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Article

Gα Solicits OsNYC4 and GW2-WG1-OsbZIP47 Modules to Regulate Grain Size in Rice (Oryza sativa L.)

by
Shiwei Ma
1,
Yiqiong Sun
2,
Xuan Chen
2,
Jiayi Guo
2,
Shuhong Wu
2,
Guofeng Wu
2,
Guanpeng Huang
2,
Manegdebwaoga Arthur Fabrice Kabore
2,
Samuel Tareke Woldegiorgis
2,
Yufang Ai
2,
Lina Zhang
2,
Wei Liu
2 and
Huaqin He
2,*
1
College of Environmental and Biological Engineering, Putian University, Putian 351100, China
2
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(7), 1514; https://doi.org/10.3390/agronomy14071514
Submission received: 12 June 2024 / Revised: 5 July 2024 / Accepted: 11 July 2024 / Published: 12 July 2024
(This article belongs to the Special Issue Genetic and Molecular Research on Rice Grain Yield)
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Abstract

:
Grain size is one of the critical factors determining rice yield. Previous studies have found the grain-size-regulating function of Gα in rice. However, the regulatory mechanism underlying the development of rice grain mediated by Gα is still unclear. To reveal the functional mechanism of Gα in grain size regulation, a mutant of Gα (Gα-Cas9) was firstly constructed through a CRISPR/Cas9 strategy and was then grown in a greenhouse and field. The results showed that the seed length, plant height, 1000-grain weight, and spike length were significantly decreased in Gα-Cas9 compared to wild-type (WT) Pi-4b. During the grain filling stage, the increase in the grain dry weight of Pi-4b occurred earlier than that of Gα-Cas9. The total starch content and amylose content of matured grains of Pi-4b were higher than those of Gα-Cas9. Secondly, transcriptome sequencing analysis of Gα-Cas9 and Pi-4b during grain filling was performed to elucidate the functional pathways regulated by Gα. In total, 2867 and 4534 differentially expressed genes (DEGs) were discovered at 5 DAF and 10 DAF, and the starch and sucrose metabolism pathway enriched by DEGs was involved in grain size regulation mediated by Gα. Gα regulated the expression of starch-synthesis-related genes during grain filling, and the Gα protein interacted with OsNYC4 to trigger the sugar signaling pathway to promote starch accumulation in grain. Additionally, the GW2-WG1-OsbZIP47 pathway was switched off by Gα to relieve the inhibition of rice grain development. In this study, the results should provide new insights into the G protein signal transduction pathway.

1. Introduction

Rice is one of the most important food crops, serving as the staple food for more than half of the population of the world [1]. With the continuous increase in population, people have had to increase rice production to meet the higher requirements. By 2050, the global population is expected to reach to 9.1 billion, meaning that one hectare of land will be needed to feed 43 people on average [2]. However, various limit factors, such as water scarcity, soil salinity, pest and disease stress, severe climate, and decreasing arable land, are constantly threatening rice production, exacerbating the world’s food shortage [3]. In face of climate change caused by global warming, it is urgent to increase rice yield per unit area to meet the food demand for the increasing population. Increasing rice yield through breeding technology is of great significance to global food security [4].
Rice yield is mainly related to the number of panicles, the number of grains per panicle, and the thousand-grain weight [4]. The number of panicles is dependent on the tillering ability of rice, while the number of grains per panicle is determined by the quantity of primary branches, secondary branches, and seed setting rate. The Ideal Plant Architecture1 (IPA1) gene is employed to confer rice high yield by reducing unproductive tillers and stronger stems [5,6]. The grain filling stage is a critical stage for grain growth and the development of rice, which directly affect rice yield and quality [7]. When both the number of panicles and the number of grains can be guaranteed, the up-regulation of grain weight is an effective strategy to increase rice yield [8]. The thousand-grain weight is determined by grain size and the degree of grain filling, while the grain size, including seed length, width, and thickness, is one of the most important yield traits of rice [9]. The alteration of grain size is controlled by cell volume and totality, and cell proliferation and growth have been proven to affect the development of rice grains [10]. Researchers have discovered and isolated grain-size-controlling genes through their participation in the transcription regulation pathway, endogenous hormone regulation pathway, ubiquitin–proteasome pathway, and G protein signal transduction pathway [11].
The heterotrimeric G protein (G protein) can sense external stimuli through receptors on the cell surface and then transduct signals to effectors to initiate various cellular behaviors [12]. Previous research has implied that the G protein in rice could regulate multiple signaling pathways, including hormone signaling, environmental sensing, ion channel regulation, disease responses, and cell death [13]. The G protein is composed of three subunits, Gα (RGA), Gβ (RGB), and Gγ (RGG), among which the Gα subunit participates in stomatal opening/closing [14], fungal defense [15], seed germination [16], sugar sensing [17], phytochrome/cryptochrome-mediated responses [18], and seedling and root development [18]. There is one Gα (RGA1), one Gβ (RGB1), and three Gγ subunits (RGG1, RGG2, and RGG3) in the rice genome. RGG3 is a homolog of AGG3, known as Dense and Erect Panicle 1 (DEP1) and G-Protein Coupled Receptor (GPCR) [19].
G proteins have also been found to be involved in the regulation of rice grain development. In rice, a mutant defect in the Gα subunit (RGA1) gene has been identified and is known as the dwarf1 (d1) mutant [20]. The knockout of RGA1 results in small rice grains [21], while over-expression of RGA1 increased grain length [22], indicating that RGA1 positively regulated grain length. Using RNA interference (RNAi) technology to suppress the expression of RGB1, rice grains were changed to be smaller [23]. Interestingly, over-expression of RGB1 also reduced the grain size [24]. The repression of RGB1 led to delayed expression of the genes related to starch biosynthesis and sucrose metabolism, reducing starch accumulation and thus decreasing grain weight. The expression of OsNF-YB1 and OsYUC11 was inhibited in RGB1 mutants, resulting in a decrease in auxin during the grain filling stage [25]. On the other hand, over-expression of RGG1 and RGG2 inhibited the growth of rice grain, resulting in small and light grains [24], and the down-regulation of RGG2 increased grain size and improved rice yield [26]. The typical Gγ genes, Grain Size 3 (GS3), DEP1, and GGC2, have been identified to be the regulators for grain size and yield in rice. GS3 acted as a negative regulator for rice yield and quality by controlling grain weight and grain length [27]. Over-expression of GS3 reduced grain size, while the grains of a GS3 knockout mutant became larger [28]. DEP1 showed an impact on both grain size and the panicle type [29]. GGC2 could trigger the growth of rice grain and increase rice yield [30]. The additive effect of DEP1 and GGC2 on grain size depended on RGB1 [23]. However, until now, the molecular mechanism underlying small grains in the Gα mutant is still unrevealed.
In this study, the knockout mutants of Gα (Gα-Cas9) in the rice cultivar Pi-4b were first constructed by using the CRISPR/Cas9 gene editing system. The difference in rice-yield-related traits and starch content were determined between the mutant Gα-Cas9 and wild-type (WT) Pi-4b. Based on the alteration in grain filling rate at the early stage, rice grains collected at 5 DAF (days after flowering) and 10 DAF were subjected to RNA-seq analysis. The differentially expressed genes (DEGs) were identified in the Gα-Cas9 mutant compared to Pi-4b and were employed to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. DEGs regulated by Gα and proteins interacting with Gα were screened to dissect the regulation of grain size. Finally, the Gα-OsNYC4 and GW2-WG1-OsbZIP47 modules participated in by Gα were depicted to reveal the production of small grains. Our research provides new insights into the G protein signal transduction pathway and elucidates the molecular mechanism for grain size regulated by Gα in rice.

