*2.2. pnfyc Are GA-Deficient with Retarded Seed Germination*

The altered storage substance proportion in *pnfyc* lines provoked us to test the roles of the five *NF-YCs* in seed germination. We carried out germination assays on the *nfyc* single mutants, *pnfyc* and NIP lines on <sup>1</sup> <sup>2</sup> MS medium for 4 days. All the five *nfyc* single mutants showed slightly lower germination rates compared with Kitaake, which was followed by retarded post-germination growth (Figure S4A–C). However, the *pnfyc* seeds showed much retarded germination (56.7–60.0%) than the NIP (90.0–96.7%) at day 4, which further confirmed the functional redundancy among the five NF-YCs. Given the key roles of ABA and GA in seed germination regulation, we subsequently investigated the seed germination under exogenous ABA, GA and PAC (GA biosynthesis inhibitor) treatments (Figure 2A–C). The application of 2 µM exogenous ABA significantly restrained the seed germination of both *pnfyc* and NIP seeds. To evaluate the relative ABA sensitivity of the seeds, we calculated the relative germination rate of the seeds under mock and ABA treatments. The results showed that the WT relative germination rates of 2 µM ABA/mock and 5 µM ABA/mock were 72.2% and 65.5%, respectively. However, for the *pnfyc* seeds, 84.5% and 51.4% were obtained. Therefore, *pnfyc* is hypersensitive to exogenous ABA treatment in seed germination (*p* < 0.05) (Figure 2D). Similar hypersensitivity to ABA inhibition effects was also observed in the post-germination growth of *pnfyc* seedlings. In contrast to the ABA treatments, the application of 2 µM exogenous GA significantly recovered the seed germination of *pnfyc*, and a more intense recovering effect was observed when 5 µM exogenous GA was applied, suggesting the GA deficiency might be the major reason for the retarded seed germination in *pnfyc*. Exogenous PAC displayed similar inhibitory effects as ABA, as 10 µM exogenous PAC decreased the NIP germination rate and post-germination growth to the *pnfyc* level. In addition, we quantified the GA3 contents in the germinative seeds of NIP and *pnfyc-1*. The result showed that the GA3 content in *pnfyc-1* was reduced

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 4 of 13

to only 30% of the NIP (*p* < 0.01), which is in agreement with the recovered germination of *pnfyc* by GA (Figure 2E).

**Figure 1.** (**A**) (a–o) Grain chalkiness phenotypical characterization (a–f), bar = 2 mm. Scanning electron microscopy (SEM) analysis (g–o). The central areas shown are indicated as red squares*.* The magnification is 30 times in (g,h,i); 2000 times in (j,k,l), and 5000 times in (m,n,o). (**B**–**F**) Quality trait parameters of mature seeds from *pnfyc* lines and NIP. Data are shown as means ± SD of at least three **Figure 1.** (**A**) (a–o) Grain chalkiness phenotypical characterization (a–f), bar = 2 mm. Scanning electron microscopy (SEM) analysis (g–o). The central areas shown are indicated as red squares. The magnification is 30 times in (g,h,i); 2000 times in (j,k,l), and 5000 times in (m,n,o). (**B**–**F**) Quality trait parameters of mature seeds from *pnfyc* lines and NIP. Data are shown as means ± SD of at least three biological replicates. (\*\* *p* < 0.01 by two-tailed Student's *t*-test).

of the five *NF-YCs* in seed germination. We carried out germination assays on the *nfyc* single mutants, *pnfyc* and NIP lines on ½ MS medium for 4 days. All the five *nfyc* single mutants showed slightly lower germination rates compared with Kitaake, which was followed by retarded post-germination growth (Figure S4A–C). However, the *pnfyc* seeds showed much retarded germination (56.7–60.0%) than the NIP (90.0–96.7%) at day 4, which further confirmed the functional redundancy among the five NF-YCs. Given the key roles of ABA and GA in seed germination regulation, we subsequently investigated the seed germination under exogenous ABA, GA and PAC (GA biosynthesis inhibitor) treatments (Figure 2A–C). The application of 2 μM exogenous ABA significantly restrained the seed germination of both *pnfyc* and NIP seeds. To evaluate the relative ABA sensitivity of the seeds, we calculated the relative germination rate of the seeds under mock and ABA treatments. The results showed that the WT relative germination rates of

biological replicates. (\*\* *p* < 0.01 by two-tailed Student's *t*-test).

