Regulatory mechanism and molecular genetic dissection of rice (Oryza sativa L.) grain size

Abstract

With the sharp increase of the global population, adequate food supply is a great challenge. Grain size is an essential determinant of rice yield and quality. It is a typical quantitative trait controlled by multiple genes. In this paper, we summarized the quantitative trait loci (QTL) that have been molecularly characterized and provided a comprehensive summary of the regulation mechanism and genetic pathways of rice grain size. These pathways include the ubiquitin-proteasome system, G-protein, mitogen-activated protein kinase, phytohormone, transcriptional factors, abiotic stress. In addition, we discuss the possible application of advanced molecular biology methods and reasonable breeding strategies, and prospective on the development of high-yielding and high-quality rice varieties using molecular biology techniques.

1. Introduction

Rice (Oryza sativa L.) is the main food crop for more than 50 % of the global population. It is estimated that by 2030, rice production needs to increase by 40 % to meet the expected requirements of the growing world population. Therefore, increasing rice production plays a critical role in ensuring world food security. There are three crucial factors of rice yield: the number of panicles per plant, the number of grains per panicle, and grain weight. The grain size of rice includes four dimensions: grain length, grain width, grain weight, and length-width ratio, which positively correlate with grain weight [1]. As a critical breeding target, the grain size of rice directly affects the yield and quality of rice [2]. Seed size is determined by the integrated signals of maternal and zygotic tissues, controls the coordinated growth of the embryo, endosperm, and seed coat. Therefore, the grain size depends on the maternal genotype [3].

In the last few decades, scientists have detected a large amount of rice grain size QTL, and cloned many rice grain size genes. These findings are valuable for illustrating the molecular regulation mechanism of rice grain and breeding new high-yield and high-quality rice varieties. In this review, we summarize recent advances in molecular identification of important QTL for rice grain size and their molecular mechanisms, conclude their signaling pathways, regulatory mechanisms, and genetic relationships in a molecular background, provide insights into the opportunities and challenges faced by breeders in the post-genomic era.

2. QTL mapping for grain size and weight of rice

Grain size mainly composed by grain size, grain length, grain width, grain thickness, aspect ratio and grain weight, is a complex quantitative trait. Up to now, more than 400 QTL controlling grain size and weight have been identified by using various mapping populations [4,5]. Among of these QTL, there are 103 QTL related to grain length, 95 QTL related to grain width and relatively few QTL related to grain aspect ratio and grain thickness. These QTL distribute on 12 chromosomes of rice. In this review, we characterize the molecular features of several major QTL/genes for grain size and explore their role in determining the regulatory mechanisms of grain size or weight (Fig. 1). GL2/GS2 regulating grain length and weight was mapped within the 700 kb region of RM13792 and RM8248, on chromosome two by using BC₃F₂ population derived from the cross between BobaiB (BBB) and RW11. GL2, an allele of OsGRF4, can potentially increase grain weight and grain yield. And it interacts with OsGSK2, a negative regulator of oleuropein lactones, which increased the expression of GRF4 and promoted seed development [6]. GW2 encodes a previously RING-type protein with E3 ubiquitin ligase activity. Functional deletion of GW2 increases the number of cells and the rate of filling, negatively regulates rice grain length and width [7].

Fig. 1.

Genes Associated with Rice Grain Length and Width. The genes associated with rice grain length and width are divided into two groups: positive regulators (black) and negative regulators (red). The positive regulators including: OsBRI1 [8], PGL2 [9], qGL3-1 [10], PGL1, GW6a [11], TGW6 [12], DEP2 [13], TID1 [14], GW2 [15], GS2 [16], GSD1 [17], GS5 [18], GR5 [19] GW5 [20], APG [21], GS6 [22], SMG11 [23], SMG1 [24], qIGL1 [25], qGL3 [26], BRD1 [27], OsRGB1 [28], OsMADS1 [29], TUD1 [30], XIAO [31], D11 [32], OsGSK2 [33], GL7 [34], GL7NR [35], OsGASR9 [36], GE [37], OsBZR1 [32], SLG [38], OsBAK1 [39], OsFIE1 [40], OsDEP1 [41], Brd 2 [42], OsGBP3 [43]; The negative regulators including: GS3 [44], RGG1 [45], SMG2 [46], OsSRS3 [47], CLG1 [2], OsGBP1 [43], qGW8 [48], WTG1 [49], SG1 [50], SLG1 [51], OsPPKL3 [52].