2. Materials and Methods

2.1. Plant Materials and Measurement of Growth and Yield of Rice

Rice seeds of Pi-4b and mutant Gα-Cas9 were soaked in water for 3 days and then sown in soil for 20 days for seedling cultivation at room temperature. Each seedling was transplanted in paddy fields in Putian (25°26′39″ N and 119°4′32″ E), China, under natural growing conditions. In the growing season of 2023, rice seeds were sown in March, transplanted in April, and harvested in July. Each cultivar was composed of at least 120 plants with a row spacing of 30 cm × 20 cm. The average temperature during the growing season was 28 °C. Rice spikes were marked and harvested at a designated time after flowering, and then were dried at 50 °C to a constant weight. A total of 20 grains were selected randomly to be weighed accurately, and the mean of the grains was calculated to be the dry weight. At the harvesting stage in July 2023, plant height and spike length were determined by a band tape, and the number of spikes of each plant was counted by using 3 individual rice plants. The matured grain was collected from the paddy field and air-dried, and seed length and width were measured by vernier caliper. A total of 300 grains were weighed and this was converted to thousand-grain weight with 3 replicas.

2.2. The Construction of Mutant Gα-Cas9

The binary vector Cas9-Gα with two sgRNA expression cassettes was constructed as described previously [31], and was then transferred into Agrobacterium strain EHA105 by chemical transformation. EHA105 harboring the Cas9-Gα plasmid was used to be transformed into rice cv. Pi-4b, and successfully transgenic seedlings were screened on medium with 50 mg/L hygromycin [32]. Initially, T0 rice transgenic plants were confirmed by PCR using primers Hyg-F/R and were then cultivated in paddy fields for seed collection. Seeds of T0 were soaked into the screening solution with hygromycin, and the DNA fragment containing two editing targets was cloned from germinated T1 plants with primers Gα-F/R to perform Sanger sequencing for homozygous identification. Seeds were harvested in July 2022 from the homozygous mutant of the validated T1 generation and were used for further study.

2.3. Starch Content Assay

Total starch and amylose content were measured by the Starch Content Assay Kit and Amylose Content Assay Kit (Solarbio, Beijing, China), respectively. In brief, matured grains were dried and ground absolutely in a mortar. A total of 0.1 g of rice grain powder filtered by a 100-mesh screen was used for the detection of starch content and amylose content following the manufacturer’s instructions.

2.4. RNA-Seq and the Identification of DEGs

The grains of Pi-4b and mutant Gα-Cas9 at 5 DAF and 10 DAF were harvested for transcriptome sequencing. RNA extraction, library construction, and transcriptome sequencing and assembly were conducted as per our previous research [33]. Genes in mutant Gα-Cas9 with an absolute log2 ratio ≥ 1 compared with Pi-4b and a false discovery rate (FDR) < 0.05 were identified as DEGs and screened by DESeq2 (v 1.24.0) [34]. GO and KEGG enrichment analysis were performed on the DEGs by using Omicshare Tools (https://www.omicshare.com/, accessed on 10 July 2024). The heatmaps of DEGs were visualized using the ggplot package.

