1. Introduction
Rice is one of the world’s most economically crucial food crops and plays a vital role in ensuring food security for the future. The demand for high-quality rice has increased recently, with an improvement in people’s living standards [
1]. The goal of rice production has shifted from yield first to yield and quality in parallel. Rice quality consists of its appearance, processing, nutrition, and eating and cooking qualities. Appearance is amongst the most critical determinants of rice quality and is closely related to the processing quality and eating and cooking quality [
2].
The appearance of rice is mainly determined by its chalkiness. The chalky, opaque portion of the rice grain deteriorates the appearance and eating and cooking quality [
3]. Excessive chalkiness of rice will lead to poor processing quality in rice, which is due to the high chalkiness of the internal arrangement of the grain being loose, and the texture of the rice is loose [
4]. It is easy to break during the milling process, which reduces the processing quality of rice. High or low temperatures have repeatedly been demonstrated to adversely affect rice quality, especially the CGR and CD, due to changes in the biosynthesis rates of starch and other storage compounds during the grain-filling stage (GFS) [
5]. The temperature mainly affects the rice chalkiness through a certain period during the GFS. Wakamatsu et al. [
6] reported that temperature changes most significantly affected rice quality within 20 days after heading. Siddik et al. [
7] also found that rice quality was most sensitive to high or low temperatures during a 7–14 day period after heading. Moreover, high temperatures more severely affected chalkiness than low temperatures [
7]. Under high or low temperatures, the starch granules in rice endosperm were inconsistent in shape and loosely arranged, resulting in more gaps among the starch granules, thereby reflecting more light and thus resulting in chalkiness [
8].
Starch, which is a major storage carbohydrate in rice, consists of amylose and amylopectin, accounting for 80–90% of the total weight. The fine structure and physicochemical properties of starch are the major determinants that affect the rice quality [
9]. The starch fine structure is closely related to the development and occurrence of rice chalkiness [
10]. An increase in the number of short chains in amylopectin, crystallinity, and the order of crystallinity, but a decrease in the number of long chains in amylopectin and the ratio of amorphous to ordered starch structure, leads to a deterioration in rice quality by enhancing its chalkiness [
11]. In addition, the size distribution of the starch granules is also one of the critical factors affecting rice chalkiness [
12]. In a study, scanning electronic microscopy (SEM) observation showed a deficient uniformity and regularity of starch granules, resulting in the altered transmission of light through the endosperm, thereby increasing the CGR and CD [
13]. However, the fine structure and physicochemical properties of starch, which are some of the most critical determinants of chalkiness, are influenced by environmental conditions and are genetically regulated [
8,
9].
Changes in temperature greatly affect the yield and quality of rice. The ambient temperature during the GFS in rice is one of the most critical environmental factors affecting the fine structure of the starch in the endosperm [
14]. High temperatures inhibit the activities of granule-bound starch synthase (GBSS) and starch branching enzymes (SBEs). Therefore, the ratio of short to long chains of amylopectin is increased, but the AC is decreased, thereby increasing the CGR and CD [
8,
15]. Additionally, high temperatures stimulate the expression of amylase-encoding genes and increased amylase activity, leading to starch degradation and the formation of starch granules of inconsistent size [
16,
17]. Low temperatures increase the length of amylopectin short chains while decreasing the length of amylopectin long chains and relative crystallinity of the starch, thus altering its crystal structure [
18]. These modifications in the starch structure reduce the uniformity and regularity of starch granules and widen the gaps among them [
19].
Elevated ambient temperatures during GFS affect the fine structure of starch and increase the CGR and CD [
9]. Compared with the daily maximum temperature and daily average temperature, nighttime temperature has a greater effect on the formation of chalkiness in rice [
20]. Cheng et al. [
21] showed that the effect of nighttime temperature on rice chalkiness was more pronounced after a 7-day period after heading. At present, the influence of variations in temperature on the chalkiness and the physicochemical properties of starch has mainly been studied under extended treatment conditions throughout the GFS. However, the formation of rice chalkiness and its relationship with the starch fine structure and physicochemical properties under short-term low nighttime temperature (LNT) or high nighttime temperature (HNT) treatments are still unclear. Therefore, LNT and HNT treatments were performed between the 7th and 14th day after heading to evaluate them. Our results provide useful information for understanding the underlying causes of rice chalkiness occurrence under varying nocturnal temperatures at the early GFS.
2. Materials and Methods
2.1. Plant Materials and Experimental Design
The experiments were conducted at the Science and Technology Park of Jiangxi Agricultural University (115°49’53″ E, 28°46’8″ N), Nanchang, Jiangxi Province, China. Two indica rice cultivars, Jiuxiangzhan (JXZ) and Huanghuazhan (HHZ), were used. The soil was pre-experimentally air-dried and filtered through a 5 mm sieve, and 8 kg of it was added to each pot. The chemical properties of the soil were 30.12 g·kg−1 organic matter, 2.27 g·kg−1 total N, 97.71 mg·kg−1 alkaline hydrolyzable N, 100.73 mg·kg−1 available K, and 27.37 mg·kg−1 available P.
