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Article

ABA Affects Distinctive Rice Caryopses Physicochemical Properties on Different Branches

by
Yunfei Wu
1,*,
Ebenezer Ottopah Ansah
1,†,
Licheng Zhu
1,†,
Wenchun Fang
1,2,
Leilei Wang
1,
Dongping Zhang
1 and
Baowei Guo
1,*
1
Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern Production Technology of Grain Crops/Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou 225009, China
2
Biology Institute, Guangxi Academy of Sciences Co., Ltd., Nanning 530000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this article.
Agronomy 2024, 14(11), 2632; https://doi.org/10.3390/agronomy14112632
Submission received: 27 September 2024 / Revised: 1 November 2024 / Accepted: 4 November 2024 / Published: 8 November 2024
(This article belongs to the Special Issue Molecular Regulatory Network of Plant Nutrition Signaling)
Browse Figures
Versions Notes

Abstract

:
Abscisic acid (ABA) plays an important regulatory role in the grain filling process, which in turn will affect the final yield and quality of rice. The ABA biosynthesis genes of OsNCED3 and degradation gene OsABA8ox3 affect the ABA content, and then further regulate the ABA signaling. During the development of rice panicle, compared with primary grains (superior grains) growing on primary branches, secondary grains (inferior grains) growing on secondary branches exhibit characteristics. However, little is reported on the physicochemical characteristics of starch between superior and inferior grains in ABA related transgenic lines. In this study, OsNCED3 and OsABA8ox3 transgenic plants were used as materials. The results showed that compared with the WT, the OsNCED3-RNAi lines on grain weight was consistent with the trend of superior and inferior grains, while the OsABA8ox3-RNAi lines affected superior or inferior grains. The total starch and soluble sugar content of grains decreased in both OsNCED3-RNAi and OsABA8ox3-RNAi lines, and the total starch content of superior and inferior grains in OsABA8ox3-RNAi lines decreased. The starch granule size distribution of all samples showed a bimodal and increased proportion of starch grains with large granule size, in which the influence on inferior grains was greater than that of superior grains, which eventually led to a significant increase in their average granule size. The apparent amylose content of inferior grains increased significantly in most lines. The swelling power of the superior grains decreased significantly, while that of the inferior grains increased significantly. Fourier analysis showed that the order degree of starch granule surface decreased in the superior grains of the RNAi line, while it increased in the inferior grains of the OsABA8ox3-RNAi line but decreased in the OsNCED3-RNAi lines. In the superior grains, the relative crystallinity of starch decreased in the OsNCED3-RNAi lines, but remained unchanged or increased in the OsABA8ox3-RNAi line. In inferior grains, the relative crystallinity of starch decreased in the ABA synthesis RNAi line, but increased in the OsABA8ox3-RNAi line. In summary, the influence of ABA on the physicochemical properties of inferior grains is greater than that of superior grains.

1. Introduction

The phenomenon of asynchronous flowering exists in different grain positions on the rice panicle. The superior grains located in the upper half of the spikelet bloom early, fill quickly, and have good endosperm enrichment and high grain weight, whereas the inferior grains located in the lower half of the spikelet bloom late, fill slowly, and have poor endosperm enrichment and low grain weight. The difference between superior and inferior grains does not only affect yield, but also restricts the quality of rice [1].
Abscisic acid (ABA) is a plant hormone that has frequently been shown to impede plant growth during plant development [2]. ABA plays an important role in various stages of plant development, such as regulating stomatal closure caused by drought stress to increase plant stress tolerance [3,4]. Regulating crop ABA levels through genetic engineering is an effective means to improve crop yields under specific growing conditions. 9-cis-epoxy carotenoid dioxygenase (NCED), encoded by the NCED gene, is a key rate-limiting enzyme throughout the biosynthesis reaction phase of ABA [2]. The OsNCEDs gene family, which comprises five genes including OsNCED1~5, is found to encode NCEDs in rice [5,6], with OsNCED3 identified as a drought stress gene in rice that regulates ABA levels and stress tolerance in arid environments [7,8]. OsNCED1 was expressed in rice leaves, and as water stress increased, its expression steadily decreased, demonstrating a negative connection with the severity of stress [2]. OsNCED2 was more abundant in seeds [5,9]; in indirect heterologous tables, OsNCED4 and OsNCED3 changed plant size and leaf morphology, delayed seed germination, and enhanced drought tolerance [10], supporting speculation that they may have other functions in the formation of leaf morphology and vascular bundles. Studies has found that OsNCED3 mutants can significantly delay the aging of rice leaves, and the agronomic traits of osnced3 mutant lines at maturity, such as seed fullness, 1000 grain weight, and fruiting rate, were better than those of wild-type lines, which may lead to prolonged rice growth periods and ultimately affect rice yield [11]. OsNCED5 may regulate plant development and stress resistance through control of ABA biosynthesis [12]. OsNCED3, also as a drought stress gene, had better agronomic traits than wild-type lines during rice grain ripening [6]. However, the changes in the physicochemical properties of starch in the mutant grains are unknown.
When stress is relieved or the concentration of abscisic acid needs to be reduced, the ABA in the plant is mainly degraded by ABA 8′-hydroxylase (ABA8ox) to form phaseic acid (PA), and the process is irreversible [13,14]. In rice, ABA 8’-hydroxylase (ABA8ox) is encoded by the three genes OsABA8ox1, OsABA8ox2 and OsABA8ox3 [3]. The expression of OsABA8ox2 and OsABA8ox3 is induced by ABA. OsABA8ox1 is a cold-induced gene, and all three genes are induced by high salinity, drought and osmotic stress [15]. The mRNA levels of OsABA8ox1 increased significantly after the seeds on the plant were submerged for 1 h [5]. After knockdown of the three OsABA8ox genes using CRISPR/Cas9, there was no effect on the yield of the mutants, but high endogenous ABA levels in the seeds affected ABA signaling and inhibited the ability to metabolize substances in the seeds, which in turn led to higher seed dormancy [16]. The use of exogenous glucose during rice seed germination significantly inhibits the expression of OsABA8ox2 and OsABA8ox3 [4]. In addition, studies have shown that OsABA8ox3 plays an important role in regulating ABA levels and drought stress resistance in rice [17].
As the main storage substance of rice endosperm, starch accounts for about 80–90% of the mass of brown rice [18], providing a large source of energy for humans [19]. Its physicochemical properties are also directly related to applications in food and non-food industries. As an important regulatory hormone during the development of rice, ABA has an important effect on the yield and quality of mature rice. In recent years, it has been found that there is a close correlation between the grain filling process of superior and inferior grains of rice and ABA content through spraying exogenous ABA [4,20,21]. According to research, superior and inferior grains of super rice have significantly different starch physicochemical properties [22]. Although the ABA level of rice plants changed as a result of the osnced3 mutation, it is not known whether the physical and chemical characteristics of superior and inferior grain starch showed any variations or trends.
In this study, the transgenic lines of key genes in the process of ABA biosynthesis and degradation were selected as materials, the agronomic traits of superior and inferior grains in the ripening stage of ABA mutants were comprehensively analyzed, and the physical and chemical properties of superior and inferior grains starch in rice were comprehensively compared to provide a reference for the improvement of mature grain starch quality.