2.5. Quantitative Real-Time PCR Analysis

The total RNAs of grains during filling were extracted and then reverse-transcribed using the PrimeScrip RT reagent Kit with a gDNA Eraser (TaKaRa, Dalian, China). The expression of starch-synthesis-related genes, ISA1 (LOC_Os08g40930), BE1 (LOC_Os06g51084), AGPL1 (LOC_Os03g52460), ISA2 (LOC_Os05g32710), AGPS2 (LOC_Os08g25734), PUL1 (Os04g0164900), GW2 (LOC_Os02g14720), WG1 (LOC_Os02g30850), OsbZIP47 (LOC_Os06g15480), and OsNYC4 (LOC_Os05g48450), were analyzed with specific primers, listed in Table S3. qPCR reactions were conducted on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions for the TB Green® Premix Ex Taq™ II kit (TaKaRa, Dalian, China). Each qPCR assay was performed in triplicate for three independent RNA samples. OsActin1 (LOC_Os03g50885) was used as an internal reference, and the relative expression level of genes was determined using the 2−ΔΔCT method [35].

2.6. Y2H

A Y2H assay was performed according to the user manual of the Matchmaker Gold Yeast Two-Hybrid System (Clontech, Mountain View, CA, USA). In brief, the ORF sequence of Gα was constructed into pGBKT7 as the bait to screen candidate interacting proteins from the cDNA library of Pi-4b rice plant leaves and verify the interaction with partners. Primers are listed in Table S3.

2.7. Statistical Analysis

A statistical analysis was performed using SPSS 21.0 statistical software (International Business Machines, Armonk, NY, USA). Student’s t-test was used to compare and determine statistically significant differences between the two groups. SigmaPlot 14.0 software (Systat, San Jose, CA, USA) was used to make the charts and pictures.

3. Results

3.1. Gα Positively Regulated the Yield-Related Traits of Rice

To develop the knockout mutants of Gα, the vector Cas9-OsBWR1 was transformed into rice cultivar Pi-4b. The genotypes of the CRISPR/Cas9-edited mutant Gα-Cas9 (M-1 to M-5) are shown in Figure S1, and all transgenic lines had a 1 bp insertion in target 1, resulting in an early stop codon ahead of target 2. One of Gα-Cas9 homozygote mutants, M-1, was selected for further studies. The Gα-Cas9 mutant, and its WT Pi-4b, were grown in both the field and greenhouse. The biomass of WT plants was obviously larger than that of Gα-Cas9 plants (Figure 1a), and the grains of Gα-Cas9 plants were shorter than those of WT plants (Figure 1b). However, no significant difference was observed for the grain width between Gα-Cas9 and WT (Figure 1c). Compared to WT, the grain length, plant height, 1000-grain weight, and spike length of Gα-Cas9 were decreased by 24.7%, 52.4%, 43.5%, and 32%, respectively, but a similar seed width and number of spikes were exhibited on Gα-Cas9 and Pi-4b (Figure 1d–i). These results showed that Gα could positively regulate grain length, plant height, thousand grain weight, and spike length.

3.2. Gα Regulated the Grain Filling Rate at Early Stage

During the grain filling process, the grain shape was measured, which is shown in Figure 2a. The grain shape of Pi-4b was gradually full from 5 days after flowering (DAF) to 10 DAF, and no longer enlarged during the period of 15–25 DAF (Figure 2a). Similar trends were found in Gα-Cas9 except for a smaller seed length. With the prolongation of grain filling, the grain dry weight of WT and Gα-Cas9 increased continuously (Figure 2b). The grain filling rate of Gα-Cas9 was decreased at the early stage (0–10 DAF). Although the dry weight of each grain of WT Pi-4b was higher than that of mutant Gα-Cas9, the grain filling rates of those two plants were almost the same at the later stage of grain filling (10–25 DAF). Starch was mainly accumulated by grain filling, and starch content was determined by using mature seeds. Compared with the WT, a lower content of total starch and amylose were revealed in mutant Gα-Cas9 (Figure 2c). These results indicated that Gα could regulate starch accumulation and grain shape through the reduction in grain filling rate at the early stage.

3.3. DEGs Were Identified between Mutant Gα-Cas9 and Pi-4b at 5 DAF and 12 DAF

To elucidate the regulating mechanism of Gα on grain filling rate, grain samples of Gα-Cas9 and its WT at 5 and 12 DAF were collected for RNA-seq analysis. A total of 548,789,980 clean reads (Q30 > 97.0%) were obtained from 12 samples (Table S1). After comparison, 2867 and 4534 DEGs were, respectively, discovered in the tested samples at 5 DAF and 10 DAF, and the number of up-regulated genes was more than down-regulated ones (Figure 3a). In total, 1316 common DEGs were screened from the sample pairs at 5 DAF and 10 DAF, and the unique DEGs at 5 DAF and 10 DAF were 1551 and 3218 (Figure 3b). RT-qPCR was used to detect the expression of 5 randomly selected DEGs, including LOC_Os08g39860, LOC_Os05g45720, LOC_Os06g15480, LOC_Os02g51070, and LOC_Os01g63270, to confirm the RNA-seq data analysis results. The first 3 DEGs showed down-regulated expression levels in mutant Gα-Cas9, and the other 2 DEGs were up-regulated with the mutation of Gα, which were consistent with the results of RNA-seq data analysis (Figure 3c–e).

