 F3 Maia, NETZSCH) following a previously described method (Gu et al., 2018). Each sample (5 mg, dry weight) was loaded onto an aluminum pan (25/40 ml, D = 5 mm) and distilled water was added to achieve a starch−water suspension containing 66.7% water. Samples were hermetically sealed and allowed to stand for 24 h at 4°C before heating in the DSC. The DSC analyzer was calibrated using an empty aluminum pan as the reference. Sample pans were heated at a rate of 10°C/min from 20°C to 100°C. Thermal transitions of starch were defined as onset temperature (T_o), peak gelatinization temperature (T_p), conclusion temperature (T_c), and gelatinization enthalpy (ΔH_gel). Samples were stored at 4°C for 7 days after conducting thermal analysis for retrogradation investigations. Retrogradation enthalpy (ΔH_ret) was automatically evaluated and retrogradation percentage (%R) was calculated as %R = 100 × ΔH_ret/ΔH_gel.

Statistical analysis

Data presented in tables and figures are the average value of three replications. Analysis of variance was performed with Data Processing System (DPS version 7.05) to determine the least significant difference at the p < 0.05 level (Tang & Feng, 2007).

RESULTS AND DISCUSSION Morphology of starch granules

Waxy maize starch granules presented irregular or oval polytopes under different treatments due to mutual oppresses (Figure 1). Postsilking HS increased the ratio of granules with pits or holes when plant growth under AT is used as the control. Holes were larger and deeper, especially under long HS durations (HS10), than those under short HS durations (HS5). Increased pits or holes under HS were also reported in rice (Lin et al., 2020; Yao et al., 2020), wheat (Hurkman & Wood, 2011), blackgram (Partheeban & Vijayaraghavan, 2020) and normal (Commuri & Jones, 1999) and waxy (Lu et al., 2013) maize. Pits or holes likely due to the high activity of α- and β-amylase under HS (Figure S1) corroded starch granules. Studies on rice (Hakata et al., 2012) and wheat (Impa et al., 2020; Liu, Ma, et al., 2017; Liu, Zhao, et al., 2017) demonstrated that the upregulated expression of α- and β-amylase under HS increases the susceptibility of endosperm starch granules to enzymatic hydrolysis or degradation and ultimately induces cellular structures (Kato et al., 2019).

FIGURE 1. Morphology of starch granules for two waxy maize hybrids under ambient temperature, 5- and 10-day heat stress conditions (SYN5, Suyunuo5; YN7, Yunuo7; AT, ambient temperature; HS5, 5-day heat stress; HS10, 10-day heat stress)

Size distribution of starch granules

The size distribution of starch granules presents typical dual-peak profiles (Figure 2). The percentage of small starch granules with a diameter d < 5 μm ranged from 9.1% to 10.1% among different treatments (Table S1). Compared with AT, HS significantly increased the percentage of large granules (d > 15 μm) and average starch granule size as well as gradually enlarged the size with the extended HS duration in both hybrids. Our previous studies also reported that the size of starch granules enlarges due to post‒silking HS (Gu et al., 2018; Lu et al., 2014). The number of endosperm starch granules in the maize grain development stage begin to increase in the first 2 weeks after enlargement of the granule size (Li et al., 2007). Endosperm cell division and amyloplast formation were disturbed by postsilking HS, and the decreased number of endosperm starch granules was severe under long HS durations (Commuri & Jones, 1999). The substrate transferred to existing starch granules and enlarged them because HS restricted the formation of starch granules; this phenomenon also demonstrated that starch granules are loosely packed in the endosperm (Lo et al., 2016). A study on barley demonstrated that the proportion of small B-granules decreases but that of large A-granules increases by HS and the restriction of B-granule initiation by precocious senescence (Cuesta-Seijo et al., 2019). A-type starch granules in wheat increased in number and decreased in size, while B-type starch granules decreased in both number and size (Hurkman & Wood, 2011). Studies on rice also reported that the starch granule size enlarges with HS (Liu, Ma, et al., 2017; Liu, Zhao, et al., 2017; Wang et al., 2020). However, a field study reported that the average starch granule in rice decreases by HS (Yao et al., 2020). This discrepancy may be due to the difference between field and chamber studies performed.

FIGURE 2. Volume distributions of starch granules for two waxy maize hybrids under ambient temperature, 5- and 10-day heat stress conditions (AT, ambient temperature; HS5, 5-day heat stress; HS10, 10-day heat stress. Data in the bracket are the value of average granule size)