*2.2. pnfyc Are GA-Deficient with Retarded Seed Germination* 

agreement with the recovered germination of *pnfyc* by GA (Figure 2E).

2 μM ABA/mock and 5 μM ABA/mock were 72.2% and 65.5%, respectively. However, for the *pnfyc* seeds, 84.5% and 51.4% were obtained. Therefore, *pnfyc* is hypersensitive to exogenous ABA treatment in seed germination (*p* < 0.05) (Figure 2D). Similar hypersensitivity to ABA inhibition effects was also observed in the post-germination growth of *pnfyc* seedlings. In contrast to the ABA treatments, the application of 2 μM exogenous GA significantly recovered the seed germination of *pnfyc*, and a more intense recovering effect was observed when 5 μM exogenous GA was applied, suggesting the GA deficiency might be the major reason for the retarded seed germination in *pnfyc*. Exogenous PAC displayed similar inhibitory effects as ABA, as 10 μM exogenous PAC decreased the NIP germination rate and post-germination growth to the *pnfyc* level. In addition, we quantified the GA3 contents in the germinative seeds of NIP and *pnfyc-1*. The result showed that

**Figure 2.** (**A**) The germination rate of NIP and *pnfyc* lines under different concentrations of exogenous hormones. (**B**) The plant morphology of NIP and *pnfyc* lines treated with different exogenous hormones at 7 days after germination. (**C**) The seedling height of NIP and *pnfyc* lines under different exogenous hormones at 7 days after germination. Bar = 1 cm. (**D**) The relative germination of the NIP and *pnfyc* seeds under ABA treatments were determined after 4 days and expressed as a percentage of those grown under 'mock' conditions. (**E**) Quantification of GA3 derivatives in NIP and *pnfyc* seeds germinated for 6 h was analyzed with liquid chromatography-tandem mass spectrometry. Data are shown as means ± SD of at least three biological replicates. (\*\* *p* < 0.01 by two-tailed **Figure 2.** (**A**) The germination rate of NIP and *pnfyc* lines under different concentrations of exogenous hormones. (**B**) The plant morphology of NIP and *pnfyc* lines treated with different exogenous hormones at 7 days after germination. (**C**) The seedling height of NIP and *pnfyc* lines under different exogenous hormones at 7 days after germination. Bar = 1 cm. (**D**) The relative germination of the NIP and *pnfyc* seeds under ABA treatments were determined after 4 days and expressed as a percentage of those grown under 'mock' conditions. (**E**) Quantification of GA3 derivatives in NIP and *pnfyc* seeds germinated for 6 h was analyzed with liquid chromatography-tandem mass spectrometry. Data are shown as means ± SD of at least three biological replicates. (\*\* *p* < 0.01 by two-tailed Student's *t*-test).

Student's *t*-test). To test the involvement of the five *NF-YCs* in ABA and GA biosynthesis and signaling, we examined the transcription of the genes in the germinative seeds of various ABA or GA-related genetic lines by qRT-PCR. *SAPK8*, *9* and *10* are core elements of ABA To test the involvement of the five *NF-YCs* in ABA and GA biosynthesis and signaling, we examined the transcription of the genes in the germinative seeds of various ABA or GA-related genetic lines by qRT-PCR. *SAPK8*, *9* and *10* are core elements of ABA signaling, and over-expression of the genes conferred plants ABA hypersensitivity [33]. We found that *NF-YC10* was significantly up-regulated by *SAPK8* and *10*, while *NF-YC8* and *NF-YC12* were up-regulated by *SAPK10* and *SAPK9*, respectively. However, *NF-YC9* and *NF-YC11* were down-regulated in all the three *OxSAPK* lines (Figure 3A). In the GA-deficient mutant *sd1* [34], the transcription of *NF-YC8*, *9*, *10* and *11* were all severely repressed, indicating the five *NF-YCs* are highly responsive to endogenous GA level (Figure 3B). Taken together, we proposed that *NF-YC8*, *9*, *10*, *11* and *12* may serve as key regulators mediating the balance of GA and ABA.

tors mediating the balance of GA and ABA.