3.4. GO and KEGG Enrichment of DEGs in Gα-Cas9 and WT at 5 and 10 DAF

In total, 22, 14, and 12 second-level GO terms in biological process, cellular component, and molecular function were enriched by DEGs in the tested sample pair at 5 DAF, respectively (Figure 4a). The top three GO terms were metabolic process, cellular process, and biological process in biological process. Cell, cell part, and membrane were the three GO terms in cellular component containing the most DEGs, while binding, catalytic activity, and transport activity were enriched in molecular function. On the other hand, the top three enriched GO terms with the most DEGs at 10 DAF were consistent with the enrichment results at 5 DAF, except organelle was in place of membrane in biological process (Figure S2).
Pathways regulated by Gα were searched by KEGG enrichment analysis. Six pathways were enriched by DEGs at 5 DAF, and the top three pathways containing 84 DEGs (22.4%) were plant hormone signal transduction, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism (Figure 4b, Table S2). A total of 28 pathways that were enriched by DEGs were identified at 10 DAF, and 140 DEGs (21.0%) enriched the top three pathways, including carbon metabolism, biosynthesis of cofactors, and carbon fixation in photosynthetic organisms (Figure 4c, Table S2). Moreover, plant hormone signal transduction and starch and sucrose metabolism pathways were also discovered to be enriched by 1316 common DEGs (Figure S3), indicating that DEGs enriching these two pathways regulated by Gα played important roles in the regulation of grain shape.

3.5. Starch Metabolism Participated in the Regulation of Grain Shape Mediated by Gα

In total, 22 DEGs enriched the starch and sucrose metabolism pathway, and 15 of them were up-regulated DEGs in mutant Gα-Cas9 at 5 DAF (Figure 5a). The expression level of Os4BGlu16, OsINV2, OsCel9A, Os1Bglu5, OsSTA246, OsCIN1, and Os4BGlu18 in mutant Gα-Cas9 was over 4-fold higher that in Pi-4b at 5 DAF, and LOC_Os03g25790, Os4BGlu16, Os4BGlu18, OsTPP9, and Os1Bglu5 were still elevated in mutant Gα-Cas9 at 10 DAF. The starch synthesis genes, OsSSIV4b and OsGBSSII, were repressed significantly due to the mutation of Gα at 5 DAF. As shown in Figure 5b, 36 DEGs including 31 up-regulated and 5 down-regulated genes in mutant Gα-Cas9 at 5 DAF enriched the pathway of plant hormone signal transduction. The expression level of OsPR1b, OsSAUR12, and OsSAPK1 were increased in mutant Gα-Cas9 both at 5 DAF and 10 DAF compared to Pi-4b. The reduction in OsSAUR22, OsSAUR8, OsRLCK308, OsbZIP47, and OsIAA30 only appeared in mutant Gα-Cas9 at 5 DAF.
The expression patterns of 29 starch-synthesis-related genes were analyzed due to the decrease in starch content in mutant Gα-Cas9. The results showed that the expression abundance of most starch-synthesis-related genes in mutant Gα-Cas9 at 5 DAF and 10 DAF was lower (Figure 5c) than that in WT. A total of 17 of the above 29 genes were identified to be DEGs during the grain filling period, among which 15 were down-regulated. The expression abundance of OsPho2 and OsSUT5 was increased by the mutation of Gα. These results implied that the starch content was reduced due to the inhibition of starch synthesis genes in mutant Gα-Cas9. A total of 12 genes involved in grain shape were subjected to heatmap analysis, and most of them showed a relatively higher expression level in WT Pi-4b at 5 DAF and 10 DAF, except for GS2 and GW2 (Figure 5d). Among the above 12 genes, 4 genes were identified to be DEGs, while GW2 was exclusively up-regulated in mutant Gα-Cas9 at 5 DAF.
To confirm these results, RT-qPCR was performed to test the expression patterns of the starch-synthesis- and grain-shape-related genes in Gα-Cas9 and WT during the grain filling stage. The peak expression of ISA1 and BE1 in Pi-4b was obtained at 10 DAF, which appeared earlier than that of mutant Gα-Cas9 (Figure 6a,b). In WT Pi-4b, the expression level of AGPL1 was higher than that in Gα-Cas9 at 5–10 DAF, and reached its peak at 20 DAF, which was lower than that of mutant Gα-Cas9 (Figure 6c). With the progress of grain filling, ISA2, AGPS2, and PUL1 were up-regulated and reached their peak expression level in both Pi-4b and mutant Gα-Cas9 at 20 DAF, and then fell down at 25 DAF (Figure 6d–f). However, ISA2, AGPS2, and PUL1 exhibited higher and earlier up-regulation in Pi-4b than in mutant Gα-Cas9 at the early stage of grain filling (5–15 DAF), suggesting that the delayed expression elevation of starch synthesis genes resulted in the reduction in starch content in mutant Gα-Cas9.
The pathway GW2-WG1-OsbZIP47 is known to regulate grain shape [36]; the expression level of GW2 was up-regulated by the mutation of Gα at each stage of grain filling (Figure 6g). Meanwhile, WG1 was down-regulated in mutant Gα-Cas9 compared to Pi-4b (Figure 6h). The expression level of OsbZIP47 was decreased gradually from 5 to 25 DAF, and a lower level was found in mutant Gα-Cas9 in the early stage of grain filling (Figure 6i). These results indicated that Gα regulated the grain shape of rice through the GW2-WG1-OsbZIP47 module.