Amylopectin chain length

Chain length distribution of amylopectin can be fractionated into A (DP 6–12), B1 (DP 13–24), B2 (DP 25–36) and B3 (DP ≥ 37) chains (Hsieh et al., 2019). The chain length distribution of isoamylase–debranched amylopectin was affected by postsilking HS. HS increased the average chain length of amylopectin, and the increase was higher under HS10 than that under HS5 in both hybrids (Figure 3). Although the percentage of A chain in YN7 decreased by HS, values under HS5 and HS10 were similar. The percentage of A chain in SYN5 increased under HS5 but decreased with HS10. The percentage of B1 chains decreased by HS, and a significant decrease was observed under HS5 and HS10 in both SYN5 and YN7. The percentage of B2 chains in SYN5 was increased by HS, the higher increase was with HS5, and the value in YN7 was unaffected by HS5 but was increased by HS10. The percentage of B3 chain gradually increased with extended HS durations in both hybrids (Table S2). A study on barley also showed that the frequency of A chain decreases when plants suffer from HS during the grain filling stage (Cuesta-Seijo et al., 2019). A study on rice observed that DP 6–15 decrease and DP > 18 increase under HS (Kato et al., 2019). Similar to the results of earlier studies on waxy maize (Yang et al., 2018), wheat (Zhang et al., 2017), and rice (Hu et al., 2021; Kato et al., 2019; Zhao et al., 2019), the increase of long chains may be due to the decreased activity of starch branching enzyme (SBE). This phenomenon occurs because SBE activity constantly leads to the disruption of long chains of glucose to produce two short ones (Nakamura et al., 2010). Therefore, this study showed that low SBE activity leads to the increase in the number of long chains. However, SBE can retain its level of activity based on its substrate with different amylopectin chains given that maize endosperms contain three isozymes (sbe1, sbe2a, and sbe2b). Hence, clarifying their differential expressions and activity under HS may provide additional details to changes in the amylopectin chain length (Tetlow & Emes, 2014).

FIGURE 3. Chain length distributions of debranched amylopectin for two waxy maize hybrids under ambient temperature, 5- and 10-day heat stress conditions (AT, ambient temperature; HS5, 5-day heat stress; HS10, 10-day heat stress. Data in the bracket are the value of average chain length)

FTIR and XRD pattern

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectrometry has been widely used to investigate crystalline and amorphous regions near surfaces of starch granules (Zhang et al., 2013). The 1047/1022 cm⁻¹ ratio from the deconvoluted FT-IR spectrum was used to quantify the degree of order of the starch granule surface (Lin et al., 2020). Compared with that under AT, the 1047/1022 cm⁻¹ ratio increased by HS and the increase was higher under HS10 in both hybrids (Figure 4). This finding indicated that the high temperature stressed starch forms an increasingly ordered structure. Similar results were also reported in rice (Lin et al., 2020) and in our previous study (Wang et al., 2021). The high 1047/1022 cm⁻¹ ratio may be because the starch presents a large granule size and long amylopectin chain length (Wang et al., 2021). Meanwhile, a study on high-amylose maize starch demonstrated that small starch granules exhibit a high 1047/1022 cm⁻¹ ratio (Lin et al., 2016). This discrepancy may be due to the likely participation of amylose in the control of starch structures.

FIGURE 4. Starch FTIR profiles of two waxy maize hybrids under ambient temperature, 5- and 10-day heat stress conditions (SYN5, Suyunuo5; YN7, Yunuo7; AT, ambient temperature; HS5, 5-day heat stress; HS10, 10-day heat stress. Data in the bracket are the value of 1047/1022 cm−1 ratio)

The XRD pattern was unaffected by the HS and all samples presented a typical “A”-shaped pattern (Figure 5). The increase of relative crystallinity (RC) of SYN5 due to HS was high under HS10. RC of YN7 decreased with HS5 but increased with HS10. Studies on rice also presented that HS at primordial differentiation and pollen-filling stages increases the RC of starches (Lin et al., 2020; Wang et al., 2020). However, another field study reported that the RC in response to HS is different among various varieties based on thermotolerance (Yao et al., 2020). The change in RC under HS may be due to the large granules and high proportions of long amylopectin chains in starch (Lin et al., 2020).

FIGURE 5. Starch XRD profiles of two waxy maize hybrids under ambient temperature, 5- and 10-day heat stress conditions (AT, ambient temperature; HS5, 5-day heat stress; HS10, 10-day heat stress. Data in the bracket are the value of relative crystallinity)

Pasting properties

Starch pasting characteristics were affected by HS in both hybrids (Figure 6, Table S3). The pasting temperature (P_temp) of YN7 was unaffected by HS, and the decrease of pasting viscosities due to HS was similar under HS5 and HS10. Peak (PV) and setback (SB) viscosities of SYN5 were unaffected by HS5 but decreased under HS10. The decrease of trough viscosity (TV) due to HS was similar under HS5 and HS10. Breakdown viscosity (BD) decreased with HS10 but increased under HS5. Final viscosity (FV) decreased by HS, and the decrease was severe under HS10. P_temp decreased under HS5 but increased with HS10. Our previous studies showed that pasting viscosities decrease by HS and P_temp is dependent on hybrids (Gu et al., 2018; Lu et al., 2016; Wang et al., 2021). Reduced pasting viscosities under HS may be due to the large granules (Gu et al., 2018; Lu et al., 2016) and long amylopectin chains (Zhang et al., 2008) of starch. Meanwhile, pitted and eroded granules under HS may be due to the increased amylase activity induced starch hydrolysis and suppressed starch swelling (Asiri et al., 2019; Zhang et al., 2021). Studies on rice (Fahad et al., 2016; Siddik et al., 2019) also demonstrated that pasting properties deteriorate by HS during grain filling. Fan et al. (2019) observed that HS increases the rice starch TV, FV, and P_temp but decreases PV and BD in waxy rice and increases PV and BD in nonwaxy rice. Yao et al. (2020) revealed that HS decreases SB and TV and increases BD while improving pasting properties.