A major QTL for grain weight and grain length, GS3, was mapped on rice chromosome 3 by using a backcross population crossing between Mingchuan 63 (large grain) and Chuan 7 (small grain). The GS3 locus can explain 80–90% of grain weight and length variation in the BC₃F₂ population. These findings suggest that GS3 may be a negative regulator that controls grain size growth [53]. GS3 encodes a transmembrane protein containing a plant-specific organ size regulation (OSR) domain. At the cellular level, GS3 regulates seed length by controlling cell number in the upper epidermis of the glumes [54]. FAZ1 (small grain indica variety) and WY3 (larger grain indica variety) rice varieties were selected as parents to map a new major QTL, GL3.1, which of grain length. GL3.1 encodes a protein phosphatase kelch (PPKL) family-Ser/Thr phosphatase. GL3.1-WY3 influences the phosphorylation of proteins in spikelets and accelerates the cell division rate, increasing grain length and crop yield [55]. GWAS detects Ninety-nine QTL for grain length. OsLG3, has been identified, encoding an APETALA2/ethylene-responsive element binding protein, which can regulate grain length positively and improve rice yield without influencing grain quality [56]. qTGW3/TGW3/GL3.3, is also located on chromosome 3 and encodes an Osk41/OsGSK5 protein kinase involved in phosphorylating the auxin inhibitor factor OsARF4, negatively regulating grain length. Expression analysis showed that qTGW3 was primarily expressed in young rice spikelet and regulated rice grain length negatively by interacting with and phosphorylating OsARF4, a transcriptional repressor in the growth factor pathway, and inhibiting the expression of genes related to the growth factor signaling pathway during grain development [57,58].

A major grain length QTL, GL4 controlling rice grain length was mapped on the region between RM3335 and RM5608 on chromosome 4 by using An F₂ population containing 186 lines crossing between the progressive line GIL25 with and the African cultivar IRGC102305. It was shown that single nucleotide polymorphism (SNP) mutations in the GL4 results in the early stop termination of codons in the premature coding region, which results in small grain and seed shattering in African wild rice [59].

GW5, a major QTL controlling grain width and grain weight in rice, codes a novel nuclear protein, and plays a role in the ubiquitin-proteasome pathway, regulates cell division during seed growth [60]. GW5 protein is localized at the plasma membrane. and inhibits the activity of the GSK2 kinase, resulting in the accumulation of unphosphorylated OsBZR1 and DLT proteins in the nucleus, which mediates the expression of brassinosteroid (BR) responsive genes and the growth responses, thereby negatively regulates the grain width of rice seeds [20]. qGL5/OsAUX3,a major gene negatively regulating grain length and weight were identified by using a recombinant inbred line population crossing between 9311 and Nipponbare. The growth factor OsARF6 can directly bind to the AuxRE motif of the qGL5/OsAUX3 promoter and positively regulate the expression of qGL5/OsAUX3 [61].

qTGW6 for 1000-grain weight were analyzed under different environments in backcross inbred lines of Nipponbare and Kasalath. TGW6 can enhance the accumulation of carbohydrates in plants, thereby increasing rice yield potential [62]. TGW6 encodes a novel protein with IAA glucose hydrolase activity, affecting the transition from syncytial to cytosolic phase by controlling growth hormone activity and limiting the increase of cell number and length [63]. GW6a/OsglHAT1, associated with grain length and grain weight, encoding a novel GNAT-like protein with intrinsic histone acetyltransferase activity (OsglHAT1), was mapped on chromosome 6. GW6a/OsglHAT1 increases grain length and weight by positively regulating cell number and grain filling rate [64].