3.6. Gα Interacted with OsNYC4 to Trigger Sugar Signaling

To elucidate the regulatory mechanism of grain shape mediated by Gα, the proteins interacting with Gα (GPA1) in Arabidopsis thaliana were firstly predicted by using STRING. A total of 10 proteins were discovered to directly interact with GPA1, which was proven by wet experimental evidence (Figure 7a). Among the 10 interacting proteins, THF1 interacts with GPA1 to regulate the sugar signaling response in Arabidopsis. After that, a Y2H assay was used to verify that OsNYC4, one ortholog of Arabidopsis THF1, could interact with Gα in rice in vitro (Figure 7b). Moreover, the expression level of OsNYC4 was higher in Pi-4b than in Gα-Cas9 during the grain filling stage (Figure 7c).

4. Discussion

Improving rice yield and quality through regulating grain size is the direction of rice breeders’ efforts [37]. It is of great significance to explore grain-regulating genes and clarify their mechanisms for the development of new rice varieties with high yield and quality [26]. In this study, we found that knockout of Gα in Pi-4b caused smaller grains, dwarfed plants, decreased 1000-grain weight, and shortened panicle length (Figure 1). The same morphology induced by Gα has been reported in previous studies. The RGA1 mutant exhibited dwarfed plants, short panicles, and short and round grains of rice [20]. SNP at the splicing site of the first intron of the RGA1 gene destroyed the normal splicing of the RGA1 transcript precursor, leading to the premature appearance of a stop codon, inducing rice plants to produce small and round grains with a small degree of chalkiness [38]. Furthermore, some previous studies have found other functions of Gα. RGA1 enhanced the tolerance of rice plants to low light through promoting the transport of assimilates and providing sufficient energy for the elongation of pollen tubes in pistil tissue, which retained the seed setting rate [39].
Grain weight is an important agronomic trait for rice yield and quality formation, which is closely related to the filling degree and starch content [9]. In this study, the grain shape was developed completely at 10 DAF, and there was no significant delay between Pi-4b and the Gα-Cas9 mutant (Figure 2). However, the accumulation rate of dry matter in the mutant Gα-Cas9 was significantly lower than that in the wild type during the early filling stage. The dry weight of rice grains was mainly determined by starch [24]. Both the amylose and total starch content in the mature seeds of the wild-type Pi-4b were significantly higher than those in the mutant Gα-Cas9. These results suggested that Gα promoted the accumulation of dry matter in the early grain filling stage by increasing starch content, and ultimately raised the grain weight. The same was true for RGB1. The grain filling rate was up-regulated earlier with the presentation of RGB1, and the higher starch accumulation enhanced the grain weight [25]. The accumulation of starch during the grain filling stage involved multiple biological processes, including photosynthetic transport from photosynthetic sources (i.e., leaves and stem sheaths), sucrose degradation, transmembrane transport, and starch synthesis in grains [40]. Therefore, it was necessary to further analyze the starch accumulation pathways regulated by Gα to reveal the grain weight maintenance in Pi-4b.
Using RNA-seq technology, 2867 and 4534 DEGs were identified in the grains of Gα-Cas9 and its WT at 5 DAF and 10 DAF, respectively. RT-qPCR was used to verify the expression of five randomly selected DEGs, indicating the strong reliability of RNA-seq data analysis results. The top three GO terms enriched by DEGs in biological process, cellular component, and molecular function were the same at both 5 DAF and 10 DAF, implying that the DEGs regulated by Gα participated in similar functions in rice at 5 and 10 DAF. In KEGG analysis, the DEGs at 5 DAF and common DEGs of the two stages enriched starch and sucrose metabolism and plant hormone signal transduction pathways, suggesting that starch synthesis genes were important mediators for grain development regulated by Gα. The expression levels of most DEGs related to starch synthesis were lower in mutant Gα-Cas9 than that in WT Pi-4b, but they were all up-regulated with the progress of the grain filling stage. However, the starch-synthesis-related genes were up-regulated earlier in the WT Pi-4b during the grain filling stage, resulting in the elevation of starch accumulation and grain weight. Previous research has found that starch biosynthesis was enhanced to positively regulate grain size and weight mediated by DEP1 via increasing the number of endosperm cells [41]. The grain weight was reduced by the mutation of GIF1 through the block of starch accumulation, because GIF1 was specifically expressed in vascular bundles of developing grains to control the transport and unloading of sucrose during grain filling in rice [42]. Filling-defective and grain width 1 (FGW1) could regulate grain size and chalky rate through the promotion of starch synthesis during the grain filling stage [43].