2. Materials and Methods

2.1. Materials and Growth Conditions

ABA synthesis pathway key gene, OsNCED3 (LOC_Os03g44380) transgenic plants of OsNCED3-OX, OsNCED3 RNAi-1, OsNCED3 RNAi-2 and ABA degradation pathway key gene OsABA8ox3 (LOC_Os09g28390) RNA interference transgenic plants (OsABA8ox3 RNAi-9, OsABA8ox3 RNAi-27). The above materials are based on ZhongHua11 as the genetic background. All the transgenic lines were published by Cai et al. 2015 [17]. Seedlings were cultivated for 7 days at 28 °C under continuous light. Subsequently, 28 days after germination (DAG), they were transferred to paddy fields at Yangzhou University (119°01′ E, 32°15′ N), China, from May to October 2019, according to local conventional agriculture operations. Mature grains were separated into superior and inferior grains: the superior grains are all the grains of the upper primary stem of the rice ear, and the inferior grains are all the grains of the lower secondary stem (Figure 1A).

2.2. Observation and Size Measurement of Superior and Inferior Grains

The grains were observed by placing them on a black cloth and photographed with a digital camera. Grain weight was calculated by measuring 300 grains (100 grains per group), whereas grain size was measured by randomly choosing and measuring 30 grains (10 grains per group). Error bars represent STDEV of at least 3 samples.

2.3. Starch Isolation

Starch was extracted in reference to the method in [23]. Briefly, matured dried caryopses were ground into powder with a mortar, mixed with water and stirred. The solution was filtered through eight layers of gauze, and the filtrate was collected in a centrifuge tube. The filtrate was centrifuged to separate the supernatant from the precipitate. After discarding the supernatant, 0.2% NaOH solution was added, and the precipitate was centrifuged again. The supernatant was discarded, and the yellow material on the surface of the precipitate was scraped off. The process of adding 0.2% NaOH solution and scraping off yellow material was repeated until the surface of the precipitate was clear. Anhydrous ethanol was then added to the precipitate and centrifuged. After discarding the ethanol, the remaining extracted starch was placed in a 40 °C oven and left to dry to a constant weight. The starch was thereafter stored in a desiccator for later use.

2.4. Determination of Soluble Sugar and Total Starch Content

To determine the soluble sugar content, 30 grains (10 grains in each group) were randomly selected and ground into powder with a mortar [23]. Total starch content was determined in reference to Yu et al. (2022) [24]. Error bars represent STDEV of at least 3 samples.

2.5. Observation of Superior and Inferior Grain Starch Morphology and Starch Particle Size Distribution

The method used to observe superior and inferior grain starch morphology and starch particle size distribution was in reference to Gao et al. (2015) [23]. A small amount of starch was dissolved in 50% glycerol and well mixed in order to measure the granule size distribution. The mixture was pipetted in drops onto a glass slide, which was then covered with a coverslip. Using a light microscope (400×), the starch granules that were freely dispersed were examined and captured on camera (DMLS, Leica, Solms, Germany). The maximum length of the granules was determined by measuring through the center of individual granules using Image-Pro Plus (Media Cybernetics, Rockville, ML, USA) professional image analysis software [24]. Error bars represent STDEV of at least 3 samples.