GLW7 is a major QTL for grain length and grain width, explaining 30% of the variation in grain length and 25% in grain width in Japonica rice varieties. It was verified that OsSPL13 is the target gene of GLW7. OsSPL13, a plant-specific transcription factor, positively regulates grain length and weight by modulating rice glume cell size [65]. qSS7, a major QTL determining grain length, width, and the ratio of seed length to width, was mapped on the long arm of chromosome 7. It was shown that qSS7 can regulates grain length by modulating cell elongation in the glume lemma [66]. GW7 encodes a TONNEAU1-recruiting motif protein. Up-regulation of GW7 expression can promote the longitudinal cell division and reduce the transverse cell division. OsSPL6 (GW8), an SBP domain transcription factor that regulating grain width, directly binds to and represses the expression of the GW7 promoter [67]. qLGY3 encodes MADS domain transcription factor OsMADS1, a key downstream effector of G protein βγ dimerization. positively regulates grain length by promoting longitudinal cell extension [29].

qGW8, a primary QTL for grain width, was detected in the long arm of chromosome 8 by using 153 single-segment substitution lines (SLS). GW8, a homologous gene of OsSPL16, positively regulates cell proliferation. High expression of GW8 promotes cell division and grain filling, which increases grain width in rice [48]. GS9 encoding a protein with unknown conserved functional domain, negatively regulates rice grain size by altering cell division. GS9 interacts with the ovate family proteins OsOFP14 and OsOFP8, which is regulated by the OsGSK2 [68].

3. Regulatory mechanisms of grain size in rice

Rice grain size affects the crop's adaptability to environmental and directly determines the crop's yield. Seed size is majorly determined by Genetic factors, seed growth is coordinately controlled by both maternal tissues and zygotic tissues [3]. Research identified several signaling pathways that control grain size by regulating maternal tissue growth, including ubiquitin-proteasome pathway G protein signaling, MAPK signaling, phytohormone signaling, and transcriptional regulators; these pathways control rice grain size by affecting the development of the grain. Related studies have shown that plant abiotic stress, photosynthetic product accumulation, and grain filling rate can affect rice grain size by influencing rice grain filling. Then, we will discuss recent advances in grain size regulatory pathways in rice.

3.1. Ubiquitin-proteasome pathway

Protein ubiquitination affects many aspects of protein stability, activity, structure, and cellular metabolism. Ubiquitination usually occurs on the lysine (K) side chain of the target protein, a process that involves the coordinated action of E1-ubiquitin activating enzymes, E2-ubiquitin conjugating enzymes, and E3-ubiquitin ligases. The function of the ubiquitinated proteasome pathway in regulating the grain size of rice has received broad interest (Fig. 2).

Fig. 2.

E3 ubiquitin ligase GW2 interacts with EXPLA1, which functions as a cell relaxation protein and promotes cell expansion. When external growth signals are enhanced, WG1 binds to the transcriptional repressor ASP1, inhibiting the transcriptional activity of OsbZIP47, which negatively regulates rice cell proliferation. In addition, in the presence of enhanced external growth signals, GW2 promotes the ubiquitination of WG1, which enhances the transcriptional activation activity of OsbZIP47 and promotes cell growth. Furthermore, E3 ubiquitin ligase directly interacts with EXPLA to regulate cell expansion; GW6a ubiquitinates HDR3, a ubiquitin-containing receptor involved in seed size regulation; CLG1 ubiquitinates GS3-1/2/3, but GS3-4 is localized on the cell membrane and promotes grain length by balancing G protein signaling.