In this study, the interaction between Gα and OsNYC4 was verified by using the Y2H method, through which interaction of the ortholog pair, GPA1 and THF1, in Arabidopsis thaliana has been confirmed by using Fluorescence Resonance Energy Transfer (FRET) and Co-IP methods [44]. Moreover, the structure of OsNYC4 was homological to THF1 [45], and OsNYC4 had the same sub-cellular localization as Gα [46]. All these pieces of evidence strongly supported the interaction between Gα and OsNYC4. In Arabidopsis, THF1 interacted with Gα and played a role in the D-glucose signaling pathway at the downstream of Gα [44]. Root growth was inhibited in THF1 mutants of Arabidopsis treated with exogenous high concentrations of D-glucose, whilst over-expression of THF1 resisted the repression of root growth rate, indicating that Gα promoted the utilization of high concentrations of glucose in plants. In this research, the expression level of OsNYC4 was significantly higher in WT Pi-4b than in the mutant Gα-Cas9 during the grain filling stage. These results indicated that the utilizing efficiency of glucose produced by photosynthesis might be strengthened by the interaction of Gα with OsNYC4 and the up-regulation of OsNYC4. In the cytoplasm or plastids of endosperm cells, high concentrations of glucose could be rapidly converted into ADP-glucose through the action of isomerases and phosphotransferases [47], which was catalyzed by ADP-glucose pyrophosphorylase (AGPase) to accumulate the starch synthetic raw material [48]. Then, starch synthesis was enhanced due to the higher and earlier expression of starch-synthesis-related genes, resulting in the stronger accumulation of starch in WT Pi-4b.
At the initial stage of grain filling, the expression of grain-shape-regulating genes was inhibited in mutants, except for GW2. GW2 could bind to WG1 and degrade it through ubiquitination [36]. gw2.1, the allele of GW2, regulated the genes related to cell proliferation to affect the size of rice glumes, and rice grain width, grain length, and 1000-grain weight were heightened with the over-expression of gw2.1 [49]. Although the expression of GW2 and WG1 both showed a trend of first increasing and then decreasing, the higher expression level of GW2 and the significant down-regulation of WG1 were observed in mutant Gα-Cas9 simultaneously during the grain filling stage (Figure 6). The expression of OsbZIP47 was blocked by WG1 with the recruitment of the transcriptional repressor ASP1, and the limitation of the cell proliferation of rice grain was eliminated to elevate grain weight and grain width with the switching off of OsbZIP47 [36]. In this research, the expression of OsbZIP47 was continuously reduced in Pi-4b and mutant Gα-Cas9 during the grain filling stage, and Pi-4b showed a significantly lower expression of OsbZIP47 at the early stage of grain filling. These results suggested that Gα down-regulated the expression level of GW2 to repress OsbZIP47 through the maintenance of WG1, thereby regulating grain size and weight.
In summary, the mutation of Gα in Pi-4b led to shorter grain length, lower 1000-grain weight, and lower starch content. Transcriptome sequencing analysis of Pi-4b and mutant Gα-Cas9 during the grain filling stage showed that Gα mainly participated in the regulation of starch synthesis and the metabolism pathway. Firstly, Gα interacted with OsNYC4 to trigger the sugar-signaling pathway to induce earlier and higher expression of starch synthesis genes, resulting in starch accumulation in grain and grain weight increasing (Figure 8). Secondly, Gα repressed the expression of GW2, and then switched off the GW2-WG1-OsbZIP47 module, relieving the inhibition of grain development. Ultimately, the grain development regulated by Gα relied on the Gα-OsNYC4 and GW2-WG1-OsbZIP47 modules.