signaling, and over-expression of the genes conferred plants ABA hypersensitivity [33]. We found that *NF-YC10* was significantly up-regulated by *SAPK8* and *10*, while *NF-YC8* and *NF-YC12* were up-regulated by *SAPK10* and *SAPK9*, respectively. However, *NF-YC9* and *NF-YC11* were down-regulated in all the three *OxSAPK* lines (Figure 3A). In the GAdeficient mutant *sd1* [34], the transcription of *NF-YC8*, *9*, *10* and *11* were all severely repressed, indicating the five *NF-YCs* are highly responsive to endogenous GA level (Figure

**Figure 3.** (**A**) The expression level of *NFYC8* to *NFYC12* in the seeds of wild-type-NIP, *OxSAPK8*, *OxSAPK9,* and *OxSAPK10* mutants that germinated for 6 h. (**B**) The expression level of *NFYC8* to *NFYC11* in the seeds of wild-type-ZH11 and *sd1* mutant that germinated for 6 h (\* *p* < 0.05, \*\* *p* < 0.01 by two-tailed Student's *t*-test). **Figure 3.** (**A**) The expression level of *NFYC8* to *NFYC12* in the seeds of wild-type-NIP, *OxSAPK8*, *OxSAPK9,* and *OxSAPK10* mutants that germinated for 6 h. (**B**) The expression level of *NFYC8* to *NFYC11* in the seeds of wild-type-ZH11 and *sd1* mutant that germinated for 6 h (\* *p* < 0.05, \*\* *p* < 0.01 by two-tailed Student's *t*-test).

Although *NF-YC10* and *NF-YC12* have been reported as key regulators of rice seed development [8,10,32], the roles of the five seed-specific NY-YCs are still not very clear so far, given the potential functional redundancy among them. By simultaneously knocking out the five genes, we revealed their functions in grain quality and seed germination, and the GA-deficiency in *pnfyc* might be the major reason for the observed phenotype. Aside from the well-known function as a seed germination promoter, GA has been recently found to regulate endosperm development as well. Cui et al. (2020) reported that application of exogenous GA4+7 significantly altered the content of other phytohormones such as auxin, zeatin and ABA, increased the activities of superoxide dismutase, catalases, and peroxidases and reduced the malondialdehyde content, which finally improved grain filling and yield in maize [35]. In Arabidopsis, *NF-YC3*, *NF-YC4* and *NF-YC9* have redundant Although *NF-YC10* and *NF-YC12* have been reported as key regulators of rice seed development [8,10,32], the roles of the five seed-specific NY-YCs are still not very clear so far, given the potential functional redundancy among them. By simultaneously knocking out the five genes, we revealed their functions in grain quality and seed germination, and the GA-deficiency in *pnfyc* might be the major reason for the observed phenotype. Aside from the well-known function as a seed germination promoter, GA has been recently found to regulate endosperm development as well. Cui et al. (2020) reported that application of exogenous GA4+7 significantly altered the content of other phytohormones such as auxin, zeatin and ABA, increased the activities of superoxide dismutase, catalases, and peroxidases and reduced the malondialdehyde content, which finally improved grain filling and yield in maize [35]. In Arabidopsis, *NF-YC3*, *NF-YC4* and *NF-YC9* have redundant roles in the regulation of GA-ABA-mediated seed germination [36]. NF-YCs bind RGL2, a repressor during GA signaling, and then in the form of NF-YC–RGL2 module targets *ABI5*, a key factor in ABA signaling [37,38]. NF-YC can bind to the CCAAT-box on the *ABI5* promoter to regulate ABI5 gene expression. The NF-YC–RGL2–ABI5 module integrates ABA and GA signaling to regulate seed germination [36].

#### roles in the regulation of GA-ABA-mediated seed germination [36]. NF-YCs bind RGL2, *2.3. NF-YCs Regulates the Transcription of ABA and GA Pathway Genes*