Arabidopsis thaliana DA1, encoding a predicted ubiquitin receptor, regulating seed size by limiting cell proliferation [69]. A grain width-regulated gene with ubiquitinates activity, OsUBP15, was identified by map-based cloning by using the mutant material lg1-D and the wild type as parents. OsUBP15 positively regulates grain width in rice [70]. GW2 encoding a RING-type protein with E3 ubiquitin ligase activity, negatively regulates grain width and weight in rice. GW2 can elevate rice yield without compromising seed quality, exhibiting significant potential-for increased productivity [15]. CLG1, which encodes an E3 ligase, positively regulates grain size and interacts with GS3. CLG1 ubiquitinates GS3, which is subsequently degraded via the endosomal degradation pathway, leading to an increased grain size [71]. It was shown that GW6a interacts with HDR3, the ubiquitin response factor of DA1, regulates grain size by altering cell proliferation in spikelet hulls [15]. Mechanistically, HDR3 interacts with and stabilizes GW6a in an ubiquitin-dependent way, delaying protein degradation by the 26S proteasome, positively regulating the grain size [72]. The 26S proteasome is a multi catalytic proteasome composing of a 20S core protease and a 19S regulatory particle (RP). Studies have shown that a subunit of RP, RPT2, is involved in controlling seed size in Arabidopsis. The Arabidopsis RPT2 subunit is mainly encoded by two homologous genes, AtRPT2a and AtRPT2b. Lossing of the function of the subunit-regulated granule AAA ATPase (RPT2a) leads to a decrease in 26S proteasome activity and results in enlarged leaves, stems, flowers, fruits, seeds, and embryos [73]. WTG1 determines grain size and shape mainly by affecting cell expansion. WTG1 encodes an otubain-like protease with ubiquitination, and the lossing of ubiquitination activity of the mutant protein (wtg1-1) results in decreased rice seed size [49]. GSE5, encoding a plasma membrane-associated protein, interacts with the rice calmodulin OsCaM1-1. GSE5 negatively regulates grain width and grain weight in rice by affecting cell proliferation in the glumes [74].

3.2. G-protein signaling

Heterotrimeric GTP-binding proteins (G proteins) are signal transduction elements that mediate membrane receptor action in eukaryotic organisms [75]. G proteins are composed of alpha (α), beta (β), and gamma (γ) subunits, usually localized at the cytoplasmic face of the plasma membrane (Fig. 3). G protein promotes GDP release and GTP binding, and plays a crucial role in plant growth and development [76]. In Arabidopsis, three genes encoding Gγ subunits (AGGs): AGG1, AGG2, and AGG3 were identified. G protein c-subunit (AGG3) regulates plant morphological development in Arabidopsis thaliana. The homologs of AGG3 in rice have been identified as crucial genes for grain size and yield [77].

Fig. 3.

G protein signaling controls grain size in rice. Gβ (RGB1) interacts with GS3/DEP1 and regulates the expression of the downstream gene OsMADS1, which regulates the proliferation and expansion of rice cells to change the rice grain size. Meanwhile, GS3/DEP1 antagonizes Gγ (GGC2) and competitively binds to Gβ (RGB1) to regulate seed grain size.

There are one Gα (RGA1), one Gβ (RGB1) and five Gγ homologs (RGG1, RGG2, GS3, qPE9-1/DEP1 and GGC2) in the rice. Three Gγ proteins, DEP1, GGC2, and GS3, antagonistically regulate grain size. DEP1 and GGC2 complexed with Gβ increase grain length. GS3 having no effect on grain size by itself but reduces grain length through a competitive interaction with Gβ [78]. qLGY3 encodes a MADS domain transcription factor, OsMADS1, a critical downstream effector of G-protein βγ dimer. The Gγ subunits GS3 and DEP1 function in direct interactions with the MADS transcription factors to enhance the transcriptional activity of OsMADS1 and promote synergistic transactivation of common target genes to regulate grain size and shape [29].