FIGURE 6. Starch pasting profiles of two waxy maize under ambient temperature, 5- and 10-day heat stress conditions (AT, ambient temperature; HS5, 5-day heat stress; HS10, 10-day heat stress)

Thermal properties

Starch ΔH_gel in YN7 was unaffected by HS but that in SYN5 significantly decreased by HS, and the decrease was similar in both HS5 and HS10 (Table 1). T_o and T_p in SYN5 increased by HS, and the increase was higher under HS10 than that under HS5. Meanwhile, T_c in SYN5 decreased by HS5 but increased with HS10. Gelatinization temperatures in YN7 significantly increased by HS. T_o and T_c values were similar under HS5 and HS10, while T_p was higher under HS5 than that under HS10. Studies on rice (Fan et al., 2019; Yao et al., 2020), maize (Gu et al., 2018; Lu et al., 1996, 2014), barley (Cuesta-Seijo et al., 2019), and wheat (Thitisaksakul et al., 2012) also showed that gelatinization temperatures increase by HS. However, ΔH_gel in response to HS is dependent on hybrids and stress stages (Gu et al., 2018; Lu et al., 2016; Wang et al., 2021). High gelatinization temperatures may be due to the high ratio of long amylopectin chains, high relative crystallinity, and increasingly ordered form of starch (Hsieh et al., 2019).

TABLE 1 Starch thermal characteristics of waxy maize under different treatments^a

Variety	Treatments	ΔHgel (J/g)	To (℃)	Tp (℃)	Tc (℃)	ΔHret (J/g)	%R (%)
Suyunuo5	Control	11.6a	67.6e	74.0d	81.7bc	4.3d	37.6c
	HS 2–6	8.6c	70.3b	75.6b	79.8d	4.75cd	55.6a
	HS 2–11	9.1c	71.8a	76.6a	83.6a	5.1bc	55.7a
Yunuo7	Control	10.9b	68.3d	73.4e	80.7cd	5.3bc	48.2b
	HS 2–6	11.3ab	69.8bc	74.7c	82.8ab	5.6b	49.7ab
	HS 2–11	12.0ab	69.1c	74.2d	82.2abc	6.3a	52.9ab

Abbreviations: %R, retrogradation percentage; AT, ambient temperature; HS10, 10-day heat stress; HS5, 5-day heat stress; T_c, conclusion temperature; T_o, onset temperature; T_p, peak gelatinization temperature; ΔH_gel, gelatinization enthalpy; ΔH_ret, retrogradation enthalpy.

^aMean values in the same column followed by different letters are significantly different (p < 0.05).

Retrogradation occurred after storing gelatinized samples at 4°C for 7 days. ΔH_ret in both hybrids was unaffected by HS5 but increased by HS10. The %R increased by HS, and the increase was similar between HS5 and HS10 in both hybrids. Our earlier studies also reported that %R increases when plants suffer from short or long HS durations at the grain filling stage (Gu et al., 2018; Lu et al., 2013, 2014). The high %R under HS may be due to long amylopectin chains (Bertofta et al., 2016; Hsieh et al., 2019; Matalanis et al., 2009), large granule size, and high crystallinity (Lu et al., 2014; Shi & Seib, 1995) of starch.

CONCLUSION

The present study demonstrated the deterioration of starch physicochemical properties of waxy maize due to postsilking HS. Our results showed that HS enlarges the starch granule size and increases the amylopectin chain length and 1047/1022 cm⁻¹ ratio while presenting a stronger influence under HS10 than that under HS5. The starch granule surface exhibited more pits or holes under HS due to the enhanced activity of the amylolytic enzyme. RC increased with HS10 in both hybrids but increased under HS5 in SYN5 and decreased in YN7. Pasting and thermal properties deteriorated by HS. The deterioration was more severe under HS10, presenting as lower viscosities and higher retrogradations, but it also probably useful for waxy maize production with specific purpose. Proactive investigations should focus on developing feasible approaches (adjusting sowing dates or fertilization strategies) and key technologies (application of exogenous plant growth regulators) to reduce quality deterioration in waxy maize production under warmer climates. Moreover, genotypic variation in thermal tolerance and its association with enhanced quality under warm climates can provide a reference for minish the negative influence of high temperatures.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (31771709, 32071958), Jiangsu Agriculture Science and Technology Innovation Fund (CX[20]3147), Postgraduate Scientific Research Innovation of Jiangsu (KYCX_2108), Priority Academic Program Development of Jiangsu Higher Education Institutions, and High-end Talent Support Program of Yangzhou University.

CONFLICT OF INTEREST

The authors have stated explicitly that there are no conflicts of interest in connection with this article.

AUTHOR CONTRIBUTIONS

XG and SY performed the experiments. XG wrote the manuscript. GL and WL participated design and supervised the experiments. DL conceptualized the study and finalized the manuscript. All authors contributed and approved the manuscript.