a repressor during GA signaling, and then in the form of NF-YC–RGL2 module targets *ABI5*, a key factor in ABA signaling [37,38]. NF-YC can bind to the CCAAT-box on the *ABI5* promoter to regulate ABI5 gene expression. The NF-YC–RGL2–ABI5 module integrates ABA and GA signaling to regulate seed germination [36]. *2.3. NF-YCs Regulates the Transcription of ABA and GA Pathway Genes*  RNA sequencing experiments on the 6 HAI (6 h after imbibition) germinating seeds of *pnfyc* and WT were carried out to identify the potential target genes of the five NF-YCs. As a result, there were a total of 469 differentially expressed genes (DEGs) as shown in (Supplementary Table S1). KEGG analysis revealed predominant enrichment of the DEGs on the pathways of 'phenylpropanoid biosynthesis', 'protein processing in endoplasmic reticulum' and 'plant hormone signal transduction' (Figure S5A). We further carried out a qRT-PCR analysis on eight randomly selected DEGs to verify the RNA-seq results, and RNA sequencing experiments on the 6 HAI (6 h after imbibition) germinating seeds of *pnfyc* and WT were carried out to identify the potential target genes of the five NF-YCs. As a result, there were a total of 469 differentially expressed genes (DEGs) as shown in (Supplementary Table S1). KEGG analysis revealed predominant enrichment of the DEGs on the pathways of 'phenylpropanoid biosynthesis', 'protein processing in endoplasmic reticulum' and 'plant hormone signal transduction' (Figure S5A). We further carried out a qRT-PCR analysis on eight randomly selected DEGs to verify the RNA-seq results, and the results showed that seven of those genes had a similar transcriptional level inclination to that detected by the RNA-seq, indicating the high reliability of our RNA-seq results (Figure S5B). Notably, a series of genes reported as critical regulators of the ABA signal pathway were found to be down-regulated in *pnfyc* (Figure 4B), including positive ABA signaling factors like *OsbZIP46* [39,40], *OsbZIP12* [41,42] and *OsNAC52* [43] as well as the *OsHSP24.1* encoding an ABA-responsive heat shock protein [44]. Furthermore, we conducted the qRT-PCR and results showed that the expression of most GA biosynthesis genes was down-regulated in *pnfyc* plants, while the expression of ABA biosynthesis and negative signal pathway-related genes was up-regulated (Figure 4A,B).

the results showed that seven of those genes had a similar transcriptional level inclination to that detected by the RNA-seq, indicating the high reliability of our RNA-seq results (Figure S5B). Notably, a series of genes reported as critical regulators of the ABA signal

signaling factors like *OsbZIP46* [39,40], *OsbZIP12* [41,42] and *OsNAC52* [43] as well as the

negative signal pathway-related genes was up-regulated (Figure 4A,B).

*OsHSP24.1* encoding an ABA-responsive heat shock protein [44]. Furthermore, we conducted the qRT-PCR and results showed that the expression of most GA biosynthesis genes was down-regulated in *pnfyc* plants, while the expression of ABA biosynthesis and

**Figure 4.** (**A**) The expression level of GA synthesis and metabolism-related genes in the seeds of wild-type-NIP and *pnfyc* mutants that germinated for 6 h. (**B**) The expression level of ABA biosynthesis and negative signal pathway-related genes in the seeds of wild-type-NIP and *pnfyc* mutants that germinated for 6 h. (**C**) The expression level of starch synthesis-related genes in wild-type-NIP and *pnfyc* mutant seeds 7 days after pollination. Data are shown as means ± SD of at least three biological replicates. (\*\* *p* < 0.01 by two-tailed Student's *t*-test). **Figure 4.** (**A**) The expression level of GA synthesis and metabolism-related genes in the seeds of wildtype-NIP and *pnfyc* mutants that germinated for 6 h. (**B**) The expression level of ABA biosynthesis and negative signal pathway-related genes in the seeds of wild-type-NIP and *pnfyc* mutants that germinated for 6 h. (**C**) The expression level of starch synthesis-related genes in wild-type-NIP and *pnfyc* mutant seeds 7 days after pollination. Data are shown as means ± SD of at least three biological replicates. (\*\* *p* < 0.01 by two-tailed Student's *t*-test).

Given the severely affected starch qualities in *pnfyc* seeds, we also examined the transcriptional levels of several starch biosynthesis enzyme or regulator genes in the developing seeds of *pnfyc* and NIP. It was found that, except for *ISA2*, all of these ADP-glucose pyrophosphorylase, granule-bound starch synthase, starch synthase and starch branching enzyme were mostly down-regulated in 7 DAP endosperm of *pnfyc* lines (Figure 4C) [45– Given the severely affected starch qualities in *pnfyc* seeds, we also examined the transcriptional levels of several starch biosynthesis enzyme or regulator genes in the developing seeds of *pnfyc* and NIP. It was found that, except for *ISA2*, all of these ADP-glucose pyrophosphorylase, granule-bound starch synthase, starch synthase and starch branching enzyme were mostly down-regulated in 7 DAP endosperm of *pnfyc* lines (Figure 4C) [45–48].

