**1. Introduction**

Rice (*Oryza sativa* L.) is a major cereal crop in the world, as it is consumed as a staple food by more than half of the world's population [1]. Rice seed is a complex organ that is comprised of a maternal caryopsis coat, a diploid embryo and a triploid endosperm. The nutrients such as starch, protein and lipids are accumulated in the endosperm underpinning seed germination or grain yield and quality for human consumption. It has been known that phytohormones are extensively involved in the regulation of plant seed development [2–5]. The action of GAs and ABA on seed development is strictly correlated and antagonistic [2]. Through the investigation of the rice seed hormonal dynamics during the grain filling stage, Yang et al. (2001) revealed that GAs play key roles in embryogenesis, while the ABA content reached the peak at a much later stage, thus it seemed to be more relevant to the seed maturation [6]. So far, numerous pieces of literature about genes controlling rice

**Citation:** Xu, H.; Li, S.; Kazeem, B.B.; Ajadi, A.A.; Luo, J.; Yin, M.; Liu, X.; Chen, L.; Ying, J.; Tong, X.; et al. Five Rice Seed-Specific *NF-YC* Genes Redundantly Regulate Grain Quality and Seed Germination via Interfering Gibberellin Pathway. *Int. J. Mol. Sci.* **2022**, *23*, 8382. https://doi.org /10.3390/ijms23158382

Academic Editor: Cristina Martínez-Villaluenga

Received: 26 May 2022 Accepted: 26 July 2022 Published: 29 July 2022

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seed development have been published, and these genes are involved in transcriptional regulation, the ubiquitin–proteasome pathway, plant hormone response, and so on [7–9]. Specifically, Yao et al. (2017) found that *NF-YC8* to *NF-YC12* are five important genes involved in starch synthesis, seed storage protein and the stress response [7]. The study shows that *NF-YC12* is a key transcription factor in regulating endosperm development [8] and storage material accumulation in rice seeds [10]. A novel transcription factor subunit *NF-YC13* was identified in indica rice, which can respond to salt stress signals by interacting with the B-subunit [11]. It is studied that NF-YC2 and NF-YC4 proteins can interact with three flowering-time genes to regulate the photoperiodic flowering response under long sunlight conditions [12].

Nuclear Factor Y (NF-Y) is a family of transcription factors that are found in vast quantities in higher eukaryotes. The NF-Y protein complex consists of three subunits: NF-YA (CBF-B/HAP2), NF-YB (CBF-A/HAP3), and NF-YC (CBF-C/HAP5) which usually forms a heterotrimer to regulate the transcription of the target genes [13,14]. For yeast and animals, each NF-Y subunit is encoded by a single gene. However, the situation in plants is more complicated with multiple members of each subunit, which dramatically expanded the diversity of *NF-Y*s' gene function in the plant kingdom [13,15]. In rice, each NF-Y subunit covers more than 10 gene members as reported, and many of them have been identified to participate in extensive developmental processes like nutrient accumulation in the endosperm, flowering regulation and ABA signal response [16–19]. *NF-YBs* are well-documented among those three subunits and have been implicated in plant height regulation, grain yield, carbon assimilation, photoperiodic flowering and other processes [20–24]. For example, *NF-YB2*, *NF-YB3* and *NF-YB4* are close homologs that are functionally redundant in regulating chloroplast biogenesis in rice [25]. It is noteworthy that several *NF-YBs* and *NF-YCs* were found to be specifically expressed in rice endosperm. Some seed-specific *NF-YBs* control rice seed development by affecting the nutrition accumulation and the loading of sucrose into developing seeds [26–28]. In addition, endosperm-specific *NF-YBs* and *NF-YCs* may also form heterotrimer complexes with other non-NF-Y transcription factors, hence regulating grain filling and quality via the ubiquitin–proteasome pathway [8,29]. For example, AtNF-YB9-YC12-bZIP67 can activate the expression of *SUS2* and promote seed development [30]; OsNF-YB1 interacts with OsNF-YC12, and OsNF-YC12 can bind to the promoter of *FLO6* and *OsGS1;3* to regulate grain weight and chalky endosperm [10].