2.6. Determination of Apparent Amylose Content, Swelling Potential and Solubility

The apparent amylose content was determined with reference to [25]. The method used to determine solubility and swelling power was in reference to Yu et al. (2022) [24]. Error bars represent STDEV of at least 3 samples.

2.7. Fourier Transform Infrared (FTIR) Analysis of Starch

Briefly, 30 mg of starch was prepared in a milky form with 200 µL distilled water and applied to obtain the original spectrum of the starch at the attenuated total reflection mode sample stage of a Fourier transform infrared spectrometer (7000, Varian, Agilent, Cary, NC, USA) with a background of distilled water in a wavenumber range from 800 cm−1 to 4000 cm−1. The deconvoluted spectra of the samples were obtained by deconvolution of the spectra [26]. Error bars represent STDEV of at least 3 samples.

2.8. X-Ray Diffraction Analysis

The dried starch sample was placed on a centrally grooved glass piece, compressed, and scanned on an X-ray diffractometer (D8 Advance, Bruker AXS, Karlsruhe, Germany) from 2θ angles of 3° to 40° (step size: 0.3 s) at the Yangzhou University Test Center. The XRD spectrum of the obtained starch sample was used to calculate the relative crystallinity of the starch [27] by using Photoshop CS6.0 and Image-Pro-Plus image analysis software. Error bars represent STDEV of at least 3 samples.

2.9. Statistical Analysis

Statistical analysis was performed using SPSS 19.0 data analysis software, and the difference between the measured values of different starch samples was tested at the p < 0.05 level (LSD method).

3. Results and Discussion

3.1. Effects of ABA Transgenic Lines on the Appearance Quality in Rice

Previously, it was reported that OsNCED3 and OsABA8ox3 mediated ABA synthesis and degradation, separately [8,17]. Here, we isolated superior grains and inferior grains from mature grains from six different lines, and then analyzed the agronomic traits (Figure 1A). Within the same line, the grain length, grain width, grain thickness and 10-grain weight of the superior grains were greater than those of the inferior grains. Compared with the superior grains of ZhongHua11, the grain length of the OsNCED3-OX lines increased, while that of the OsNCED3 RNAi-1, OsNCED3 RNAi-2 and OsABA8ox3 RNAi-27 lines decreased. The width of the grain decreased, except that of the OsNCED3-OX line. The grain thickness of the OsABA8ox3 RNAi-9 lines increased. The 10-grain weight of the OsNCED3-OX lines increased, and that of the OsNCED3 RNAi-2 and OsABA8ox3 RNAi-27 lines decreased. Compared with the inferior grains of ZhongHua11, the grain length of the OsNCED3-OX line increased. The grain width of the OsNCED3-OX line increased, and that of the OsABA8ox3 RNAi-2 line decreased. The grain thickness of the OsNCED3 RNAi-2 and OsABA8ox3 RNAi-9 lines decreased. The 10-grain weight of OsNCED3-OX and OsABA8ox3 RNAi-9 increased, and that of OsNCED3 RNAi-2 decreased. In summary, the transcript level of ABA synthesis or degradation pathway genes might involve grain development by affecting the balance between superior and inferior.

3.2. OsNCED3 and OsABA8ox3 Affect Total Starch and Soluble Sugar in Superior and Inferior Grains

Starch and protein make up the majority of the storage material in rice grain. We determined the total starch and soluble content in the superior and inferior grains of wild-type and six rice lines, as shown in Table 1. In the same line, the total starch content of superior grains was higher than that of inferior grains except in OsABA8ox3 RNAi-27, and there were significant differences in ZhongHua11, OsNCED3-OX, OsNCED3 RNAi-1 and OsSNCED3 RNAi-2, but almost no change in OsABA8ox3 RNAi-9 and OsABA8ox3 RNAi-27. Compared with the total starch of superior and inferior grains of ZhongHua11, the total starch content was significantly lower than that of ZhongHua11, except for the OsNCED3 RNAi-2 transgenic line. In the same line, the soluble sugar content of ZhongHua11 superior and inferior grains remained unchanged, and the soluble sugar content of superior grains was significantly higher than that of inferior grains in the OsABA8ox3 RNAi-9 lines, but the soluble sugar content of superior grains was significantly lower than that of inferior grains in OsNCED3-OX, OsNCED3 RNAi-1, OsNCED3 RNAi-2 and OsABA8ox3 RNAi-27 rice lines. Compared with ZhongHua11 superior grains, the soluble sugar content in the superior grains of the five lines was significantly reduced, except for OsNCED3 RNAi-1 and OsABA8ox3 RNAi-9. Compared with ZhongHua11 inferior grains, the soluble sugar content in the inferior grains of OsNCED3-OX and OsNCED3 RNAi-1 lines was significantly increased. Studies have found that spraying exogenous ABA at flowering can increase the content of total starch and soluble sugar in grains [28], and can significantly increase the average filling speed and grain weight of inferior grains [29]. However, it was found that higher ABA levels in inferior grains inhibited sucrose synthase (SUS) expression, which was not conducive to starch synthesis [12]. In general, the total starch content and soluble sugar content of superior grains in the lines were mainly reduced, while the total starch content decreased and the soluble sugar content increased in inferior grains. These results indicated that ABA synthesis and degradation might affect the accumulation of storage materials in superior and inferior grains in rice.