#### 48]. *2.4. Potential Interactive Proteins of the Five NF-YCs*

*2.4. Potential Interactive Proteins of the Five NF-YCs*  We tested the protein–protein interaction between the 5 NF-YCs and 10 SAPKs which are ABA signaling components. A total of 50 NF-YC-SAPK combinations were tested by yeast-two-hybrid, and results showed that NF-YC10 binds to SAPK4, 6 and 10, while all the other combinations were negative. Hence, the suggestion is that NF-YC10 perceives We tested the protein–protein interaction between the 5 NF-YCs and 10 SAPKs which are ABA signaling components. A total of 50 NF-YC-SAPK combinations were tested by yeast-two-hybrid, and results showed that NF-YC10 binds to SAPK4, 6 and 10, while all the other combinations were negative. Hence, the suggestion is that NF-YC10 perceives the ABA signal from SAPK4, 6 and 10 (Figure 5A).

the ABA signal from SAPK4, 6 and 10 (Figure 5A). Our previous study has demonstrated that NF-YB1-YC12 dimer binds to bHLH144 to form a heterotrimer complex that regulates rice grain quality [8]. To identify other components that may interact with NF-YB1-YC12 dimer, we performed yeast-three hybrid (Y3H) experiments to screen a seed-derived prey library using NF-YB1-YC12-pBRIDGE as bait. We finally obtained three interactive proteins LOC\_Os01g68950, LOC\_Os07g46160 and LOC\_Os11g38670 which are annotated as ubiquitin domain-containing protein, BTB/POZ domain-containing protein and dead-box ATP-dependent RNA helicase, respectively. Interestingly, the interactions are valid only on the SD/-Met/-Leu/-Trp/-Ade/-His/+X-α-Gal medium, in which the drop-out of methionine drove the expression of NF-YC12 under Met25 promoter. Meanwhile, the interactions were compromised on SD/-Leu/-Trp/- Ade/-His/+X-α-Gal medium, in which NF-YC12 was suppressed by the supplemented methionine in the medium. Hence, the binding of NF-YB1-YC12 is necessary for the formation of heterotrimer complexes with the three proteins (Figure 5B–D).

In conclusion, we report a rice pentuple gene mutant *pnfyc,* which knocked out five homologous genes *OsNF-YC8, OsNF-YC9, OsNF-YC10, OsNF-YC11* and *OsNF-YC12*

simultaneously. The expression of starch synthesis genes decreased in *pnfyc,* resulting in the decrease of starch content and the increase of protein content, the change of grain quality and the significant increase of chalkiness trait. The results showed that *NF-YC8* to *NF-YC12* could regulate grain quality traits by regulating starch synthesis. In addition, *NF-YC8-12* also inhibited seed germination by affecting the expression of GA-related genes, and the phenotype is significantly restored by applying exogenous GA. Finally, the expression of ABA-related genes in *pnfyc* increased, and *pnfyc* seeds were hypersensitive to exogenous ABA. NFYC10 could interact with SAPK to regulate ABA expression. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 8 of 13

**Figure 5.** (**A**) Y2H assay of interaction between NF-YC10 and SAPKs. BD: pDEST32; AD: pDEST22; EV: empty vector, pDEST32 or pDEST22; P: positive control, pGBKT7-53/pGADT7-T; N: positive control, pGBKT7-Lam/pGADT7-T. (**B**) Y3H analysis of NF-YB1, NF-YC12, and Ubiquitin protein domain. (**C**) Y3H analysis of NF-YB1, NF-YC12, and BTB/POZ protein domain. (**D**) Y3H analysis of NF-YB1, NF-YC12, and Dead-box protein domain. **Figure 5.** (**A**) Y2H assay of interaction between NF-YC10 and SAPKs. BD: pDEST32; AD: pDEST22; EV: empty vector, pDEST32 or pDEST22; P: positive control, pGBKT7-53/pGADT7-T; N: positive control, pGBKT7-Lam/pGADT7-T. (**B**) Y3H analysis of NF-YB1, NF-YC12, and Ubiquitin protein domain. (**C**) Y3H analysis of NF-YB1, NF-YC12, and BTB/POZ protein domain. (**D**) Y3H analysis of NF-YB1, NF-YC12, and Dead-box protein domain.