The pot-planting method was used to cultivate the rice plants, as described previously [
22]. Briefly, the seedlings were transplanted to the pots (length, 27.5 cm; width, 21.0 cm; height, 32.0 cm) 28 days after sowing. The seedlings were planted in 2 holes per pot, with 2 seedlings per hole, and 40 pots were used for each treatment, which was performed in triplicate. The N was applied at a rate of 2.61 g N pot
−1, and the basal, tillering and panicle fertilizer were applied in 5:2:3 ratio. The application of N, P (as P
2O
5), and K (as K
2O) was in the ratio of 2:1:2. P was applied only as a basal fertilizer, but K was applied as 70% basal and 30% as panicle fertilizer. Other management techniques, such as pest and disease control, were implemented based on the high-yield and -quality cultivation program.
On the 7th day after heading, the rice plants were moved to artificially controlled-climate chambers for temperature treatment, with each chamber divided into two halves for the two cultivars, JXZ and HHZ. Temperature settings during the grain-filling stages were set in reference to the manner of Du et al. [
23]. The rice plants were treated with different nighttime temperatures (18:00–06:00 h) for 7 days as follows: (i) medium temperature (MT = 30 °C–25 °C); (ii) low nighttime temperature (LNT = 25 °C–20 °C); and (iii) high nighttime temperature (HNT = 35 °C–30 °C). The daytime (06:00–18:00 h) temperatures were 27 °C–32 °C on all days. The divergent temperatures varied at intervals of 1 °C linearly during the temperature treatment period. The temperature settings are detailed in
Table A1.
2.2. Determination of CGR and CD
The determination of chalkiness was performed using the Wanshen Rice Analysis System. Refined, whole rice grains from the triplicates of each treatment were analyzed, and their average values were obtained to determine the CGR and CD as follows:
2.3. Determination of AC
The AC was determined based on the methods described by the National Standard of Rice Quality Evaluation “GB/T17871-2017” [
24], the People’s Republic of China.
2.4. Ultrastructural Observations of Starch Granules
The ultrastructural properties of the amyloplasts in the endosperm were determined based on a previously described method [
11]. The arrangement and surface structure of starch granules were observed on a GeminiSEM 300 instrument (Zeiss, Oberkochen, Germany).
2.5. Determination of Granule Size
The granule size was determined using the Mastersizer 3000 (Malvern Panalytical Ltd., Worcestershire, UK) according to a previously described method [
25]. Based on the volume, surface area, and proportion, starch granules were categorized into small and medium (<10 μm) or large (>10 μm).
2.6. Analysis of the X-ray Diffraction (XRD) Patterns and Determination of the Branching Degree
The XRD of the starch molecules was performed with an X’Pert Pro X-ray diffractometer (PANalytical, EA Almelo, The Netherlands) under the following conditions: power at 1600 W (40 kW × 40 mA) using a scanning range of 4°–60°, step size of 0.02°, and scan speed of 4°/min. The parameters of the degree of crystallinity (%), crystalline morphology, and diffraction peaks at 2θ value (angle) were calculated using the software MDI Jade 6 (Materials Data, Inc, Livermore, CA, USA) Jade 6.0. The starch branching distribution was determined via 1H NMR analysis (Bruker BioSpin GmbH, Ettlingen, Germany) with a scan number of 32, a resonance RF of 500.23 MHz, and an NMR spectrum of 1H. The data were analyzed using the MestReNova 14.0 software.
A and B indicate the peak areas of the α-1,6 and α-1,4 linkages, respectively.
2.7. Molecular Weight Distribution of Starch
The starch dissolved in a DMSO-LiBr solution was debranched using isoamylase according to previous methods [
26], and the components’ molecular weights were ascertained using a U3000 GPC (Thermo Fisher Scientific, Waltham, MA, USA) and an OPTILAB
® T-rEX™ differential detector (Wyatt Technology, Goleta, CA, USA). The chromatographic data were processed using the ASTRA 6.1 software. Pullulan was used as a molecular weight standard consisting of 342, 3650, 21,000, 131,400, 610,500, 821,700, and 3,755,000 Da.
2.8. Measurement of Thermal Properties
The DSC 200 F3 differential calorimetric scanner (NETZSCH, Waldkraiburg, Germany) was employed to ascertain the changes in the enthalpy of the starch by scanning the heat at a temperature-change rate of 10 °C/min from 30 °C–105 °C. The heat change data were then analyzed using Universal Analysis/Proteus Thermal Analysis 2000 software. The parameters of onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy of gelatinization (ΔH) during the process of the phase change in the sample were calculated separately [
25].
2.9. Determination of Pasting Properties
The pasting properties of starch were determined using a Starch Master TM 17,133 Rapid Viscosity Analyzer (Newport Scientific Pvt. Ltd., Warier Wood, Australia). The RVA spectral characteristics included peak viscosity (PV), trough viscosity (TV), final viscosity (FV), breakdown viscosity (BD), setback viscosity (SB), pasting temperature (PaT), and pasting time (PT).
2.10. Statistical Analysis
All data for each treatment are represented as the means of three replicates. Statistical analyses were performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) using the least significant differences (LSD) method at the p < 0.05 level. Pearson correlation analysis (PCA) was performed to assess the correlation between chalkiness and the fine structure and the physicochemical properties of rice starch. The differences at p levels < 0.05 or <0.01 were considered statistically significant.