3.3. OsNCED3 and OsABA8ox3 Promote the Enlargement of Starch Granules

After the grains of the six rice lines had ripened, the starch was extracted and examined under a microscope (Supplemental Figure S1). It can be observed that the starch grains of the six rice lines exhibit an irregular polyhedral shape for the most part under the microscope, and a small part appears as smooth spheres, which is consistent with the observations [30,31]. Image-Pro Plus software (Media Cybernetics, Rockville, ML, USA) was used to measure the diameter of granules, and origin software was used to plot the distribution histogram (Figure 2). The figure illustrates bimodal distribution of granule sizes among the 14 starch samples, each with a distinctive peaking. The superior and inferior grains of ZhongHua11 were 5.4 μm and 5.7 μm, those of OsNCED3-OX were at 6.9 μm and 6.3 μm, those of OsNCED3 RNAi-1 were at 6.6 μm and 6.6 μm, those of OsNCED3 RNAi-2 were both at 7.2 μm, those of OsABA8ox3 RNAi-9 were both at 6.9 μm, and those of OsABA8ox3 RNAi-27 were at 5.1 μm and 6.6 μm, respectively. In the same line, the starch granule size corresponding to the main peak of the superior grains is generally larger than that of the inferior grains. Compared with ZhongHua11, the starch particle size at the main peak of both the superior and inferior grains of the line increased except for the superior grains of OsABA8ox3 RNAi-27. Further statistics were compiled on starch granules to plot starch granule size distribution tables (Table 2). In the same rice line, the proportion of small starch granules in superior grains of ZhongHua11 and OsABA8ox3 RNAi-9 in the range of 0–1.5 μm was significantly smaller than that of inferior grains, while the proportion of superior grain starch was significantly larger than that of inferior grains in the other five rice lines. The average granule size of rice in OsABA8ox3 RNAi-27 showed that the superior grains were significantly smaller than the inferior grains, while the other rice lines showed that the superior grains were significantly larger than the inferior grains. Compared with ZhongHua11, the proportion of superior and inferior starch granules in the range of 1.5–10 μm of ABA-related pathway lines increased significantly, which in turn affected the average starch granule size of superior and inferior grains of lines. The above phenomenon shows that the increase in ABA content after gene mutation of ABA synthesis or degradation pathway in rice will promote the formation of large starch granules.

3.4. Effect of OsNCED3 and OsABA8ox3 on Apparent Amylose Content Is Reversed in Superior and Inferior Grains

Apparent amylose content (AAC) is based on the fact that starch can be combined with iodine to produce different absorption spectra at different positions in the spectrum, and the optical density ratio (OD620/550) at 620 nm and 550 nm is used as the content value [32]. The taste of cooked rice is significantly influenced by the apparent amylose content of the rice grains [23]. The apparent amylose content of superior and inferior grains of six rice lines is shown in Table 2. With the exception of the inferior grains of the OsNCED3-OX line, whose apparent amylose concentration is significantly greater than that of superior grains, the apparent amylose content of the inferior grains in all six other lines was lower than that of the superior grains, significantly in ZhongHua11 and OsNCED3 RNAi-2 lines. Compared with ZhongHua11, the apparent amylose content of superior grains in the line remained unchanged or significantly decreased, while the apparent amylose content of inferior grains remained unchanged or significantly increased. This suggests that after mutations in key genes in the ABA synthesis and degradation pathway, the effect on the apparent amylose content of superior and inferior grains is reversed, and greater in inferior grains. The GBSSI gene controls amylose synthesis in rice [33], and its expression in rice grains is up-regulated after spraying exogenous ABA [34]. Teng et al. reported that the ABA content of inferior grains increased significantly after OsABA8ox2 was knocked out [35]. Thus, it might be that ABA content increases in inferior grains after OsABA8ox3 is silenced, which causes up-regulation of the OsGBSSI transcript level, leading to an increase in the biosynthesis of amylose.