GR5, a transcriptional activator, determines grain size by affecting cell proliferation and expansion. GR5 physically interacts with five Gγ subunit proteins, including RGG1, RGG2, DEP1, GS3, and GGC2, and works in downstream of the G-protein complex to regulate the expression of downstream target genes which determine grain size [19]. GTPase-activating proteins are closely related to a family of G-protein-associated receptors regulating G-protein binding and signaling with other proteins. GRAIN SIZE AND WEIGHT 3 (GSW3), a GTPase-regulated protein, negatively regulates grain length and width by promoting grain glume cell division and cell growth. Mutations in a crucial SNP in the coding region of GSW3 result in an amino acid substitution from Gln to Arg at position 161 of GSW3, which reduced the grain size [79].

3.3. Mitogen-activated protein kinase (MAPK) signaling

Mitogen-activated protein kinase (MAPK) cascade is a generic signal transduction module in eukaryotes [80]. Each MAPK cascade consists of a set of three sequentially acting protein kinases: MAPK, MAPK kinase (MKK), and kinase of MAPK kinase (MKKK) [81]. OsMKK10-OsMKK4-OsMPK6 participate in rice panicle morphogenesis and act in a common pathway. GSN1 encodes OsMKP1, a mitogen-activated protein kinase phosphatase. As a negative regulator of the OsMKKK10-OsMKK4-OsMPK6, the GSN1-MAPK module coordinates the balance between grain number and grain size by integrating localized cell differentiation and proliferation [46] (Fig. 4). OsMKK4, the upstream kinase of OsMPK1, phosphorylates OsMPK1 through wounding in vivo. OsMPK1 directly interacts with the rice defense-related transcription factor OsWRKY53. The injury signaling pathway, OsMKK4-OsMPK1-OsWRKY53, may be important in regulating the interplay between abiotic and biotic stresses in plants [82].

Fig. 4.

Mitogen-activated protein kinase signaling pathway regulates the seed size pathway; OsMKKK10, OsMKK4, and OsMAPK6 negatively regulate rice grain length as a response. OsWRKY53 is a target of the OsMKKK10-OsMKK4-OsMAPK6 module to regulate grain growth by promoting the expansion of cells in the husk of the spikelet. OsMKKK70, which interacts with OsMKK4, promotes the phosphorylation of OsMAPK6, which positively regulates grain size in rice.

The Oryza sativa Mitogen-Activated Protein Kinase Kinase OsMKKK70 regulates rice grain size and leaf angle by activating with OsMAPK6. OsMKKK70 is an active kinase, interacts with OsMKK4 and promotes the phosphorylation of OsMAPK6 [83].

3.4. Phytohormone signaling

Plant hormones have multiple functions in plant growth and development, responsing to stress, and metabolism. Brassinosteroids (BR), auxin, gibberellin, and cytokinin have been proven to regulate grain growth through maternal tissues. In addition, the development of endosperm is also regulated by phytohormones, consequently affecting grain size (Fig. 5).

Fig. 5.

Phytohormone regulation of rice seed grain size: XIAO is a regulator of Brassinosteroids signaling and cell division, and the reduction of BR deficiency results in a lower rate of cell division, leading to a reduction in cell number. OsPPKL, encoded by qGL3/qGL3.1, regulates grain size by restricting cell proliferation in the spikelet hulls; The activation decreased of GSK2 can result in reduced grain length in rice, and there may be a cooperative relationship between BR and CK in the regulation of grain size in rice; GW6 encodes a GA-sensitive GAST family gene, which affects GA signaling and regulates grain width and grain weight in rice. OsGASR9 regulates rice grain size through the GA pathway. qTGW3/GL3.3 encodes an OsSK41/OsGSK protein that interacts with OsARF4 to negatively regulate rice grain size and grain weight by affecting cell expansion in the spikelet hulls. The OsPIL15-OsPUP7 module affects grain size by increasing the number of divisions of the endosperm cells to enhance grain filling. The OsPIL15-OsPUP7 module negatively regulated grain size and grain weight in rice.