#### Our previous study has demonstrated that NF-YB1-YC12 dimer binds to bHLH144 **3. Materials and Methods**

#### to form a heterotrimer complex that regulates rice grain quality [8]. To identify other com-*3.1. Plant Growth Conditions and Phenotype Measurement*

ponents that may interact with NF-YB1-YC12 dimer, we performed yeast-three hybrid (Y3H) experiments to screen a seed-derived prey library using NF-YB1-YC12-pBRIDGE as bait. We finally obtained three interactive proteins LOC\_Os01g68950, LOC\_Os07g46160 and LOC\_Os11g38670 which are annotated as ubiquitin domain-containing protein, BTB/POZ domain-containing protein and dead-box ATP-dependent RNA helicase, respectively. Interestingly, the interactions are valid only on the SD/-Met/-Leu/-Trp/-Ade/- To generate the CRISPR/Cas9-derived knock-out mutants, the specific target smallguide RNA (sgRNA) of each gene was designed and assembled in a pYLCRISPR/CAS9- MH vector system according to a previous report [49], and subsequently transformed into Nipponbare and Kitaake (*Oryza sativa*, ssp. japonica) backgrounds. All the plants were grown in the experimental greenhouse and field of the China National Rice Research

His/+X-α-Gal medium, in which the drop-out of methionine drove the expression of NF-YC12 under Met25 promoter. Meanwhile, the interactions were compromised on SD/-

mented methionine in the medium. Hence, the binding of NF-YB1-YC12 is necessary for

In conclusion, we report a rice pentuple gene mutant *pnfyc,* which knocked out five homologous genes *OsNF-YC8, OsNF-YC9, OsNF-YC10, OsNF-YC11* and *OsNF-YC12* simultaneously. The expression of starch synthesis genes decreased in *pnfyc,* resulting in the

the formation of heterotrimer complexes with the three proteins (Figure 5B–D).

Institute (CNRRI). Agronomic traits were analyzed with 10 replicates. Panicle length, number of primary branch panicles, number of effective panicles per plant, seed setting rate (%), and plant height were measured manually. Rice seed grains were harvested and air-dried at room temperature for at least 2 weeks. The thousand-grain-weight, seed length, width and chalkiness were examined by a seed phenotyping system (Wan Sheng, Hangzhou, China). Grain thickness was determined at the same time for each grain using an electronic digital calliper.

#### *3.2. Physicochemical Properties of Seed Grain*

Total starch content of the dried brown seeds was measured using a starch assay kits Megazyme K-TSTA and KAMYL (Megazyme, Ireland, UK, http://www.megazyme. com/ accessed on 6 May 2020). The total amylose and protein contents in the grains were measured by following a previous report [50]. The content is expressed as the percentage of total sample weight on an oven-dry basis. To analyze the gelatinization temperature, DSC assay was conducted on a differential scanning calorimeter DSC1 STARe system (METTLER-TOLEDO, Zurich, Switzerland). Briefly, 5 mg rice powder was sealed and placed in an aluminum sample cup, mixed with 10 µL distilled water, and then the samples were analyzed by the differential scanning calorimeter (METTLER-TOLEDO, Zurich, Switzerland). The heating rate was 10 ◦C min−<sup>1</sup> over a temperature range of 40 ◦C to 100 ◦C [51].

#### *3.3. Scanning Electron Microscopy (SEM) Assay*

Prepare two types of milled rice, one was wild-type NIP and the other was the *pnfyc* mutant. The whole grains were cut transversely with a sharp blade and then sputtered with gold in order to increase electrical conductivity. Fractured rice grains were mounted on the copper stage and then viewed with a scanning electron microscope at 30, 2000 and 5000 times magnification. The analysis was performed based on three biological replicates at least. The experiment was conducted in institute of Agriculture and Biotechnology, ZheJiang University as described previously using a HITACHI S-3400N scanning electron microscope (HITACHI, Tokyo, Japan).