3.5. OsNCED3 and OsABA8ox3 Affect the Chemical Properties of Superior and Inferior Grain Starch in Rice

The swelling power and solubility of starch refer to the ability and degree of water absorption expansion and dissolution of starch after it is heated in water. The swelling power and solubility of superior and inferior grain starch in six rice lines are shown in Table 3. In the same line, the swelling power of superior and inferior grains showed different phenomena. In ZhongHua11 and OsNCED3 RNAi-2, the swelling power of superior grains was significantly higher than that of inferior grains, while there was no significant difference in OsNCED3 RNAi-1 and OsABA8ox3 RNAi-9. However, in OsNCED3 RNAi-2 and OsABA8ox3 RNAi-27 lines, the swelling power of the superior grains was significantly lower than that of the inferior grains. Compared with ZhongHua11, except for the swelling power of superior grains in OsNCED3 RNAi-2, which was significantly increased, the swelling power of superior grains in other lines was significantly reduced. The swelling power of the inferior grains of the OsNCED3-OX and OsABA8ox3 RNAi-27 lines increased significantly, and the swelling power of the OsNCED3 RNAi-2 lines decreased significantly. In addition, the swelling power of the superior grains of OsABA8ox RNAi-27 was significantly decreased. The above results show that the swelling power of superior grain starch in rice plants is significantly reduced and that of the inferior grains is significantly increased. It has been pointed out that the shape and granule size of starch can significantly affect the swelling power and solubility of starch [36]. The proportion of large and medium starch grains in the RNAi line is significantly larger than that of ZhongHua11, so the swelling power and solubility of the superior and inferior grain starch in the above RNAi line may be affected by the particle size of the starch.

3.6. OsNCED3 and OsABA8ox3 Affect the Order of Superior and Inferior Grain Starch in Rice

Fourier transform infrared (FTIR) analysis of starch can analyze the external ordered structure of starch particles and study the structural changes of the surface layer of starch during processing [37]. Among them, the two peak intensity ratios of 1045/1022 cm−1 and 1022/995 cm−1 were used to indicate the content of ordered structure and amorphous structure outside starch granules, respectively. The lower the ratio of 1045/1022 cm−1, the lower the orderliness outside the starch granules and the lower the acid-resistant hydrolysis energy of starch [38]. In order to investigate the effect on the order of starch granules in rice grains in ABA synthesis and degradation pathway gene transgenic plants, we performed FTIR spectroscopy analysis on 12 collected starch samples. As shown in Figure 3A, the rice starch sample had similar spectra in the 800–1200 cm−1 region, but there were certain differences at 995 cm−1, 1022 cm−1 and 1045 cm−1. The relative peak intensities from baseline to peak height at 995 cm−1, 1022 cm−1 and 1045 cm−1 were recorded, and the peak–height ratios of 1022/995 cm−1 (disordered/ordered ratio) and 1045/1022 (degree of ordinality) were calculated to plot a histogram (Figure 3B,C). Except for the ZhongHua11 lines, where the superior grains were significantly higher than the inferior grains, the superior grains of the other lines were significantly lower than the inferior grains at 1022/995 cm-1 (Figure 3B). Compared with ZhongHua11, the ratio of superior grain starch was significantly reduced except for that of the OsABA8ox3 RNAi-9 line. The ratio at 1022/995 cm−1 showed different trends in the inferior grains of the RNAi line, with OsNCED3-OX decreased and OsNCED3 RNAi-1 and OsABA8ox3 RNAi-9 increased. The ratio of ZhongHua11 superior grain starch at 1045/1022 cm−1 was significantly higher than that of inferior grains, whereas OsNCED3 RNAi-1, OsNCED3 RNAi-2 and OsABA8ox3 RNAi-9 showed a reduction in superior grains compared to the inferior grains. At the ratio of 1045/1022 cm−1, compared with ZhongHua11, the superior grains in the OsNCED3-OX, OsNCED3 RNAi-1, and OsNCED3 RNAi-2 lines were reduced. The ratio of inferior grain starch at 1045/1022 cm−1 was increased, with the exception of the OsNCED3-OX line.
The above phenomena show that at the ratio of 1045/1022 cm−1 (Figure 3C), the superior grains of the OsNCED3 RNAi line decreased but the inferior grains remained unchanged, and the superior grains of the OsABA8ox3 RNAi line did not change but the inferior grains increased. At the ratio of 1022/995 cm−1 (Figure 3B), the superior and inferior grains of the OsNCED3 RNAi lines decreased, and the superior and inferior grains of the OsABA8ox3 RNAi lines increased. This indicates that the proportion of ordered structure of superior grain starch granules in rice decreases. The higher the external order, the stronger its resistance to acid hydrolysis [37]. In conclusion, the increase in ABA content in rice plants mainly enhanced the hydrolysis resistance of starch of rice inferior grains.