3.4.1. Brassinosteroids (BR)

Both BR deletion and BR insensitive mutants in Arabidopsis and rice produce short-grained. SLG (BAHD acyltransferase-like protein gene) is an important regulator of BR homeostasis. Slg-D mutant showed mild BR-deficient phenotypes, including shorter grains [38]. XIAO encoding an LRR kinase co-expressed with many genes involving in the cell cycle. XIAO is a regulator of BR signaling and cell division. Reducing the expression of XIAO leads to a decrease in the rate of cell division and a reduction in the number of cells, which leads to smaller grain size [31].

In Arabidopsis, the GSK3/shaggy-like kinase brassinosteroid-insensitive 2 (BIN2) regulates the expression of downstream BR-responsive genes. It plays a critical role in the BR signaling pathway. The activation of rice GSK3/SHAGGY-like kinase (GSK2), one of the orthologs of BIN2, results in a typical BR-deficient phenotype in rice and involves a signaling pathway that regulates seed size in rice [84]. qGL3 encodes a putative protein phosphatase (OsPPKL1) with a Kelch-like repeat structural domain. qGL3 inhibits BR signaling by regulating the phosphorylation and stability of OsGSK3, ultimately resulting in shorter grains [85]. GS5 positively regulates rice grain width by binding to the extracellular leucine-rich repeat (LRR) domain of OsBAK1-7, which competitively inhibits the interaction between OsBAK1-7 and OsMSBP1 and enhances BR signaling [86]. GLW10 encoding a putative BR signaling kinase (OsBSK2), regulates grain size by affecting cell expansion. OsBSK2 directly interacts with the OsBRI1 (BR receptor kinase), regulating grain size independently of the BR signaling pathway [87].

3.4.2. Gibberellins

Gibberellin regulates seed germination, flowering, fruiting and grain size by regulating cell growth. However, present studies have mainly focused on the effects of blocking GA biosynthesis or signaling in the GA pathway on grain size, the specific signaling pathways are still unclear. GW6 encoding a GA-regulated GAST family protein, positively regulates grain width and weight by promoting the expansion of spikelet shell cells. GW6 is induced by GA, knockout of GW6 downregulates the expression of GA biosynthetic genes and reduces GA content in the young panicles [88]. OsGASRs (Plant-specific GA-stimulated transcription gene family) are critical for plant growth and development. Ten OsGASR family members have been characterized in rice. OsGASR9 is a positive regulator responding to GA, regulates plant height, grain size, and yield through the GA pathway [36].

WRKYs are transcription factors that regulate plant resistance growth and development, including height and grain size. The sgsd3 mutant reduced sensitivity to GA and accumulates higher levels of SLR1 (a DELLA-like inhibitor of GA signaling). OsWRKY36 protects SLR1 from GA-mediated degradation, suppressing GA signaling by stabilizing the expression of SLR1 and negatively regulating grain size in rice [89].

3.4.3. Auxin

Auxin regulates the growth process of plants by determining the rate of cell division and expansion or cell development. Growth hormone signaling is mediated by a combination of growth hormone/indole-3-acetic acid transcriptional repressor and growth hormone response factor (ARF) transcription factors. Most ARF proteins contain three conserved structural domains: the N-terminal DNA-binding domain (DBD), the mid-domain (MD), and the C-terminal dimerization domain (CTD). The DBD binds specifically to AuxRE and regulates the expression of growth hormone response genes [90].

qTGW3 encodes OsSK41 (OsGSK5), a member of the GLYCOGEN SYNTHASE KINASE 3/SHAGGY-like protein family. OsSK41 interacts with and phosphorylates AUXIN RESPONSE FACTOR 4 (OsARF4), loss-of-function mutations of OsARF4 result in larger grain size in rice [26]. The dominant mutant big grain 1 (Bg1-D) exhibits an oversized grain. Overexpression of BG1 leads to a significant increase in grain size, and the mutant has an increased sensitivity to auxin and N-1-naphthylphthalamic acid (an auxin transporter inhibitor), whereas knockdown of BG1 results in a decreased sensitivity and smaller grains [91]. TGW6 encodes a protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. During grain development, the tgw6 allele influences grain size by controlling IAA synthesis, limiting cell number, and influencing the transition from the syncytium to the cell phase [12].