#### *3.4. Seed Germination and Phenotypic Assay*

Briefly, 100 dehusked seeds were surface-sterilized in 75% ethanol for 2 min, then in 50% bleach for 30 min, and then cleaned with sterilized ddH2O 5–8 times for 3 min each time. The sterilized seeds were air dried and sown on a <sup>1</sup> 2 strength MS medium containing different concentrations of ABA (0, 2, 5 µM), GA (0, 2, 5 µM) and PAC (0, 2, 5, 10 µM). Germination rates were recorded every 12 h. The seedlings' height above ground was measured and the growth status of seedlings was photographed after 7 days. Germination is established with the appearance of the emergence of 2 mm embryos through the seed coat. The data are the mean of 3 biological triplicates.

#### *3.5. RNA-Seq Analysis and RT-PCR Analysis*

For RNA-seq, total RNA of germinative seeds at 6 h-after-imbibition was extracted using Trizol as instructed (Yeasen, Shanghai, China). The high-throughput sequencing was performed using the Illumina HiSeq™ 2500 platform and the KEGG pathway analysis of the DEGs was ultimately done by Personalgene Technology Co (Personal, Shanghai, China). DEGs were defined as genes with |log2Fold change| ≥ 1 and FDR < 0.01 using EBSeq [52]. The endosperm of 7 days after fertilization and seeds of 6 h after germination were collected as samples for the extraction of RNA to detect the expression level of genes in starch biosynthesis and hormone synthesis, and the RNA was extracted by Trizol according to the kit manufacturer's instructions (Yeasen, Shanghai, China).

For the RT-PCR analysis, the first-strand cDNA was synthesized using M-MLV reverse transcriptase according to the manufacturer's instructions (Takara, Dalian, China). The expression levels of different samples were determined using CFX96 touch real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Expression was assessed by evaluating threshold cycle (CT) values. The relative expression level of tested genes was normalized to ubiquitin gene and calculated by the 2−∆∆CT method. The experiment was performed in two biological replicates with three technical triplicates of each. Primer sequences are listed in Supplementary Table S3.

### *3.6. Yeast-Two-Hybrid Assay*

The yeast two-hybrid assay was conducted based on the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). The coding sequence of *NF-YC8* to *NF-YC12* and *SAPK1* to *SAPK10* were amplified and cloned into bait vector pdest32 and prey vector pdest22, respectively. Two vectors, pDEST32-NFYCs and pDEST-SAPKs, were co-transfected into the Y2H Gold strain, and then yeast cells were grown on SD/-Trp/-Leu and SD/-Trp/- Leu/-His/-Ade medium for screening. pGBKT7-53 and pGADT7-T were used as positive controls, pGBKT7-Lam and pGADT7-T were used as negative controls. The primers used are listed in Supplementary Table S3.

### *3.7. Yeast-Three-Hybrid Assay*

*NF-YB1* CDS was cloned to fuse with GAL4 BD domain, and NF-YC12 was driven by a methionine-responsive promoter Met25 in pBRIDGE (Clontech, Dalian, China). NF-YB1-NF-YC12-pBRIDGE in strain Y2H Gold was mated with an AD domain-fused seed cDNA library in Y187 strain. The mated transformants were first selected on SD/-Leu/-Trp. Positive colonies were then transferred to SD/-Leu/-Trp/-His/-Ade/-Met/+X-a-Gal and SD/-Leu/-Trp/-His/-Ade/+X-a-Gal, respectively. The interaction was confirmed by the visualization of blue colonies on the medium.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23158382/s1.

**Author Contributions:** Conceptualization, J.Z.; Formal analysis, S.L.; Investigation, J.L.; Methodology, L.C., X.L., M.Y. and X.T.; Project administration, J.Z.; Resources, B.B.K., J.Y. and J.Z.; Software, C.C., B.N., A.A.A. and X.L.; Supervision, Y.W. and X.Z.; Writing—original draft, H.X.; Writing review & editing, J.Z. All the authors read and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Natural Science Foundation of Zhejiang province (Grant No. 343 LZ21C130001), National Natural Science Foundation of China (Grant No. 32071986 and U20A2030), CNRRI key research and development project (CNRRI-2020-01), and ASTIP program of CAAS.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and within Supplementary Material.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