3.7. OsNCED3 and OsABA8ox3 Affect the Crystal Structure of Superior and Inferior Grain Starch in Rice

The crystallinity of starch is a parameter that reflects the properties of starch granules, and changes in crystallinity will directly affect the performance of starch in application [39]. The crystalline characteristics of starch can be determined using X-ray analysis. Types A, B, and C of starch can be distinguished based on starch crystalline levels, with type A starch being more stable and retaining a tight double helix packing [40]. The dried starch sample was scanned with an X-ray diffractometer with a step size of 0.3 s from 4°–40° 2θ to obtain the XRD pattern of the starch sample (Figure 4A). It can be seen from the figure that there is no obvious difference between the XRD patterns in the superior and inferior grains of ZhongHua11 and the RNAi line. The determined XRD patterns were similar and indicated A-type diffraction properties, which are distinctive of XRD patterns of typical cereal starches, with intense diffraction peaks at around 15° and 23° and an unresolved doublet at about 17° and 18° 2θ [17]. The diffraction patterns from the 12 starches were used to calculate the area of the crystalline and amorphous regions, and a histogram was created after determining the relative crystallinity of each starch sample (Figure 4B). It can be seen from the histogram that the relative crystallinity of superior grain starch in the OsNCED3 RNAi-2 line is significantly greater than that of the inferior grain starch, while the relative crystallinity of the superior grain starch in ZhongHua11 and other RNAi lines is significantly smaller than that of the inferior grain starch. In addition, compared with ZhongHua11, the relative crystallinity of superior grain starch of OsNCED3-OX, OsNCED3 RNAi-1 and OsNCED3 RNAi-2 lines was significantly reduced, but it was significantly increased in OsABA8ox3 RNAi-9 lines. Compared with ZhongHua11, the relative crystallinity of inferior grain starch in OsNCED3-OX, OsNCED3 RNAi-1, and OsNCED3 RNAi-2 lines was significantly reduced, but significantly increased in OsABA8ox3 RNAi-9 and OsABA8ox3 RNAi-27 lines. The above phenomena show that the relative crystallinity of superior and inferior grain starch decreases in the ABA synthesis pathway. Starch’s crystalline structure is crucial for its physical and chemical characteristics, which affect its application qualities like cold water insolubility, gelatinization, swelling, and sensitivity to enzymatic hydrolysis. It also has an impact on how starch is digested in both human and animal digestive systems [41].

4. Conclusions

In this study, the key genes of OsNCED3 and OsABA8ox3 related to abscisic acid synthesis and degradation pathway were analyzed, and the effects on the agronomic traits, starch accumulation and physicochemical properties of superior and inferior grains of rice were studied. It was found that the key genes of the ABA synthesis or degradation pathway would reduce the accumulation of stored substances in rice superior and inferior grains. Additionally, the influence on the physicochemical properties of rice inferior grain starch was greater than that of superior grain starch. During the process of rice grain filling, ABA content or signal could accelerate grain filling, leading to premature aging. Therefore, by adjusting the ABA content reasonably at different stages, it might modify grain weight and quality.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14112632/s1, Figure S1. Observation of OsNCED3 and OsABA8ox3 transgenic lines strong and weak starch grain morphology.

Author Contributions

Y.W.: Writing—review and editing, Investigation, Formal analysis. E.O.A.: Writing—original draft, Data curation, Investigation, Formal analysis. L.Z.: Data curation, Investigation, Resources. L.W.: Data curation. W.F.: Editing of the manuscript. D.Z.: Conceptualization. B.G.: Data curation. Y.W. and B.G.: Project administration, Funding acquisition, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the open funds of the Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding (NO.PL202302), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX24_2326).