3.4.4. Cytokinins

Cytokinins influence cell proliferation and expansion by controlling the cell cycle of the apical meristem. It is important in regulating grain number and size in rice [92]. Big grain 3 (bg3-D) exhibits a significant increase in grain size caused by the activation of the PURINE PERMEASE gene OsPUP4. OsPUP4 is localized in the plasma membrane, whereas OsPUP7 is localized in the endoplasmic reticulum. OsPUP4 and OsPUP7 direct the transport of cytokinins between cells in a linear pathway that affects the long-range movement and local distribution of cytokinin [93]. The division and proliferation of endosperm cells can be promoted by the exogenous application of cytokinins, leading to an enhancement in the grain filling rate. In rice, six photoreceptor action factors have been identified. OsPIL15, a member of the essential helix-loop-helix transcription factor families, interacting with OsPUP7. Study has shown that the OsPIL15/OsPUP7 module can regulate rice grain morphology by enhancing endosperm cell division and grain filling [94].

3.5. Transcriptional regulatory pathway

Transcriptional regulation is crucial for many plant growth and developmental processes, some transcription factors regulate seed grain type. The SPL (SQUAMOSA Promoter Binding Protein-Like) family, a plant-specific group of transcription factors, has been confirmed to participate in various developmental processes and physiological activities, exerting a significant role in rice yield-related traits. GLW7 encoding the plant-specific transcription factor OsSPL13, increases the length and yield of rice grains by positively regulating the size of cells in the husk of rice [84]. GS2 is a semi-dominant gene controlling grain size and weight in rice. GS2 encodes the transcription factor OsGRF4 (GROWTH REGULATING FACTOR 4), regulated by OsmiR396. A 2 bp substitution mutation in GS2 disrupts the directional regulation of GS2 by OsmiR396, resulting in bigger and heavier grain size [95]. One SNP (SNP1066) in the SPL12 transcriptional activation structural domain is strongly associated with grain width and increases transcriptional activity. OsSPL12 directly binds to the 1212-bp fragment of the Indica rice. GW5 promoter and induces the expression of GW5, which determines the difference in grain size between Indica and Japonica rice through enhancer-promoter structure [96]. GW8 an allele of OsSPL16, is a positive regulator of cell proliferation. Overexpression of GW8 can promote grain width and yield in rice [48]. GW7 encodes a TONNEAU-recruiting motif protein and positively regulates grain length in rice by increasing longitudinal and decreasing transverse cell divisions. OsSPL6 (GW8), an SBP domain transcription factor that regulates grain width, directly binds to and represses the expression of the GW7 promoter [67]. Variation in the OsAUX3 promoter resulted in the function decreased of OsAUX3, and increased grain length and width. It was demonstrated that OsARF6 is an upstream transcription factor regulating the expression of OsAUX3. OsARF6 directly binds with the auxin-responsive element of the OsAUX3 promoter and controls grain length by changing the longitudinal expansion of the glume cells and auxin distribution/content [97].

The MAD-box gene is a widely existing transcription factor in plants, which has essential functions in controlling spike development. OsMADS1, a MADS-box transcriptional regulator gene family member, directly regulates seed development and grain size in rice, mainly caused by abnormal lemma and palea development [98].

4. Other pathways for regulating grain size in rice

Rice grain size is also affected by abiotic stresses. GSA1 encoding a UDP glucosyltransferase, regulates grain size by affecting cell proliferation and expansion, which are regulated by flavonoid-mediated growth auxin content and expression of related genes. Overexpression of GSA1 results in larger rice grains and enhanced the abiotic stress tolerance of rice [99]. Small heat shock proteins (sHsps) are ATP-independent chaperone proteins, widely expressed in response to diverse environmental stresses. A single hairpin construct was designed to generate all silenced CI-sHsp rice lines [100]. Rice RNAiCI-sHsp seedlings showed reduced heat tolerance and significantly decreased seed grain length. The 14-3-3 family were widely involved in regulating plant growth and development [101]. Research demonstrates that Os14-3-3 genes may regulate grain size in JA signaling [102,103].