5. Conclusions

In conclusion, Gα could maintain the grain length, 1000-grain weight, and starch content in rice through the regulation of starch synthesis and the metabolism pathway during the grain filling stage. The earlier and higher up-regulation of starch synthesis genes were triggered by the interaction between Gα and OsNYC4, and then starch accumulation was enhanced to increase starch content and grain weight. Moreover, the module GW2-WG1-OsbZIP47, inhibiting grain development, was switched off due to the repression of GW2 caused by Gα. The preliminary mechanism for grain size regulated by Gα in rice has been revealed in this research. The exact molecular links between Gα and GW2 are unclear, requiring further in-depth investigation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14071514/s1, Supplementary File 1, Table S1. Statistics of grain transcriptome sequencing data of Gα-Cas9 mutant and its wild type Pi-4b during grain filling stage. Table S2. KEGG enrichment analysis of DEGs. Table S3. Sequence of primers. Figure S1. The genotype of Gα mutants mediated by CRISPR/Cas9. Dash and red bases represent the base deletion and insertion in target sites, respectively. The insertion of one base results in an early stop codon ahead of target 2. Figure S2. GO enrichment analysis of DEGs identified from sample grains at 12 DAF. Figure S3. KEGG analysis of common DEGs of tested sample pairs.

Author Contributions

Writing—review and editing, H.H.; writing—original draft preparation, S.M.; resources, Y.S., X.C. and J.G.; methodology, S.W., G.W., G.H. and W.L.; data curation, M.A.F.K., S.T.W., Y.A. and L.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Natural Science Foundation of China (No. 32370709 and 32302372), Natural Science Foundation of Fujian (No. 2021J05240 and 2022J02020), Key Science Project of Fujian (No. 2022N5006 and 2022NZ030014), and Startup Fund for Advanced Talents of Putian University (No. 2020004).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Plant height and grain traits of Gα-Cas9 and its WT Pi-4b. (a) Plants grown in greenhouse. Bar = 20.0 cm. (b,c) The photograph of matured grain. Bar = 1.0 cm. Statistical analysis of grain length (d), width (e), plant height (f), thousand-grain weight (g), spike length (h), and the number of spikes (i) of Gα-Cas9 and Pi-4b. Six rice plants on harvesting day were randomly selected for statistical analysis. Thousand-grain weight was calculated by 300 grains. Grain length and width were obtained from 50 grains. Error bars indicate standard deviation (SD) of biological replicates, and statistical analysis was performed by Student’s t-test (** p < 0.01).
Figure 1. Plant height and grain traits of Gα-Cas9 and its WT Pi-4b. (a) Plants grown in greenhouse. Bar = 20.0 cm. (b,c) The photograph of matured grain. Bar = 1.0 cm. Statistical analysis of grain length (d), width (e), plant height (f), thousand-grain weight (g), spike length (h), and the number of spikes (i) of Gα-Cas9 and Pi-4b. Six rice plants on harvesting day were randomly selected for statistical analysis. Thousand-grain weight was calculated by 300 grains. Grain length and width were obtained from 50 grains. Error bars indicate standard deviation (SD) of biological replicates, and statistical analysis was performed by Student’s t-test (** p < 0.01).
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Figure 2. Mutation of Gα showed impacts on grain development and starch accumulation. (a) The grain morphology of mutant Gα-Cas9 and its wild type during grain filling stage. Bar, 1.0 cm. (b) The alteration in dry weight of tested cultivars during grain filling stage. The significantly increased rate of dry weight appeared earlier in Pi-4b than in mutant Gα-Cas9. (c) Total starch and amylose content in matured grains of Gα-Cas9 mutant and its wild type Pi-4b. Error bars indicate SD of biological replicates, and asterisks indicate significant differences between sample pair (Student’s t-test, ** p < 0.01).
Figure 2. Mutation of Gα showed impacts on grain development and starch accumulation. (a) The grain morphology of mutant Gα-Cas9 and its wild type during grain filling stage. Bar, 1.0 cm. (b) The alteration in dry weight of tested cultivars during grain filling stage. The significantly increased rate of dry weight appeared earlier in Pi-4b than in mutant Gα-Cas9. (c) Total starch and amylose content in matured grains of Gα-Cas9 mutant and its wild type Pi-4b. Error bars indicate SD of biological replicates, and asterisks indicate significant differences between sample pair (Student’s t-test, ** p < 0.01).
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Figure 3. DEGs in Gα-Cas9 and its WT Pi-4b at 5 and 10 DAF. (a) The number of DEGs identified from mutant Gα-Cas9 compared with Pi-4b during grain filling stage. (b) Venn diagrams showing the total overlapping DEGs between the 2 sample pairs. (c) The log2fold change in 5 randomly selected DEGs at 5 DAF and 10 DAF. (d,e) The expression patterns of the above 5 DEGs in Pi-4b and mutant Gα-Cas9 at 5 DAF and 10 DAF, respectively. The expression level of DGEs was determined by using RT-qPCR and normalized by OsActin1. Error bars indicate SD of three biological replicates, and asterisks indicate significant differences between sample pair (Student’s t-test, ** p < 0.01).
Figure 3. DEGs in Gα-Cas9 and its WT Pi-4b at 5 and 10 DAF. (a) The number of DEGs identified from mutant Gα-Cas9 compared with Pi-4b during grain filling stage. (b) Venn diagrams showing the total overlapping DEGs between the 2 sample pairs. (c) The log2fold change in 5 randomly selected DEGs at 5 DAF and 10 DAF. (d,e) The expression patterns of the above 5 DEGs in Pi-4b and mutant Gα-Cas9 at 5 DAF and 10 DAF, respectively. The expression level of DGEs was determined by using RT-qPCR and normalized by OsActin1. Error bars indicate SD of three biological replicates, and asterisks indicate significant differences between sample pair (Student’s t-test, ** p < 0.01).
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Figure 4. GO and KEGG enrichment of the DEGs in Gα-Cas9 and its WT at 5 and 10 DAF. (a) GO enrichment of DEGs at 5 DAF. KEGG pathways were enriched by DEGs in the tested cultivars at 5 DAF (b) and 10 DAF (c).