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

Author Wenchun Fang was employed by the company Guangxi Academy of Sciences Co., Ltd. The remaining 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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Figure 1. Position of superior spikelet and inferior spikelet in panicle (A), statistics of different rice lines (E), S: superior grain I: inferior grain. (B) grain length; (C) grain with; (D) grain thickness; (E) 10-grain weight. Error bars represent STDEV of at least 3 samples. Values in the same column with different letters are significantly different (p < 0.05).
Figure 1. Position of superior spikelet and inferior spikelet in panicle (A), statistics of different rice lines (E), S: superior grain I: inferior grain. (B) grain length; (C) grain with; (D) grain thickness; (E) 10-grain weight. Error bars represent STDEV of at least 3 samples. Values in the same column with different letters are significantly different (p < 0.05).
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Figure 2. Observation of OsNCED3 and OsABA8ox3 transgenic lines’ superior and inferior starch particle size statistics. (A–F): Rice starch particle size distribution of superior grain, scale: 50 μm; (a–f): Rice starch particle size distribution map of inferior grain. (A) ZhongHua11 superior grain; (a) ZhongHua11 inferior grain; (B) OsNCED3-OX superior grain; (b) OsNCED3-OX inferior grain; (C) OsNCED3 RNAi-1 superior grain; (c) OsNCED3 RNAi-1 inferior grain; (D) OsNCED3 RNAi-2 superior grain; (d): OsNCED3 RNAi-2 inferior grain; (E) OsABA8ox3 RNAi-9 superior grain; (e) OsABA8ox3 RNAi-9 inferior grain; (F) OsABA8ox3 RNAi-27 superior grain; (f) OsABA8ox3 RNAi-27 inferior grain.
Figure 2. Observation of OsNCED3 and OsABA8ox3 transgenic lines’ superior and inferior starch particle size statistics. (A–F): Rice starch particle size distribution of superior grain, scale: 50 μm; (a–f): Rice starch particle size distribution map of inferior grain. (A) ZhongHua11 superior grain; (a) ZhongHua11 inferior grain; (B) OsNCED3-OX superior grain; (b) OsNCED3-OX inferior grain; (C) OsNCED3 RNAi-1 superior grain; (c) OsNCED3 RNAi-1 inferior grain; (D) OsNCED3 RNAi-2 superior grain; (d): OsNCED3 RNAi-2 inferior grain; (E) OsABA8ox3 RNAi-9 superior grain; (e) OsABA8ox3 RNAi-9 inferior grain; (F) OsABA8ox3 RNAi-27 superior grain; (f) OsABA8ox3 RNAi-27 inferior grain.
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Figure 3. Far-infrared spectroscopy analysis of RNAi line superior and inferior grain starch. (A) The red light spectrum of Fourier far away, a: ZhongHua11 superior grain, b: ZhongHua11 inferior grain, c: OsNCED3-OX superior grain, d: OsNCED3-OX inferior grain, e: OsNCED3 RNAi-1 superior grain, f: OsNCED3 RNAi-1 inferior grain, g: OsNCED3 RNAi-2 superior grain, h: OsNCED3 RNAi-2 inferior grain, i: OsABA8ox3 RNAi-9 superior grain, j: OsABA8ox3 RNAi-9 inferior grain, k: OsABA8ox3 RNAi-27 superior grain, l: OsABA8ox3 RNAi-27 inferior grain. (B,C) The histogram of the ratio of 1022/995 cm−1 and 1045/1022 cm−1, S: superior grain, I: inferior grain. The number of samples is in triplicate, with different lowercase letters indicating significant differences between samples (p < 0.05).
Figure 3. Far-infrared spectroscopy analysis of RNAi line superior and inferior grain starch. (A) The red light spectrum of Fourier far away, a: ZhongHua11 superior grain, b: ZhongHua11 inferior grain, c: OsNCED3-OX superior grain, d: OsNCED3-OX inferior grain, e: OsNCED3 RNAi-1 superior grain, f: OsNCED3 RNAi-1 inferior grain, g: OsNCED3 RNAi-2 superior grain, h: OsNCED3 RNAi-2 inferior grain, i: OsABA8ox3 RNAi-9 superior grain, j: OsABA8ox3 RNAi-9 inferior grain, k: OsABA8ox3 RNAi-27 superior grain, l: OsABA8ox3 RNAi-27 inferior grain. (B,C) The histogram of the ratio of 1022/995 cm−1 and 1045/1022 cm−1, S: superior grain, I: inferior grain. The number of samples is in triplicate, with different lowercase letters indicating significant differences between samples (p < 0.05).
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Figure 4. X-ray diffraction analysis and calculation of relative crystallinity of superior and inferior grain starch of rice. (A) The X-ray diffraction spectrum, a: ZhongHua11 superior grain, b: ZhongHua11 inferior grain, c: OsNCED3-OX superior grain, d: OsNCED3-OX inferior grain, e: OsNCED3 RNAi-1 superior grain, f: OsNCED3 RNAi-1 inferior grain, g: OsNCED3 RNAi-2 superior grain, h: OsNCED3 RNAi-2 inferior grain, i: OsABA8ox3 RNAi-9 superior grain, j: OsABA8ox3 RNAi-9 inferior grain, k: OsABA8ox3 RNAi-27 superior grain, l: OsABA8ox3 RNAi-27 inferior grain. (B) The relative crystallinity, S: superior grain, I: inferior grain. The number of samples is in triplicate, with different lowercase letters indicating significant differences between samples (p < 0.05).