Several previous works have revealed that OsNF-YC8 (LOC\_Os01g01290), OsNF-YC9 (LOC\_Os01g24460), OsNF-YC10 (LOC\_Os01g39850), OsNF-YC11 (LOC\_Os10g23910) and OsNF-YC12 (LOC\_Os05g11580) are close homologs with similar seed-specific expression pattern, implying that they play a role in rice seed development [7,8,29,31]. So far, it is known that the NF-YC9 controls cell proliferation to influence grain width, and NF-YC11 regulates the accumulation of storage substances in rice seeds [10,32]. In 2019, our lab reported that NF-YC12 forms a heterotrimer complex with NF-YB1 and bHLH144 to regulate rice grain quality [8]. Nevertheless, knowledge about the function of the five genes is rather fragmented, given that the high similarity of the genes may give rise to functional redundancy. Here, we report the systematic functional analysis of NF-YC8, 9, 10, 11 and 12 using single gene or pentuple gene mutants. The five genes may work redundantly to regulate ABA and GA response, thus determining grain quality and seed germination. This work sheds new insight into the functional roles of the seed-specific OsNF-YCs.

### **2. Results and Discussion**

#### *2.1. Five Seed-Specific NY-YCs Works Redundantly to Regulate Grain Quality*

To specify the biological functions of *OsNF-YC8*, *OsNF-YC9*, *OsNF-YC10*, *OsNF-YC11* and *OsNF-YC12*, we generated a single mutant of each gene in the background of Kitaake (*Oryza sativa* ssp. Japonica), respectively. Sanger sequencing further confirmed the mutations of the corresponding genes with insertion or deletions, which should have shifted the open reading frame and disrupted the resulting protein functions (Figure S1). Compared

with Kitaake, *nfyc8* exhibited increased percentage of grains with chalkiness (PGWC), *nfyc12* had higher degree of chalkiness (DEC), while *nfyc9* and *nfyc10* exhibited increased PGWC and higher DEC (Figure S2A–C). We used the CRISPR/Cas9 technology to simultaneously knock out all the five *NF-YC* genes in Nipponbare (NIP, *Oryza sativa* ssp. japonica) to generate the pentuple *nf-yc* mutants (hereafter referred to as *pnfyc*) to assess the potential functional redundancy among the genes. Two representative lines *pnfyc-1* and *pnfyc-2* were selected for the followed genotyping and genetic analysis. As shown in Figure S3, Sanger sequencing detected various types of homozygous insertion or deletion mutations in each of the *NF-YC8-12*, suggesting all the five genes were successfully knocked-out. During the vegetative growth stage, no visible differences were observed in major agronomic traits such as plant height, flowering date, seed setting and spikelets per panicle in the *pnfyc* lines (Table S1). However, the milled grains of *pnfyc* lines showed obvious chalkiness. As revealed by the cross-sections of the *pnfyc* seeds, the starchy endosperm of *pnfyc* was floury-white when compared with NIP. Scanning electron microscopy (SEM) images of transverse sections indicated that the starch granule of NIP and *pnfyc* grains had different morphologies, shape and packaging densities. Unlike the regular shape of the starch granule of NIP, *pnfyc* had irregular, loosely packed starch granules, which might be responsible for the observed chalkiness (Figure 1A). Furthermore, we examined the contents of storage substances in the brown seeds, and found that the total starch and amylose contents of *pnfyc* were significantly lower than that of the NIP. Conversely, *pnfyc* had relatively higher crude protein contents than NIP (Figure 1B–D). The PGWC in *pnfyc* reached over 90%, while that of NIP was less than 10% (Figure 1E). Following the change in the starch contents, differential scanning calorimetry (DSC) analysis demonstrated that the gelatinization characteristics including the onset, peak as well as end gelatinization temperatures of *pnfyc* were also significantly altered (Figure 1F and Table S2). The results above indicated that *OsNF-YC8*, *9*, *10*, *11* and *12* work redundantly to positive regulate rice grain quality, particularly the grain chalkiness.