Figure 4. GO and KEGG enrichment of the DEGs in Gα-Cas9 and its WT at 5 and 10 DAF. (a) GO enrichment of DEGs at 5 DAF. KEGG pathways were enriched by DEGs in the tested cultivars at 5 DAF (b) and 10 DAF (c).
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Figure 5. Heatmaps showing the expression abundance of DEGs related to grain size regulation during grain filling stage. The expression patterns of DEGs enriching starch and sucrose metabolism pathway (a) and plant hormone signal transduction pathway (b). The heatmap of genes related to starch synthesis (c) and grain-shape-controlling (d). Green and red gene names represent down-regulated and up-regulated DEGs.
Figure 5. Heatmaps showing the expression abundance of DEGs related to grain size regulation during grain filling stage. The expression patterns of DEGs enriching starch and sucrose metabolism pathway (a) and plant hormone signal transduction pathway (b). The heatmap of genes related to starch synthesis (c) and grain-shape-controlling (d). Green and red gene names represent down-regulated and up-regulated DEGs.
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Figure 6. Expression patterns of starch-synthesis-related genes and GW2-WG1-OsbZIP47 during grain filling stage. Genes involved in starch synthesis were up-regulated to a relatively higher level in Pi-4b compared to in mutant Gα-Cas9 at the early stage of grain filling. GW2-WG1-OsbZIP47 was repressed with the presentation of Gα in Pi-4b during the grain filling stage. The relative expression level of ISA1 (a), BE1 (b), AGPL1 (c), ISA2 (d), AGPS2 (e), PUL1 (f), GW2 (g), WG1 (h), and OsbZIP47 (i) was detected by using RT-qPCR and normalized by OsActin1. Error bars indicate SD of three biological replicates, and asterisks indicate significant differences between sample pair (Student’s t-test, ** p < 0.01, and * p < 0.05).
Figure 6. Expression patterns of starch-synthesis-related genes and GW2-WG1-OsbZIP47 during grain filling stage. Genes involved in starch synthesis were up-regulated to a relatively higher level in Pi-4b compared to in mutant Gα-Cas9 at the early stage of grain filling. GW2-WG1-OsbZIP47 was repressed with the presentation of Gα in Pi-4b during the grain filling stage. The relative expression level of ISA1 (a), BE1 (b), AGPL1 (c), ISA2 (d), AGPS2 (e), PUL1 (f), GW2 (g), WG1 (h), and OsbZIP47 (i) was detected by using RT-qPCR and normalized by OsActin1. Error bars indicate SD of three biological replicates, and asterisks indicate significant differences between sample pair (Student’s t-test, ** p < 0.01, and * p < 0.05).
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Figure 7. OsNYC4 interacts with Gα protein to trigger sugar signaling. (a) The interacting network of GPA1 predicted in Arabidopsis thaliana by using STRING with advanced settings. GAP1 in Arabidopsis thaliana is the homologous gene of Gα (RGA1) in rice. The pink connecting lines mean the interaction was verified by wet experiment. (b) The interaction of OsNYC4 and Gα was confirmed by Y2H assay. The indicators 1, 1/10, 1/100, and 1/1000 represent a 1-, 10-, 100-, and 1000-fold dilution of yeast suspension, respectively. (c) The expression pattern of OsNYC4 in test samples during the grain filling stage was measured by using RT-qPCR. Error bars indicate SD of three biological replicates, and asterisks indicate significant differences between sample pair (Student’s t-test, ** p < 0.01).
Figure 7. OsNYC4 interacts with Gα protein to trigger sugar signaling. (a) The interacting network of GPA1 predicted in Arabidopsis thaliana by using STRING with advanced settings. GAP1 in Arabidopsis thaliana is the homologous gene of Gα (RGA1) in rice. The pink connecting lines mean the interaction was verified by wet experiment. (b) The interaction of OsNYC4 and Gα was confirmed by Y2H assay. The indicators 1, 1/10, 1/100, and 1/1000 represent a 1-, 10-, 100-, and 1000-fold dilution of yeast suspension, respectively. (c) The expression pattern of OsNYC4 in test samples during the grain filling stage was measured by using RT-qPCR. Error bars indicate SD of three biological replicates, and asterisks indicate significant differences between sample pair (Student’s t-test, ** p < 0.01).
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Figure 8. Module for grain development regulated by Gα. Gα protein interacts with OsNYC4 to trigger sugar signaling and then up-regulate the expression of genes related to starch synthesis earlier during grain filling stage to promote starch accumulation. GW2-WG1-OsbZIP47 pathway was switched off by Gα to relieve its inhibition for rice grain development.
Figure 8. Module for grain development regulated by Gα. Gα protein interacts with OsNYC4 to trigger sugar signaling and then up-regulate the expression of genes related to starch synthesis earlier during grain filling stage to promote starch accumulation. GW2-WG1-OsbZIP47 pathway was switched off by Gα to relieve its inhibition for rice grain development.
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Ma, S.; Sun, Y.; Chen, X.; Guo, J.; Wu, S.; Wu, G.; Huang, G.; Kabore, M.A.F.; Woldegiorgis, S.T.; Ai, Y.; et al. Gα Solicits OsNYC4 and GW2-WG1-OsbZIP47 Modules to Regulate Grain Size in Rice (Oryza sativa L.). Agronomy 2024, 14, 1514. https://doi.org/10.3390/agronomy14071514

AMA Style

Ma S, Sun Y, Chen X, Guo J, Wu S, Wu G, Huang G, Kabore MAF, Woldegiorgis ST, Ai Y, et al. Gα Solicits OsNYC4 and GW2-WG1-OsbZIP47 Modules to Regulate Grain Size in Rice (Oryza sativa L.). Agronomy. 2024; 14(7):1514. https://doi.org/10.3390/agronomy14071514

Chicago/Turabian Style

Ma, Shiwei, Yiqiong Sun, Xuan Chen, Jiayi Guo, Shuhong Wu, Guofeng Wu, Guanpeng Huang, Manegdebwaoga Arthur Fabrice Kabore, Samuel Tareke Woldegiorgis, Yufang Ai, and et al. 2024. "Gα Solicits OsNYC4 and GW2-WG1-OsbZIP47 Modules to Regulate Grain Size in Rice (Oryza sativa L.)" Agronomy 14, no. 7: 1514. https://doi.org/10.3390/agronomy14071514

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