Figure 4. X-ray diffraction analysis and calculation of relative crystallinity of superior and inferior grain starch of rice. (A) The X-ray diffraction spectrum, a: ZhongHua11 superior grain, b: ZhongHua11 inferior grain, c: OsNCED3-OX superior grain, d: OsNCED3-OX inferior grain, e: OsNCED3 RNAi-1 superior grain, f: OsNCED3 RNAi-1 inferior grain, g: OsNCED3 RNAi-2 superior grain, h: OsNCED3 RNAi-2 inferior grain, i: OsABA8ox3 RNAi-9 superior grain, j: OsABA8ox3 RNAi-9 inferior grain, k: OsABA8ox3 RNAi-27 superior grain, l: OsABA8ox3 RNAi-27 inferior grain. (B) The relative crystallinity, S: superior grain, I: inferior grain. The number of samples is in triplicate, with different lowercase letters indicating significant differences between samples (p < 0.05).
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Table 1. Changes in soluble sugar and total starch content of superior and inferior grains in rice.
Table 1. Changes in soluble sugar and total starch content of superior and inferior grains in rice.
SampleSoluble SugarTotal Starch
ZhongHua11Superior grain3.06 ± 0.12 b84.1 ± 2.13 a
Inferior grain2.88 ± 0.13 bc74.9 ± 4.04 b
OsNCED3-OXSuperior grain2.60 ± 0.13 c69.5 ± 0.85 c
Inferior grain4.23 ± 0.12 a64.4 ± 3.55 d
OsNCED3 RNAi-1Superior grain2.78 ± 0.25 bc77.7 ± 2.00 b
Inferior grain4.48 ± 0.23 a64.4 ± 2.07 d
OsNCED3 RNAi-2Superior grain2.01 ± 0.16 d83.5 ± 2.04 a
Inferior grain2.75 ± 0.14 bc78.0 ± 1.64 b
OsABA8ox3 RNAi-9Superior grain3.15 ± 0.08 b69.7 ± 3.35 c
Inferior grain2.21 ± 0.15 cd69.3 ± 2.96 c
OsABA8ox3 RNAi-27Superior grain2.38 ± 0.17 c67.5 ± 0.86 cd
Inferior grain3.23 ± 0.19 b67.6 ± 3.24 cd
Data are expressed as the mean ± SD (n = 3). Values in the same column with different letters are significantly different (p < 0.05).
Table 2. Apparent amylose content and starch granule size distribution in superior and inferior grains of rice.
Table 2. Apparent amylose content and starch granule size distribution in superior and inferior grains of rice.
Sample AAC0–1.5 (μm)1.5–10 (μm)Average Size
ZhongHua11Superior grain15.16 ± 0.47 a7.86 ± 0.15 d91.49 ± 1.31 b4.65 ± 0.051 i
Inferior grain12.85 ± 0.14 cd12.52 ± 0.20 a86.90 ± 2.25 d4.47 ± 0.031 h
OsNCED3-OXSuperior grain12.56 ± 0.38 d8.12 ± 0.11 cd89.93 ± 1.59 c5.56 ± 0.059 b
Inferior grain14.90 ± 0.29 ab7.01 ± 0.13 e91.60 ± 2.33 b5.27 ± 0.043 e
OsNCED3 RNAi-1Superior grain15.50 ± 0.57 a8.54 ± 0.09 c89.72 ± 2.59 c5.64 ± 0.041 b
Inferior grain15.19 ± 0.59 a7.73 ± 0.15 d90.58 ± 1.79 bc5.45 ± 0.031 cd
OsNCED3 RNAi-2Superior grain14.87 ± 0.26 ab4.82 ± 0.07 f92.67 ± 2.01 a5.80 ± 0.022 a
Inferior grain12.56 ± 0.46 d7.15 ± 0.17 e91.73 ± 2.51 b5.37 ± 0.043 d
OsABA8ox3 RNAi-9Superior grain14.78 ± 0.15 ab8.46 ± 0.16 c91.15 ± 2.51 b5.27 ± 0.041 e
Inferior grain14.33 ± 0.54 b10.17 ± 0.12 b89.13 ± 1.35 c4.96 ± 0.031 g
OsABA8ox3 RNAi-27Superior grain13.51 ± 0.27 c7.34 ± 0.11 e90.01 ± 1.22 c5.45 ± 0.054 c
Inferior grain13.29 ± 0.24 cd4.97 ± 0.05 f93.53 ± 0.99 a5.61 ± 0.072 b
AAC: Apparent amylose content. Data are expressed as the mean ± SD (n = 3). Values in the same column with different letters are significantly different (p < 0.05).
Table 3. Changes in swelling potential and solubility of starch from superior and inferior grains in rice.
Table 3. Changes in swelling potential and solubility of starch from superior and inferior grains in rice.
Sample Swelling PowerSolubility
ZhongHua11Superior grain31.01 ± 0.16 b31.48 ± 1.65 a
Inferior grain24.67 ± 0.27 c31.02 ± 1.73 a
OsNCED3-OXSuperior grain19.52 ± 0.66 d32.43 ± 1.26 a
Inferior grain29.86 ± 1.23 b32.93 ± 0.71 a
OsNCED3 RNAi-1Superior grain24.46 ± 0.39 c30.95 ± 1.22 a
Inferior grain24.30 ± 0.13 c31.07 ± 1.09 a
OsNCED3 RNAi-2Superior grain35.27 ± 0.24 a32.79 ± 0.81 a
Inferior grain22.16 ± 1.37 d33.37 ± 1.19 a
OsABA 8ox3 RNAi-9Superior grain26.70 ± 1.11 c32.43 ± 0.62 a
Inferior grain25.47 ± 1.96 c33.19 ± 1.05 a
OsABA 8ox3 RNAi-27Superior grain21.65 ± 1.27 d29.51 ± 2.20 b
Inferior grain29.72 ± 0.55 b33.97 ± 3.29 a
Data are expressed as the mean ± SD (n = 3). Values in the same column with different letters are significantly different (p < 0.05).
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Wu, Y.; Ansah, E.O.; Zhu, L.; Fang, W.; Wang, L.; Zhang, D.; Guo, B. ABA Affects Distinctive Rice Caryopses Physicochemical Properties on Different Branches. Agronomy 2024, 14, 2632. https://doi.org/10.3390/agronomy14112632

AMA Style

Wu Y, Ansah EO, Zhu L, Fang W, Wang L, Zhang D, Guo B. ABA Affects Distinctive Rice Caryopses Physicochemical Properties on Different Branches. Agronomy. 2024; 14(11):2632. https://doi.org/10.3390/agronomy14112632

Chicago/Turabian Style

Wu, Yunfei, Ebenezer Ottopah Ansah, Licheng Zhu, Wenchun Fang, Leilei Wang, Dongping Zhang, and Baowei Guo. 2024. "ABA Affects Distinctive Rice Caryopses Physicochemical Properties on Different Branches" Agronomy 14, no. 11: 2632. https://doi.org/10.3390/agronomy14112632

APA Style

Wu, Y., Ansah, E. O., Zhu, L., Fang, W., Wang, L., Zhang, D., & Guo, B. (2024). ABA Affects Distinctive Rice Caryopses Physicochemical Properties on Different Branches. Agronomy, 14(11), 2632. https://doi.org/10.3390/agronomy14112632

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