The size of rice hulls directly determines the primary factor of grain size. As early as 1970, Takeda and Takahash proposed that the size of the grain husk directly determines grain size of rice [104]. OsQUA2 is essential for maintaining a high degree of methyl esterification in the cell wall of the rice stalk-screening element (SE), which is critical for efficient Suc partitioning and grain filling [105]. GF14f, a member of the 14-3-3 family of proteins, showed differences in expression patterns between SS (earlier flowering superior spikelets) and IS (later flowering inferior spikelets), negatively regulate the seed size and seed filling [106]. FGW1 encoding a protein containing the DUF630/DUF632 structural domains, directly interacts with GF14f, it positively regulating seed width by affecting glume cytokinesis and expansion [107].

5. Discussion and perspectives

Cultivated rice varieties that have domesticated from wild rice through both natural and artificial selection. In this process, some elite genes were selected while some were eliminated, resulting in the gradual decrease of rice genetic diversity. So, it is necessary to explore new genes and excellent alleles which regulating rice grain size in wild rice populations. At the same time, contemporary high-throughput sequencing technology with GWAS, BSA, and other bioinformatics tools can be used to discover new QTL/gene related to the regulation of rice grain size, and the molecular mechanism of grain size can be explored further by combining transcriptomic, proteomic and metabolomic tools.

In recent decades, researchers have characterized over 400 QTL related to rice grain size, and the molecular mechanisms of regulating rice grain size have been studied. The main pathways include Ubiquitination-proteasome, G proteins, mitogen-activated protein kinases, transcription factors, etc. In the previous section, we have reviewed the progress of the different pathways, and the connection between these pathways has not yet been revealed. Rice grain size is mainly determined by its genetic factors and is also affected by the external environment. RGA1 is a positive regulator of drought tolerance in rice, and the RGA1 allele D1 encodes the alpha subunit of GTP-binding proteins (G proteins), regulating the size of grains through GA signaling [108,109]. Researchers can focus on the critical regulator factors of the grain size pathway, finding target genes in other pathways and connecting these pathways. Meanwhile, the researchers used forward genetics to explore the critical QTL affecting grain shape under stress, further understand the mechanism of abiotic stress on rice grain shape, and comprehensively reveal the molecular mechanisms regulating rice grain size.

Variety improvement is an important practice in the agricultural production of rice. Traditional breeding mainly uses allelic variation under different background materials to select elite strains, but the result may not be as expected. Using grain size genes for variety improvement may affect other traits of rice and result in lower yields. In our opinion, the appropriate elite genes can be selected according to the need for gene polymerization to achieve precision breeding. In polymerizing different elite genes, it is important to break the negative linkage between rice yield and quality and improve rice quality effectively. Breeders can utilize the genome editing technology CRISPR/Cas9 to accurately and efficiently polymerize superior allele genes and precisely knock out unfavorable traits in rice varieties, and rapidly obtaining new materials to improve breeding efficiency. Combine traditional and modern breeding to cultivate new high-yield and high-quality rice varieties.

Grain size is significantly different among plant species. Exploring the essential genes responsible for grain size variation among different species will help us to understand the molecular mechanism of grain size regulation on the gene origination and evolution level. The evolutionary characteristics of grain size can be explored by analyzing the genetic and genomic data of species. Grain size is a yield trait mainly linked to quality and combines the breeding objectives of achieving high yields and good quality. Therefore, exploring the molecular regulation mechanism of rice grain size and making appropriate breeding strategies to achieve high yield and quality rice is important.

Data availability statement

Data availability does not apply to this article as no new data were created or analyzed in this study.

Additional information

No additional information is available for this paper.

CRediT authorship contribution statement

Yuntao Yan: Writing – original draft. Xiaoya Zhu: Writing – original draft. Hui Qi: Methodology. Haiqing Zhang: Writing – review & editing. Jiwai He: Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.